CN119451976A

CN119451976A - Mutated fragment of VZV glycoprotein E

Info

Publication number: CN119451976A
Application number: CN202480002895.4A
Authority: CN
Inventors: 宋海峰; 冀凯; 路希山
Original assignee: Suzhou Aibo Biotechnology Co ltd
Current assignee: Suzhou Aibo Biotechnology Co ltd
Priority date: 2023-05-31
Filing date: 2024-05-30
Publication date: 2025-02-14
Also published as: WO2024245317A1; WO2024245317A9

Abstract

Provided herein are fragments of the gE protein of varicella-zoster virus and therapeutic nucleic acid molecules for controlling, preventing and/or treating diseases or conditions caused by varicella-zoster virus or by infection with varicella-zoster virus. Also provided herein are therapeutic compositions, including vaccines and lipid nanoparticles, comprising the therapeutic nucleic acids, and related therapeutic methods and uses.

Description

Mutant fragments of VZV glycoprotein E

1. Technical field

The present disclosure relates generally to fragments of glycoprotein E (gE) proteins of varicella-zoster virus (VZV) and nucleic acid molecules useful for controlling, preventing, and treating diseases or conditions caused by VZV or by infection with VZV. The disclosure also relates to lipid-containing compositions (including vaccines) of the nucleic acid molecules.

2. Background art

Varicella Zoster Virus (VZV), also known as human herpesvirus type 3, is a double stranded DNA virus belonging to the alpha herpesvirus. VZV has only one serotype. VZV has a genome comprising 71 genes and encoding 67 different proteins, including 6 glycoproteins, now designated gE, gB, gH, gI, gC and gL. Glycoproteins gE, gB and gH are very abundant in infected cells and are also present in the envelope of the virion. Antibodies induced by the three major glycoproteins can neutralize the virus. Specific humoral and cellular immunity, and cytokines such as interferons, play a major role in limiting the spread and recovery of VZV, with specific cellular immunity being particularly important.

Zostavax (MSD) is an attenuated viral vaccine. It can reduce the load by 61.1% (65.5% in the population between 60 and 69 years old and 55.4% in the population 70 years old and older). It may also reduce the duration of pain and discomfort caused by the virus. Shinrix (GSK) is a subunit vaccine. It comprises gE and AS01B, AS01B being an adjuvant system for enhancing cellular immune responses. It can reach the effect of over 90%.

3. Summary of the invention

In one aspect, provided herein are fragments of the gE protein of VZV, optionally comprising mutations, e.g., substitutions.

In some embodiments, the fragment comprises a truncation of at least 1 amino acid residue and up to 49 amino acid residues from the C-terminus as compared to the mature gE protein. In some embodiments, the fragment comprises a truncation of 49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2 or 1 amino acid residue from the C-terminus compared to the mature gE protein. In some embodiments, the fragment comprises a truncation of amino acid residues from the C-terminus 11-18 (e.g., 11, 12, 13, 14, 15, 16, 17, or 18) or 34-44 (e.g., 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44) as compared to the mature gE protein. In some embodiments, the fragment comprises a truncation of from the C-terminal 12-16 (e.g., 12, 13, 14, 15, or 16) or 35-39 (e.g., 35, 36, 37, 38, or 39) amino acid residues as compared to the mature gE protein. In some embodiments, the fragment comprises a truncation of 14 or 37 amino acid residues from the C-terminus as compared to the mature gE protein.

In some embodiments, the fragment comprises the substitution Y569A. In some embodiments, the fragment comprises the substitution Y582G. In some embodiments, the fragment comprises the substitutions Y569A and Y582G. In some embodiments, the fragment comprises the substitution S593A. In some embodiments, the fragment comprises the substitution S595A. In some embodiments, the fragment comprises the substitution T596A. In some embodiments, the fragment comprises the substitution T598A. In some embodiments, the fragment comprises the substitutions S593A, S595A, T596A and T598A. In some embodiments, the fragment comprises the substitutions Y569A, Y582G, S593A, S595A, T596A and T598A. In such embodiments, the amino acid positions are numbered based on the full length gE protein.

In some embodiments, the fragment comprises a truncation of 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution Y569A.

In some embodiments, the fragment comprises a truncation of 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution Y582G, with or without the substitution Y569A. In such embodiments, the fragment comprises the substitutions Y569A and Y582G.

In some embodiments, the fragment comprises a truncation of 30 or 29 amino acid residues from the C-terminus compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution S593A, with or without the substitution Y569A and/or Y582G. In such embodiments, the fragment comprises the substitutions Y569A, Y582G and S593A. In some embodiments, the fragment comprises a truncation of 28 amino acid residues from the C-terminus compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution S595A, with or without the substitution Y569A and/or Y582G and/or S593A. In such embodiments, the fragment comprises the substitutions Y569A, Y582G, S a and S595A. In some embodiments, the fragment comprises a truncation of 27 or 26 amino acid residues from the C-terminus compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution T596A, with or without the substitution Y569A and/or Y582G and/or S593A and/or S595A. In such embodiments, the fragment comprises the substitutions Y569A, Y582G, S593A, S595A and T596A. In some embodiments, the fragment comprises a truncation of 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues or 1 amino acid residue from the C-terminus as compared to the mature gE protein. In such embodiments, the fragment optionally comprises the substitution T598A, with or without the substitution Y569A and/or Y582G and/or S593A and/or S595A and/or T596A. In such embodiments, the fragment comprises the substitutions Y569A, Y582G, S593A, S595A, T596A and T598A.

In some embodiments, the mature gE protein comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full length gE protein comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, the fragment comprises the amino acid sequence set forth in SEQ ID NO 3, 6, 8, 10, or 12, or an amino acid sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in SEQ ID NO 3, 6, 8, 10, or 12.

In some embodiments, the N-terminus of the fragment is fused to the C-terminus of the signal peptide. In some embodiments, the N-terminus of the fragment is fused to the native signal peptide of the gE protein of VZV. In some embodiments, the native signal peptide comprises the amino acid sequence shown in SEQ ID NO. 18. In some embodiments, the N-terminus of the fragment is fused to the C-terminus of the human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23.

In one aspect, provided herein are nucleic acids encoding fragments as described herein.

In some embodiments, the fragment is encoded by the nucleotide sequence set forth in SEQ ID NO. 4, 5, 7, 9, 11, or 13, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 4, 5, 7, 9, 11, or 13.

In some embodiments, the native signal peptide of the gE protein of VZV is encoded by the nucleotide sequence set forth in SEQ ID NO. 19, 20, 21 or 22, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 19, 20, 21 or 22. In some embodiments, the human tPA signal peptide is encoded by the nucleotide sequence set forth in SEQ ID NO. 28, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO. 28. In some embodiments, the human IgE signal peptide is encoded by the nucleotide sequence set forth in SEQ ID NO. 24, 25 or 26, or a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO. 24, 25 or 26.

In some embodiments, provided herein is a protein comprising a mutant of mature glycoprotein E (gE) of varicella-zoster virus (VZV), wherein the mutant comprises (a) (i) a truncation of 37 amino acid residues from the C-terminus of mature gE, and (ii) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full length VSV gE, (b) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full length VSV gE, (C) amino acid residue substitution Y569A, Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue positions 569, 582, 593, 595, 596 and 598 are amino acid residue position numbers for full length VSV gE, (d) amino acid residue substitutions Y582G, S593A, S595A, T A and T598A, wherein amino acid residue positions 582, 593, 595, 596 and 598 are amino acid residue position numbers for full length VSV gE, or (e) (i) truncations of 50 amino acid residues from the C-terminus of the mature gE protein, and (ii) amino acid residue substitutions Y569A, wherein amino acid residue position 569 is an amino acid residue position number for full length VSV gE. In some embodiments, the mutant comprises the amino acid sequence of SEQ ID NO. 6, 8, 10, 12 or 3. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO. 6, 8, 10, 12 or 3. In some embodiments, the mutant comprises the amino acid sequence of SEQ ID NO. 6. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to SEQ ID No. 6, 8, 10, or 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID No. 6, 8, 10, or 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% identical to SEQ ID No. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO. 6. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, the protein further comprises a VZV gE signal peptide. In some embodiments, the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the amino acid sequence of the human tPA signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the protein comprises the amino acid sequence set forth in SEQ ID NO. 59.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV and a human IgE signal peptide, wherein the mutant comprises the amino acid sequence set forth in SEQ ID No. 6, and wherein the amino acid sequence of the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID No. 23. In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV and a human IgE signal peptide, wherein the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID No. 6 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID No. 23. In some embodiments, provided herein is a protein consisting of a mutant of mature gE of VZV and a human IgE signal peptide, wherein the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID No. 6 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID No. 23.

In some embodiments, provided herein is a protein comprising the amino acid sequence set forth in SEQ ID NO 59. In some embodiments, provided herein is a protein comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO 59. In some embodiments, provided herein is a protein comprising an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO 59. In some embodiments, provided herein is a protein comprising an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO 59. In some embodiments, provided herein is a protein, wherein the amino acid sequence of the protein consists of the amino acid sequence set forth in SEQ ID NO. 59.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise truncations of at least one and up to 50, 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 14 or 37 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment further comprises an amino acid residue substitution Y569A, and wherein amino acid residue position 569 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, a fragment comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID No. 6, 3, 8, 10, or 12. In some embodiments, the fragment comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, or at least 85% identical to the amino acid sequence set forth in SEQ ID NO. 6, 3, 8, 10, or 12. In some embodiments, the fragment comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93% or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 6, 3, 8, 10 or 12. In some embodiments, the fragment comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 6, 3, 8, 10, or 12. In some embodiments, the fragment comprises the amino acid sequence set forth in SEQ ID NO. 6, 3, 8, 10 or 12. In some embodiments, the amino acid sequence of the fragment consists of the amino acid sequence set forth in SEQ ID NO. 6, 3, 8, 10 or 12. In some embodiments, the fragment comprises the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the amino acid sequence of the fragment consists of the amino acid sequence shown in SEQ ID NO. 6.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise truncations of up to 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment further comprises an amino acid residue substitution Y582G, and wherein amino acid residue position 582 is numbered according to the amino acid residue position of the full-length VZV gE protein. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise truncations of up to 30 or 29 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment further comprises an amino acid residue substitution S593A, and wherein amino acid residue position 593 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise a truncation of up to 28 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment comprises amino acid residue substitution S595A, and wherein amino acid residue position 595 is numbered according to the amino acid residue position of full-length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise truncations up to 27 or 26 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment further comprises an amino acid residue substitution T596A, and wherein amino acid residue position 596 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise truncations of up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5,4, 3, or 2 amino acid residues or up to 1 amino acid residue from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment further comprises an amino acid residue substitution T598A, and wherein amino acid residue position 598 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO.1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein are fragments of mature gE of VZV, wherein the fragments comprise a truncation of 37 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment comprises (1) a truncation of 37 amino acid residues from the C-terminus of the mature gE, and (2) amino acid residue substitutions Y569A and Y582G, and wherein amino acid residue positions 569 and 582 are numbered according to the amino acid residue position of the full-length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO.1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a heterologous signal peptide, wherein the N-terminus of the fragment is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the fragment comprises a truncation of at least 1 and up to 50, 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 14 or 37 amino acid residues from the C-terminus of the mature gE. In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment further comprises one, two, three, four, five or all of the following amino acid substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein the amino acid position numbering is according to full length VZV gE. In some embodiments, the fragment is a fragment described herein. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO.1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of mature gE, and the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1 and the full length VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 55.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises the amino acid sequence set forth in SEQ ID No. 6, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID No. 23, and wherein the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide. In some embodiments, the amino acid sequence of the fragment consists of the amino acid sequence set forth in SEQ ID NO. 6 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, provided herein is a fusion protein consisting of a fragment of mature gE of VZV and a human IgE signal peptide, wherein the amino acid sequence of the fragment consists of the amino acid sequence set forth in SEQ ID No. 6, wherein the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID No. 23, and wherein the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide.

In some embodiments, provided herein is a nucleic acid encoding a protein described herein. In some embodiments, provided herein is a nucleic acid encoding a fragment described herein. In some embodiments, provided herein is a nucleic acid encoding a fusion protein described herein. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises a nucleotide sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4, or 5. In some embodiments, provided herein is a nucleic acid comprising a protein described herein, a fragment described herein, or a fusion protein described herein, wherein the nucleic acid comprises the nucleotide sequence of SEQ ID No. 7.

In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 24, 25, or 26.

In some embodiments, provided herein is a nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, provided herein is a nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 24, 25, or 26.

In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a VZV gE signal peptide, wherein the nucleotide sequence of the signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22. In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a VZV gE signal peptide, wherein the nucleotide sequence of the signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NOs 19, 20, 21, or 22.

In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises the nucleotide sequence shown in SEQ ID No. 28. In some embodiments, provided herein is a nucleic acid encoding a protein described herein, wherein the protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID No. 28.

In some embodiments, provided herein is a nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises the nucleotide sequence shown in SEQ ID No. 28. In some embodiments, provided herein is a nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID No. 28.

In some embodiments, disclosed herein are vectors or cells comprising a nucleic acid as described herein. In some embodiments, the vector is preferably an IVT plasmid. In some embodiments, disclosed herein are compositions comprising a fragment as described herein or a nucleic acid as described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a vaccine.

In one aspect, provided herein are non-naturally occurring nucleic acid molecules useful for the prevention, control, and treatment of diseases or conditions caused by VZV or by infection with VZV.

In some embodiments, the non-naturally occurring nucleic acid comprises a coding nucleotide sequence encoding a fragment as described herein. In some embodiments, the fragment consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence set forth in SEQ ID NO. 1 or a fragment thereof. In some embodiments, the fragment consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO 3, 6, 8, 10 or 12. In some embodiments, the fragment consists of, consists essentially of, or comprises the amino acid sequence set forth in SEQ ID NO 3, 6, 8, 10, or 12. In some embodiments, the coding nucleotide sequence consists of, consists essentially of, or comprises a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the nucleotide sequence set forth in SEQ ID NO.2 or a fragment thereof. In some embodiments, the coding nucleotide sequence has been codon optimized for expression in a cell of the subject. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a human. In some embodiments, the coding nucleotide sequence consists of, consists essentially of, or comprises a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 4, 5, 7, 9, 11 or 13. In some embodiments, the coding nucleotide sequence consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO.4, 5, 7, 9, 11, or 13. In some embodiments, the fragment is fused to a gE native signal peptide. In some embodiments, the signal peptide consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide is encoded by a coding nucleotide sequence consisting of, consisting essentially of, or comprising a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO 19, 20, 21 or 22. In some embodiments, the fragment is fused to a heterologous polypeptide. In some embodiments, the heterologous polypeptide is selected from the group consisting of an Fc region of a human immunoglobulin, a signal peptide, and a peptide that promotes multimerization of a fusion protein. In some embodiments, the signal peptide is a signal peptide from IgE or tPA. In some embodiments, the signal peptide consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the signal peptide is encoded by a coding nucleotide sequence consisting of, consisting essentially of, or comprising a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a nucleotide sequence set forth in SEQ ID NO. 24, 25, or 26. in some embodiments, the signal peptide consists of, consists essentially of, or comprises an amino acid sequence having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the signal peptide is encoded by a coding nucleotide sequence consisting of, consisting essentially of, or comprising a nucleotide sequence having at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the nucleotide sequence set forth in SEQ ID NO. 28. In some embodiments, the multimerization is dimerization or trimerization. In some embodiments, the non-naturally occurring nucleic acid further comprises a 5' untranslated region (5 ' -UTR), wherein the 5' -UTR comprises the sequence depicted in any one of SEQ ID NOS 29-38. In some embodiments, the non-naturally occurring nucleic acid further comprises a 3' untranslated region (3 ' -UTR), wherein the 3' -UTR comprises the sequence depicted in any one of SEQ ID NOS 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO 49, 50, 51, 52, 53, 54, 60, 61, 62, 63, or 64, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO 49, 50, 51, 52, 53, 54, 60, 61, 62, 63, or 64. In some embodiments, the non-naturally occurring nucleic acid comprises one or more functional nucleotide analogs. In some embodiments, the non-naturally occurring nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine (psd), 1-methyl-pseudouridine (m 1), and 5-methylcytosine. In some embodiments, the nucleic acid is DNA or mRNA. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 63, wherein all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU.

In some embodiments, disclosed herein are vectors or cells comprising a non-naturally occurring nucleic acid molecule as described herein. In some embodiments, the vector is preferably an IVT plasmid. In some embodiments, disclosed herein are compositions comprising non-naturally occurring nucleic acid molecules as described herein.

In some embodiments, provided herein is a non-naturally occurring nucleic acid comprising a coding nucleotide sequence encoding a protein described herein. In some embodiments, provided herein is a non-naturally occurring nucleic acid comprising a coding nucleotide sequence encoding a fragment described herein. In some embodiments, provided herein is a non-naturally occurring nucleic acid comprising a coding nucleotide sequence encoding a fusion protein described herein. In some embodiments, the coding nucleotide sequence has been codon optimized for expression in a cell of the subject. In some embodiments, the coding nucleotide sequence has been codon optimized for expression in a cell of a non-human mammal. In particular embodiments, the coding nucleotide sequence has been codon optimized for expression in human cells. In some embodiments, the non-naturally occurring nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO 7, 9, 11, 13, 4 or 5. The non-naturally occurring nucleic acid comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 7, 9, 11, 13, 4 or 5. In some embodiments, the non-naturally occurring nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 7.

In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID nos. 24, 25, or 26.

In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human IgE signal peptide, and wherein the nucleotide sequence encoding the IgE signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26.

In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a VZV gE signal peptide, wherein the nucleotide sequence of the signal peptide comprises the nucleotide sequence set forth in SEQ ID NO:19, 20, 21 or 22. In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a VZV gE signal peptide, wherein the nucleotide sequence of the signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the nucleotide sequence set forth in SEQ ID NOs 19, 20, 21 or 22.

In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a mutant of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises the nucleotide sequence shown in SEQ ID NO: 28. In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a protein described herein, wherein the protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID No. 28.

In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises the nucleotide sequence shown in SEQ ID No. 28. In some embodiments, provided herein is a non-naturally occurring nucleic acid encoding a fusion protein described herein, wherein the fusion protein comprises a fragment of mature gE of VZV and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence shown in SEQ ID No. 28.

In some embodiments, provided herein is a non-naturally occurring nucleic acid comprising a nucleotide sequence of SEQ ID No. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, the non-naturally occurring nucleic acid consists of the nucleotide sequence of SEQ ID NO. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5. In some embodiments, the non-naturally occurring nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, the non-naturally occurring nucleic acid comprises a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a nucleotide sequence set forth in SEQ ID No. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5. In some embodiments, the non-naturally occurring nucleic acid consists of or consists essentially of a nucleotide sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity to a nucleotide sequence set forth in SEQ ID NO. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5. In some embodiments, the non-naturally occurring nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO 63, 51, 60, 62, or 64. In some embodiments, the non-naturally occurring nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, the non-naturally occurring nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO. 63.

In some embodiments, the non-naturally occurring nucleic acids described herein further comprise a5 'untranslated region (5' -UTR) and/or a3 'untranslated region (3' -UTR). In some embodiments, the non-naturally occurring nucleic acids described herein further comprise a 5'-UTR, wherein the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the non-naturally occurring nucleic acids described herein further comprise a 3'-UTR, wherein the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the non-naturally occurring nucleic acids described herein further comprise a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38, and wherein the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal.

In some embodiments, the non-naturally occurring nucleic acids described herein comprise DNA. In some embodiments, the non-naturally occurring nucleic acid described herein is DNA. In some embodiments, the non-naturally occurring nucleic acids described herein comprise one or more functional nucleotide analogs. In some embodiments, the non-naturally occurring nucleic acids described herein comprise mRNA, and wherein thymine is replaced by uracil or a functional analog thereof in the nucleic acid. In some embodiments, the non-naturally occurring nucleic acid described herein is an mRNA, and wherein thymine is replaced by uracil or a functional analog thereof in the nucleic acid. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with uracil (U) or N1-methyl pseudouridine. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with uracil (U) or N1-methyl pseudouridine, and the first nucleotide G is replaced with m ⁷ GpppAmpU. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with uracil (U) or N1-methyl pseudouridine. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 63, and the first nucleotide G is substituted with m ⁷ GpppAmpU. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and the first nucleotide G is substituted with m ⁷ GpppAmpU. In some embodiments, the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with N1-methyl pseudouridine and the first nucleotide G is replaced with m ⁷ GpppAmpU.

In some embodiments, provided herein is a vector comprising a nucleic acid as described herein. In some embodiments, provided herein is a vector comprising a non-naturally occurring nucleic acid as described herein. In some embodiments, the vector is an IVT (in vitro transcription) plasmid.

In some embodiments, provided herein is a host cell comprising a nucleic acid as described herein. In some embodiments, provided herein is a host cell comprising a non-naturally occurring nucleic acid as described herein. In some embodiments, provided herein is a host cell comprising a vector described herein. In some embodiments, the host cell expresses a protein described herein, a fragment described herein, or a fusion protein described herein. In some embodiments, the host cell is in vitro, ex vivo, or isolated.

In some embodiments of the compositions described herein, the composition further comprises at least one lipid described herein. In some embodiments of the compositions described herein, the composition further comprises at least a first lipid described herein (e.g., a cationic lipid) and optionally a second lipid described herein (e.g., a polymer-bound lipid).

In some embodiments, the first lipid is a compound according to series 01, 02, 03, and 04, e.g., a compound according to formula (01-I), (01-II), (02-I), (03-I), or (04-I). In some embodiments, the first lipid is a compound listed in Table 01-1, 02-1, 03-1, or 04-1. In some embodiments, the second lipid is a compound according to series 05, e.g., a compound according to formula (05-I).

In some embodiments, the composition is formulated as a lipid nanoparticle that encapsulates a nucleic acid in a lipid shell. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the composition is a vaccine.

In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a protein described herein. In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a fragment described herein. In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a fusion protein described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, the proteins, fragments or fusion proteins described herein are used to immunize a subject against VZV. In some embodiments, a nucleic acid described herein, a protein described herein, a fragment or fusion protein described herein is used to induce an immune response in a subject (e.g., such as, for example, an immune response described in section 6). In some embodiments, the immune response comprises a humoral immune response against VZV. In some embodiments, the immune response comprises a cellular response to VZV. In some embodiments, the immune response includes an antibody (e.g., a neutralizing antibody) specific for VZV gE.

In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a nucleic acid described herein. In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a non-naturally occurring nucleic acid described herein. In some embodiments, provided herein is a composition (e.g., a pharmaceutical composition) comprising a carrier as described herein. In some embodiments, the composition is a pharmaceutical composition. In some embodiments, the pharmaceutical composition is a vaccine. In some embodiments, a nucleic acid described herein, a non-naturally occurring nucleic acid, or a vector described herein is used to immunize a subject against VZV. In some embodiments, a nucleic acid described herein, a non-naturally occurring nucleic acid described herein, or a vector described herein is used to induce an immune response (e.g., such as, for example, an immune response described in section 6) in a subject. In some embodiments, the immune response comprises a humoral immune response against VZV. In some embodiments, the immune response comprises a cellular response to VZV. In some embodiments, the immune response includes an antibody (e.g., a neutralizing antibody) specific for VZV gE.

In some embodiments, provided herein is a pharmaceutical composition comprising a non-naturally occurring nucleic acid described herein and at least a first lipid. In some embodiments, the first lipid is a lipid described herein. In some embodiments, the first lipid is a compound according to formula 01-I or formula 01-II, or a compound listed in Table 01-1, or a compound according to formula 02-I, or a compound listed in Table 02-1, or a compound according to formula 03-I, or a compound listed in Table 03-1, or a compound according to formula 04-I, or a compound listed in Table 04-1. In some embodiments, the pharmaceutical composition of claim 72 further comprises a second lipid. In some embodiments, the second lipid is a compound according to formula 05-I. In some embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid shell. In some embodiments, the pharmaceutical composition is a vaccine.

In one aspect, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a fragment described herein, a therapeutically effective amount of a nucleic acid described herein, a therapeutically effective amount of a non-naturally occurring nucleic acid described herein, or a therapeutically effective amount of a pharmaceutical composition as described herein.

In some embodiments of the methods described herein, the subject is a human or non-human mammal. In some embodiments, the subject is a human adult, a human child, or a human infant. In some embodiments, the subject has a disease or disorder. In some embodiments, the subject is at risk for or susceptible to a VZV infection. In some embodiments, the subject is an elderly person. In some embodiments, the subject has been diagnosed as positive for VZV infection. In some embodiments, the subject is asymptomatic.

In some embodiments of the methods described herein, the method comprises administering to a subject a lipid nanoparticle encapsulating a nucleic acid, and wherein the lipid nanoparticle is endocytosed by a cell in the subject. In some embodiments, the nucleic acid is expressed by a cell in the subject.

In some embodiments of the methods described herein, an immune response is elicited in the subject against VZV. In some embodiments, the immune response includes cytokine production in lymphocytes. In some embodiments, the immune response includes an increase in the proportion of lymphocytes that express the cytokine. In some embodiments, the lymphocyte is a CD4 ⁺ T cell and/or a CD8 ⁺ T cell. In some embodiments, the cytokine is one or more of IFN-gamma, IL-2, and TNF-alpha. In some embodiments, cytokine production is increased in lymphocytes. In some embodiments, the immune response includes the generation of antibodies that specifically bind to viral gE proteins. In some embodiments, the antibody is a neutralizing antibody to VZV or a cell infected with VZV. In some embodiments, the serum titer of the antibodies in the subject is increased.

In some embodiments of the methods described herein, the antibody binds to a viral particle or an infected cell, and the viral particle of the infected cell is labeled for destruction by the immune system of the subject. In some embodiments, endocytosis of the virus particle bound by the antibody is induced or enhanced. In some embodiments, antibody-dependent cell-mediated cytotoxicity (ADCC) against the infected cells in the subject is induced or enhanced. In some embodiments, antibody-dependent cell phagocytosis (ADCP) is induced or enhanced in a subject against an infected cell. In some embodiments, complement Dependent Cytotoxicity (CDC) against the infected cells in the subject is induced or enhanced.

In some embodiments of the methods described herein, the disease or disorder caused by VZV is varicella and/or shingles. In some embodiments of the methods described herein, the disease or disorder caused by VZV is post-herpetic neuralgia (PHN). In some embodiments of the methods described herein, the disease or disorder caused by VZV is one or more of meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis.

In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a fragment described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a fusion protein described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a nucleic acid described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a non-naturally occurring nucleic acid described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a vector described herein. In some embodiments, provided herein is a method for controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition described herein. In some embodiments, the methods are for preventing a disease or disorder in a subject caused by VZV or by infection with VZV. In some embodiments, an immune response against VZV is elicited in the subject. In some embodiments, the immune response includes cytokine production in lymphocytes. In some embodiments, the immune response includes an increase in the proportion of lymphocytes that express the cytokine. In some embodiments, the lymphocytes are CD4 ⁺ T cells and/or CD8 ⁺ T cells, and/or wherein the cytokine is one or more of IFN-gamma, IL-2, and TNF-alpha. In some embodiments, cytokine production is increased in lymphocytes. In some embodiments, the immune response includes the generation of antibodies that specifically bind to VZV gE (e.g., neutralizing antibodies). In some embodiments, the disease or disorder caused by VZV is (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), or (c) one or more of meningoencephalitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), or (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoepitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the subject is a human. In some embodiments, the human is a human adult. In some embodiments, the adult is at least 40 years old. In some embodiments, the adult is at least 45 years old. In some embodiments, the adult is at least 50 years old. In some embodiments, the adult is at least 55 years old. In some embodiments, the adult is at least 60 years old. In some embodiments, the human is an elderly human.

In some embodiments, provided herein are proteins described herein, fragments described herein, or fusion proteins described herein for use in a method of controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject. In some embodiments, provided herein are nucleic acids described herein, non-naturally occurring nucleic acids described herein, or vectors described herein for use in a method of controlling, preventing, or treating a disease or disorder caused by VZV or by infection of VZV in a subject. In some embodiments, provided herein are pharmaceutical compositions described herein for use in a method of controlling, preventing or treating a disease or disorder in a subject caused by VZV or by infection with VZV. In some embodiments, the disease or disorder caused by VZV is (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), or (c) one or more of meningoencephalitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), or (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoepitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the subject is a human. In some embodiments, the human is a human adult. In some embodiments, the adult is at least 40 years old. In some embodiments, the adult is at least 45 years old. In some embodiments, the adult is at least 50 years old. In some embodiments, the adult is at least 55 years old. In some embodiments, the adult is at least 60 years old. In some embodiments, the human is an elderly human.

In some embodiments, provided herein is the use of a protein described herein, a fragment described herein, a fusion protein described herein for the manufacture of a medicament for controlling, preventing or treating a disease or disorder caused by VZV or by infection of VZV in a subject. In some embodiments, provided herein is a use of a nucleic acid described herein, a non-naturally occurring nucleic acid described herein, or a vector described herein for the manufacture of a medicament for controlling, preventing, or treating a disease or disorder in a subject caused by VZV or by infection with VZV. In some embodiments, provided herein is the use of a pharmaceutical composition described herein for the manufacture of a medicament for controlling, preventing or treating a disease or disorder in a subject caused by VZV or by infection with VZV. In some embodiments, the disease or disorder caused by VZV is (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), or (c) one or more of meningoencephalitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), or (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella or zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoeencephalitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the disease or disorder caused by VZV is one or more of (a) varicella and zoster, (b) Post Herpetic Neuralgia (PHN), and (c) meningoepitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In some embodiments, the subject is a human. In some embodiments, the human is a human adult. In some embodiments, the adult is at least 40 years old. In some embodiments, the adult is at least 45 years old. In some embodiments, the adult is at least 50 years old. In some embodiments, the adult is at least 55 years old. In some embodiments, the adult is at least 60 years old. In some embodiments, the human is an elderly human.

4. Description of the drawings

Figure 1 shows expression of the candidate by HEK293T transfected in vitro. The suffix psd represents the pseudo-U modification and the suffix m1 represents the 1-N-pseudo-U modification.

Figure 2 shows gE-specific IgG titers induced by the candidates. The suffix psd represents the pseudo-U modification and the suffix m1 represents the 1-N-pseudo-U modification.

Figure 3 shows the gE peptide Chi Teyi-induced T cell response by the candidate. The suffix psd represents the pseudo-U modification and the suffix m1 represents the 1-N-pseudo-U modification.

Fig. 4 shows the predicted secondary structure of full length VZV gE. A, wild type, B, comprising mutants substituted for Y569A, Y582G, S593A, S595A, T596A and T598A.

Figure 5 shows the median fluorescence intensity of the candidate transfected cells in vitro.

FIG. 6 shows the expression rate of the candidate in vitro transfected cells.

Figure 7 shows gE-specific IgG titers induced by the candidates.

Figure 8 shows the gE peptide Chi Teyi-induced T cell response by the candidate.

FIG. 9 shows the location of a target protein in a cell. Green represents gE protein, red represents golgi, and blue represents nucleus.

FIG. 10 shows an analysis of inhibition of inflammatory response in vitro by RIG-I activation and IFN- β release using mRNA.

FIG. 11 shows the expression rate of the candidate in vitro transfected cells.

Figure 12 shows the median fluorescence intensity of the candidate transfected cells in vitro.

Figure 13A shows gE-specific IgG titers after the first immunization in mice that were initially subjected to the test.

Figure 13B shows gE-specific IgG titers following boost immunization in mice that were initially subjected to the test.

Figure 13C shows gE-specific IgG titers after the first immunization in mice that underwent LAV.

Figure 13D shows gE-specific IgG titers following boost immunization in mice subjected to LAV.

Figure 14A shows the cd4+ T cell response induced by the vaccine in mice that were initially tested.

Figure 14B shows the cd4+ T cell response induced by the vaccine in mice that underwent LAV.

5. Detailed description of the preferred embodiments

Provided herein are therapeutic nucleic acid molecules useful for the prevention, control and treatment of diseases or conditions caused by VZV or by infection with VZV. Also provided herein are pharmaceutical compositions comprising therapeutic nucleic acid molecules, including pharmaceutical compositions formulated as lipid nanoparticles, and related therapeutic methods and uses for preventing, controlling, and treating diseases or disorders caused by VZV or by infection of VZV. Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of the following detailed description of specific embodiments.

5.1 General technique

The techniques and procedures described or referenced herein include those commonly employed by those skilled in the art to which conventional methods are generally well-understood and/or used, such as those described in Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001), current Protocols in Molecular Biology (Ausubel et al, 2003).

5.2 Terminology

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of explaining the present specification, the following description of terms will be applied, and terms used in the singular will also include the plural and vice versa, where appropriate. All patents, applications, published applications and other publications are incorporated by reference in their entirety. If any description set forth regarding a term conflicts with any document incorporated herein by reference, the term description set forth below controls.

As used herein and unless otherwise indicated, the term "lipid" refers to a group of organic compounds that include, but are not limited to, esters of fatty acids, and are generally characterized as poorly soluble in water, but soluble in many non-polar organic solvents. While lipids are generally poorly soluble in water, there are certain classes of lipids (e.g., lipids modified with polar groups, such as DMG-PEG 2000) that have limited water solubility and are soluble in water under certain conditions. Known types of lipids include biomolecules such as fatty acids, waxes, sterols, fat-soluble vitamins, monoglycerides, diglycerides, triglycerides and phospholipids. Lipids can be divided into at least three categories, (1) "simple lipids" which include fats and oils and waxes, (2) "complex lipids" which include phospholipids and glycolipids (e.g., DMPE-PEG 2000), and (3) "derivatized lipids", such as steroids. Further, as used herein, lipids also encompass lipid compounds. The term "lipid compound" is also referred to simply as "lipid" and refers to lipid-like compounds (e.g., amphiphilic compounds having lipid-like physical properties).

The term "lipid nanoparticle" or "LNP" refers to particles having a size (e.g., 1 to 1,000 nm) of at least one nanometer (nm) scale that contain one or more types of lipid molecules. The LNPs provided herein can further comprise at least one non-lipid payload molecule (e.g., one or more nucleic acid molecules). In some embodiments, the LNP comprises a non-lipid payload molecule partially or fully encapsulated inside a lipid shell. In particular, in some embodiments, wherein the payload is a negatively charged molecule (e.g., mRNA encoding a viral protein), and the lipid component of the LNP comprises at least one cationic lipid. Without being bound by theory, it is contemplated that the cationic lipid may interact with negatively charged payload molecules and facilitate payload binding and/or encapsulation into the LNP during LNP formation. Other lipids that may form part of the LNP as provided herein include, but are not limited to, neutral lipids and charged lipids, such as steroids, polymer-bound lipids, and various zwitterionic lipids. In certain embodiments, LNPs according to the present disclosure comprise one or more of the series 01, 02, 03, and 04 of lipids, e.g., one or more of the formula (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-types thereof) as described herein.

The term "cationic lipid" refers to a lipid that is positively charged at any pH or hydrogen ion activity of its environment, or that is capable of being positively charged in response to the pH or hydrogen ion activity of its environment (e.g., the environment of its intended use). Thus, the term "cation" encompasses both "permanent cations" and "cationizable". In certain embodiments, the positive charge in the cationic lipid results from the presence of a quaternary nitrogen atom. In certain embodiments, the cationic lipid comprises a zwitterionic lipid that is positively charged in the environment of its intended use (e.g., at physiological pH). In certain embodiments, the cationic lipid is one or more of the series 01, 02, 03, and 04 lipids, e.g., one or more of the formula (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-types thereof) as described herein. The term "anionic lipid" refers to a lipid that is negatively charged at any pH or hydrogen ion activity of its environment, or that is capable of being negatively charged in response to the pH or hydrogen ion activity of its environment (e.g., the environment of its intended use). Exemplary anionic lipids include one or more negatively charged phosphate groups, for example, at physiological pH.

The term "polymer-bound lipid" refers to a molecule that comprises both a lipid moiety and a polymer moiety. An example of a polymer-bound lipid is a pegylated lipid (PEG-lipid), wherein the polymer moiety comprises polyethylene glycol.

The term "neutral lipid" encompasses any lipid molecule that exists in an uncharged form or in a neutral zwitterionic form at or within a selected pH range. In some embodiments, the useful pH or range selected corresponds to the pH conditions in the environment of the intended lipid use, such as physiological pH. As non-limiting examples, neutral lipids that may be used in connection with the present disclosure include, but are not limited to, phosphatidylcholine, such as1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dipalmitoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-dimyristoyl-sn-glycero-3-phosphorylcholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), phosphatidylethanolamine, such as1, 2-dioleoyl-sn-glycero-3-phosphorylethanolamine (DOPE), 2- ((2, 3-bis (oleoyloxy) propyl) dimethylammonium) ethyl hydrogen phosphate (DOCP), sphingomyelin (SM), ceramides, such as sterols and derivatives thereof. Neutral lipids provided herein may be synthetic or derived from (isolated or modified from) natural sources or compounds.

The term "charged lipid" encompasses any lipid molecule that exists in a positively or negatively charged form at or within a selected pH. In some embodiments, the selected pH or range corresponds to pH conditions in the environment of the intended lipid use, such as physiological pH. As non-limiting examples, neutral lipids that may be used in connection with the present disclosure include, but are not limited to, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, sterol hemisuccinate, dialkyltrimethylammonium-propane (e.g., DOTAP, DOTMA), dialkyldimethylaminopropane, ethylcholine phosphate, dimethylaminoethane carbamoyl sterols (e.g., DC-Chol), 1, 2-dioleoyl-sn-glycerol-3-phosphate-L-serine sodium salt (DOPS-Na), 1, 2-dioleoyl-sn-glycerol-3-phosphate- (1' -rac-glycerol) sodium salt (DOPG-Na), and 1, 2-dioleoyl-sn-glycerol-3-phosphate sodium salt (DOPA-Na). Charged lipids as provided herein may be synthetic or derived from (isolated or modified from) natural sources or compounds.

As used herein and unless otherwise indicated, the term "alkyl" refers to a saturated straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms. In one embodiment, the alkyl group has, for example, one to twenty four carbon atoms (C ₁-C₂₄ alkyl), four to twenty carbon atoms (C ₄-C₂₀ alkyl), six to sixteen carbon atoms (C ₆-C₁₆ alkyl), six to nine carbon atoms (C ₆-C₉ alkyl), one to fifteen carbon atoms (C ₁-C₁₅ alkyl), one to twelve carbon atoms (C ₁-C₁₂ alkyl), one to eight carbon atoms (C ₁-C₈ alkyl), or one to six carbon atoms (C ₁-C₆ alkyl) and is attached to the remainder of the molecule by a single bond. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, 1-methylethyl (isopropyl), n-butyl, n-pentyl, 1-dimethylethyl (t-butyl), 3-methylhexyl, 2-methylhexyl, and the like. Unless otherwise indicated, alkyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "alkenyl" refers to a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, which contains one or more carbon-carbon double bonds. As will be appreciated by those of ordinary skill in the art, the term "alkenyl" also includes groups having "cis" and "trans" configurations, or having "E" and "Z" configurations. In one embodiment, the alkenyl group has, for example, two to twenty-four carbon atoms (C ₂-C₂₄ alkenyl), four to twenty carbon atoms (C ₄-C₂₀ alkenyl), six to sixteen carbon atoms (C ₆-C₁₆ alkenyl), six to nine carbon atoms (C ₆-C₉ alkenyl), two to fifteen carbon atoms (C ₂-C₁₅ alkenyl), two to twelve carbon atoms (C ₂-C₁₂ alkenyl), two to eight carbon atoms (C ₂-C₈ alkenyl), or two to six carbon atoms (C ₂-C₆ alkenyl), and is linked to the remainder of the molecule by a single bond. Examples of alkenyl groups include, but are not limited to, vinyl, prop-1-enyl, but-1-enyl, pent-1, 4-dienyl, and the like. Unless otherwise indicated, alkenyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "alkynyl" refers to a straight or branched hydrocarbon chain group consisting of only carbon and hydrogen atoms, which contains one or more carbon-carbon triple bonds. In one embodiment, the alkynyl group has, for example, two to twenty-four carbon atoms (C ₂-C₂₄ alkynyl), four to twenty carbon atoms (C ₄-C₂₀ alkynyl), six to sixteen carbon atoms (C ₆-C₁₆ alkynyl), six to nine carbon atoms (C ₆-C₉ alkynyl), two to fifteen carbon atoms (C ₂-C₁₅ alkynyl), two to twelve carbon atoms (C ₂-C₁₂ alkynyl), two to eight carbon atoms (C ₂-C₈ alkynyl) or two to six carbon atoms (C ₂-C₆ alkynyl) and is attached to the remainder of the molecule by a single bond. Examples of alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, pentynyl, and the like. Unless otherwise indicated, alkynyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "alkylene" or "alkylene chain" refers to a straight or branched divalent hydrocarbon chain that connects the remainder of the molecule to a group, consisting of only carbon and hydrogen, and being saturated. In one embodiment, the alkylene group has, for example, one to twenty-four carbon atoms (C ₁-C₂₄ alkylene), one to fifteen carbon atoms (C ₁-C₁₅ alkylene), one to twelve carbon atoms (C ₁-C₁₂ alkylene), one to eight carbon atoms (C ₁-C₈ alkylene), one to six carbon atoms (C ₁-C₆ alkylene), two to four carbon atoms (C ₂-C₄ alkylene), one to two carbon atoms (C ₁-C₂ alkylene). Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, n-butylene, and the like. The alkylene chain is linked to the rest of the molecule by a single bond and to the group by a single bond. The point of attachment of the alkylene chain to the remainder of the molecule and to the group may be through one carbon or any two carbons within the chain. Unless otherwise indicated, the alkylene chain is optionally substituted.

As used herein and unless otherwise indicated, the term "alkenylene" refers to a straight or branched divalent hydrocarbon chain that connects the rest of the molecule to a group, consisting of only carbon and hydrogen and containing one or more carbon-carbon double bonds. In one embodiment, the alkenylene group has, for example, two to twenty-four carbon atoms (C ₂-C₂₄ alkenylene), two to fifteen carbon atoms (C ₂-C₁₅ alkenylene), two to twelve carbon atoms (C ₂-C₁₂ alkenylene), two to eight carbon atoms (C ₂-C₈ alkenylene), two to six carbon atoms (C ₂-C₆ alkenylene), or two to four carbon atoms (C ₂-C₄ alkenylene). Examples of alkenylene groups include, but are not limited to, ethenylene, propenylene, n-butenyl, and the like. Alkenylene is attached to the remainder of the molecule by a single or double bond and to a group by a single or double bond. The point of attachment of the alkenylene group to the remainder of the molecule and to the group may be through one carbon or any two carbons within the chain. Unless otherwise indicated, alkenylene groups are optionally substituted.

As used herein and unless otherwise indicated, the term "cycloalkyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting of only carbon and hydrogen atoms and being saturated. Cycloalkyl groups may include fused or bridged ring systems. In one embodiment, cycloalkyl has, for example, 3 to 15 ring carbon atoms (C ₃-C₁₅ cycloalkyl), 3 to 10 ring carbon atoms (C ₃-C₁₀ cycloalkyl), or 3 to 8 ring carbon atoms (C ₃-C₈ cycloalkyl). Cycloalkyl groups are linked to the rest of the molecule by single bonds. Examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of polycyclic cycloalkyl groups include, but are not limited to, adamantyl, norbornyl, decalinyl, 7-dimethyl-bicyclo [2.2.1] heptyl, and the like. Cycloalkyl groups are optionally substituted unless otherwise indicated.

As used herein and unless otherwise indicated, the term "cycloalkylene" is a divalent cycloalkyl group. Unless otherwise indicated, cycloalkylene groups are optionally substituted.

As used herein and unless otherwise indicated, the term "cycloalkenyl" refers to a non-aromatic monocyclic or polycyclic hydrocarbon group consisting of only carbon and hydrogen atoms and containing one or more carbon-carbon double bonds. Cycloalkenyl groups may include fused or bridged ring systems. In one embodiment, cycloalkenyl has, for example, 3 to 15 ring carbon atoms (C ₃-C₁₅ cycloalkenyl), 3 to 10 ring carbon atoms (C ₃-C₁₀ cycloalkenyl), or 3 to 8 ring carbon atoms (C ₃-C₈ cycloalkenyl). The cycloalkenyl group is linked to the rest of the molecule by a single bond. Examples of monocyclic cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl, and the like. Unless otherwise indicated, cycloalkenyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "cycloalkenyl" is a divalent cycloalkenyl group. Unless otherwise indicated, cycloalkenyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "heterocyclyl" refers to a monocyclic or polycyclic moiety of a non-aromatic radical containing one or more (e.g., one or two, one to three, or one to four) heteroatoms independently selected from nitrogen, oxygen, phosphorus, and sulfur. The heterocyclyl may be attached to the main structure at any heteroatom or carbon atom. The heterocyclyl may be a monocyclic, bicyclic, tricyclic, tetracyclic or other polycyclic ring system, wherein the polycyclic ring system may be a fused, bridged or spiro ring system. The heterocyclyl-based multicyclic system may contain one or more heteroatoms in one or more rings. The heterocyclyl groups may be saturated or partially unsaturated. Saturated heterocycloalkyl groups may be referred to as "heterocycloalkyl groups". Partially unsaturated heterocycloalkyl groups may be referred to as "heterocycloalkenyl" when the heterocyclyl contains at least one double bond, or as "heterocycloalkynyl" when the heterocyclyl contains at least one triple bond. In one embodiment, the heterocyclyl has, for example, 3 to 18 ring atoms (3 to 18 membered heterocyclyl), 4 to 18 ring atoms (4 to 18 membered heterocyclyl), 5 to 18 ring atoms (3 to 18 membered heterocyclyl), 4 to 8 ring atoms (4 to 8 membered heterocyclyl), or 5 to 8 ring atoms (5 to 8 membered heterocyclyl). When appearing herein, a numerical range such as "3 to 18" means each integer in the given range, for example, "3 to 18 ring atoms" means that a heterocyclyl can consist of 3 ring atoms, 4 ring atoms, 5 ring atoms, 6 ring atoms, 7 ring atoms, 8 ring atoms, 9 ring atoms, 10 ring atoms, etc. (up to and including 18 ring atoms). Examples of heterocyclyl groups include, but are not limited to, imidazolyl, imidazolidinyl, oxazolyl, oxazolidinyl, thiazolyl, thiazolidinyl, pyrazolidinyl, pyrazolyl, isoxazolidinyl, isothiazolidinyl, isothiazolyl, morpholinyl, pyrrolyl, pyrrolidinyl, furyl, tetrahydrofuryl, thienyl, pyridyl, piperidyl, quinolinyl, and isoquinolinyl. Unless otherwise indicated, the heterocyclyl groups are optionally substituted. Unless otherwise indicated, the heterocyclyl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "heterocyclyl" is a divalent heterocyclyl. Unless otherwise indicated, the heterocyclylene groups are optionally substituted.

As used herein and unless otherwise indicated, the term "aryl" refers to a monocyclic aromatic group and/or a polycyclic monovalent aromatic group containing at least one aromatic hydrocarbon ring. In certain embodiments, aryl groups have 6 to 18 ring carbon atoms (C ₆-C₁₈ aryl), 6 to 14 ring carbon atoms (C ₆-C₁₄ aryl), or 6 to 10 ring carbon atoms (C ₆-C₁₀ aryl). Examples of aryl groups include, but are not limited to, phenyl, naphthyl, fluorenyl, azulenyl, anthracenyl, phenanthrenyl, pyrenyl, biphenyl, and biphenyl. The term "aryl" also refers to bicyclic, tricyclic, or other polycyclic hydrocarbon rings in which at least one ring is aromatic and the other rings may be saturated, partially unsaturated, or aromatic, such as dihydronaphthyl, indenyl, indanyl, or tetrahydronaphthyl (tetrahydronaphthyl/tetralinyl). Unless otherwise indicated, aryl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "arylene" is a divalent aryl group. Unless otherwise indicated, arylene groups are optionally substituted.

As used herein and unless otherwise indicated, the term "heteroaryl" refers to a monocyclic aromatic group and/or polycyclic aromatic group containing at least one aromatic ring, wherein at least one aromatic ring contains one or more (e.g., one or two, one to three, or one to four) heteroatoms independently selected from O, S and N. Heteroaryl groups may be attached to the main structure at any heteroatom or carbon atom. In certain embodiments, heteroaryl groups have 5 to 20, 5 to 15, or 5 to 10 ring atoms. The term "heteroaryl" also refers to bicyclic, tricyclic, or other polycyclic rings in which at least one ring is aromatic, and the other rings may be saturated, partially unsaturated, or aromatic, in which at least one aromatic ring contains one or more heteroatoms independently selected from O, S and N. Examples of monocyclic heteroaryl groups include, but are not limited to, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, thiadiazolyl, isothiazolyl, furanyl, thienyl, oxadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, and triazinyl. Examples of bicyclic heteroaryl groups include, but are not limited to, indolyl, benzothiazolyl, benzoxazolyl, benzothienyl, quinolinyl, tetrahydroisoquinolinyl, isoquinolinyl, benzimidazolyl, benzopyranyl, indolizinyl, benzofuranyl, isobenzofuranyl, chromonyl, coumarin, cinnolinyl, quinoxalinyl, indazolyl, purinyl, pyrrolopyridinyl, furopyridinyl, thienopyridinyl, dihydroisoindolyl, and tetrahydroquinolinyl. Examples of tricyclic heteroaryl groups include, but are not limited to, carbazolyl, benzindolyl, phenanthrolinyl, acridinyl, phenanthridinyl, and xanthenyl. Unless otherwise indicated, heteroaryl groups are optionally substituted.

As used herein and unless otherwise indicated, the term "heteroarylene" is a divalent heteroaryl group. Unless otherwise indicated, heteroarylene is optionally substituted.

When a group described herein is referred to as "substituted," it may be substituted with one or more of any suitable substituent. Illustrative examples of substituents include, but are not limited to, the substituents found in the exemplary compounds and embodiments provided herein, as well as halogen atoms such as F, cl, br, or I, cyano, oxo (=o), hydroxy (-OH), alkyl, alkenyl, alkynyl, cycloalkyl, aryl ;-(C＝O)OR';-O(C＝O)R';-C(＝O)R';-OR';-S(O)_xR';-S-SR';-C(＝O)SR';-SC(＝O)R';-NR'R';-NR'C(＝O)R';-C(＝O)NR'R';-NR'C(＝O)NR'R';-OC(＝O)NR'R';-NR'C(＝O)OR';-NR'S(O)_xNR'R';-NR'S(O)_xR';, and-S (O) _x NR ' R ', where R ' is, independently at each occurrence, H, C ₁-C₁₅ alkyl or cycloalkyl, and x is 0, 1, or 2. In some embodiments, the substituent is a C ₁-C₁₂ alkyl group. In other embodiments, the substituent is cycloalkyl. In other embodiments, the substituent is a halo group, such as a fluoro group. In other embodiments, the substituent is oxo. In other embodiments, the substituent is hydroxy. In other embodiments, the substituent is an alkoxy (-OR'). In other embodiments, the substituent is a carboxyl group. In other embodiments, the substituent is an amino group (-NR 'R').

As described herein and unless otherwise indicated, the terms "optional" or "optionally" (e.g., optionally substituted) mean that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. For example, "optionally substituted alkyl" means that the alkyl group may or may not be substituted, and the description includes both substituted alkyl groups and unsubstituted alkyl groups.

As used herein and unless otherwise indicated, the term "prodrug" of a bioactive compound refers to a compound that can be converted to the bioactive compound under physiological conditions or by solvolysis. In one embodiment, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a biologically active compound. Prodrugs may be inactive when administered to a subject in need thereof, but are converted in vivo to the biologically active compound. Prodrugs are typically rapidly converted in vivo to produce the parent bioactive compound, for example, by hydrolysis in the blood. Prodrug compounds generally provide solubility, histocompatibility or delayed release advantages in mammalian organisms (see Bundgard, h., design ofProdrugs (1985), pages 7-9, pages 21-24 (Elsevier, amsterdam)). Discussion of prodrugs is provided in Higuchi, T.et al, A.C.S. symposium Series, volume 14, and Bioreversible CARRIERS IN Drug Design, edward B.Roche, eds., americanPharmaceutical Association andPergamon Press, 1987.

In one embodiment, the term "prodrug" is also intended to include any covalently bonded carrier that releases the active compound in vivo when such prodrug is administered to a mammalian subject. Prodrugs of the compounds may be prepared by modifying functional groups present in the compound in such a way that the modification may be cleaved, either in routine manipulation or in vivo, to yield the parent compound. Prodrugs include compounds wherein a hydroxyl, amino, or sulfhydryl group is bonded to any group that, when the prodrug of the compound is administered to a mammalian subject, cleaves to form a free hydroxyl, free amino, or free sulfhydryl group, respectively.

Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol functional groups or amide derivatives of amine functional groups in the compounds provided herein.

As used herein and unless otherwise indicated, the term "pharmaceutically acceptable salt" includes both acid addition salts and base addition salts.

Examples of pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like; an organic acid is used to prepare the organic acid, such as, but not limited to, acetic acid, 2-dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid-10-sulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclic benzoic acid (CYCLAMIC ACID), dodecylsulfuric acid, ethane-1, 2-disulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutarate, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1, 5-disulfonic acid, naphthalene-2-sulfonic acid, 1-hydroxy-naphthoic acid, nicotinic acid, oleic acid, orotic acid, pamoic acid, oxalic acid, palmitic acid, propionic acid, glutamic acid, pyruvic acid, salicylic acid, 4-aminosalicylic acid, sebacic acid, succinic acid, sulfanilic acid, tartaric acid, tricarboxylic acid, undecylenic acid, and the like.

Examples of pharmaceutically acceptable base addition salts include, but are not limited to, salts prepared by adding an inorganic or organic base to the free acid compound. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. In one embodiment, the inorganic salts are ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, dantol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine (procaine), hydrabamine, choline, betaine, phenethylbenzylamine, benzathine, ethylenediamine, glucosamine, methylglucamine, theobromine, triethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. In one embodiment, the organic base is isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline, and caffeine.

The compounds provided herein may contain one or more asymmetric centers and thus may produce enantiomers, diastereomers, and other stereoisomeric forms, which may be defined as (R) -or (S) -or (D) -or (L) -for amino acids, depending on the absolute stereochemistry. Unless otherwise indicated, the compounds provided herein are intended to include all such possible isomers, as well as their racemic and optically pure forms. Optically active (+) and (-), (R) -and (S) -or (D) -and (L) -isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques, such as chromatography and fractional crystallization. Conventional techniques for preparing/separating individual enantiomers include chiral synthesis from suitable optically pure precursors or resolution of the racemate (or of a salt or derivative) using, for example, chiral High Pressure Liquid Chromatography (HPLC). When a compound described herein contains an olefinic double bond or other geometric asymmetric center, the compound is intended to include both the E and Z geometric isomers unless specified otherwise. Also, all tautomeric forms are intended to be included.

As used herein and unless otherwise indicated, the term "isomer" refers to different compounds having the same molecular formula. "stereoisomers" are isomers that differ only in the arrangement of atoms in space. "atropisomers" are stereoisomers resulting from a hindered rotation about a single bond. "enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of each other. A mixture of any ratio of a pair of enantiomers may be referred to as a "racemic" mixture. "diastereomers" are stereoisomers which have at least two asymmetric atoms and which are not mirror images of each other.

"Stereoisomers" may also include E and Z isomers or mixtures thereof, as well as cis and trans isomers or mixtures thereof. In certain embodiments, the compounds described herein are isolated as E or Z isomers. In other embodiments, the compounds described herein are mixtures of E and Z isomers.

"Tautomer" refers to the isomeric forms of a compound that are balanced with each other. The concentration of the isomeric forms will depend on the environment in which the compound is located and may vary depending on, for example, whether the compound is in a solid or in an organic or aqueous solution.

It should also be noted that the compounds described herein may contain non-natural proportions of atomic isotopes at one or more atoms. For example, the compounds may be radiolabeled with a radioisotope, such as tritium (³ H), iodine-125 (¹²⁵ I), sulfur-35 (³⁵ S) or carbon-14 (¹⁴ C), or may be isotopically enriched, such as deuterium (² H), carbon-13 (¹³ C) or nitrogen-15 (¹⁵ N). As used herein, "isotopologue" is an isotopically enriched compound. The term "isotopically enriched" refers to an atomic isotopic composition that differs from the natural isotopic composition of the atom. "isotopically enriched" may also mean that the isotopic composition of at least one atom contained in a compound is different from the natural isotopic composition of said atom. The term "isotopic composition" refers to the amount of each isotope present for a given atom. Radiolabeled and isotopically enriched compounds are useful as therapeutic agents, e.g., cancer therapeutic agents, research reagents, e.g., binding assay reagents, and diagnostic agents, e.g., in vivo imaging agents. All isotopic variations of the compounds described herein, whether radioactive or not, are intended to be encompassed within the scope of the embodiments provided herein. In some embodiments, isotopologues of the compounds described herein are provided, e.g., isotopologues are deuterium, carbon-13, and/or nitrogen-15 enriched. As used herein, "deuterated" means that at least one hydrogen (H) in the compound has been replaced with deuterium (represented by D or ² H), that is, the compound is deuterium-enriched in at least one position.

It should be noted that if there is a difference between the depicted structure and the name of the structure, the depicted structure should be subject to.

As used herein and unless otherwise indicated, the term "pharmaceutically acceptable carrier, diluent or excipient" includes, but is not limited to, any adjuvant, carrier, excipient, glidant, sweetener, diluent, preservative, dye/colorant, flavor enhancer, surfactant, wetting agent, dispersing agent, suspending agent, stabilizer, isotonizing agent, solvent or emulsifier approved by the U.S. food and drug administration for use in humans or livestock.

The term "composition" is intended to encompass products containing the specified ingredients (e.g., mRNA molecules provided herein) in the optionally specified amounts.

As used interchangeably herein, the term "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes, for example, DNA and RNA. The nucleotide may be a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and/or analogue thereof, or any substrate that can be incorporated into the polymer by a DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may comprise modified nucleotides, such as methylated nucleotides and the like. The nucleic acid may be in single-stranded or double-stranded form. As used herein and unless otherwise indicated, "nucleic acid" also includes nucleic acid mimics, such as Locked Nucleic Acids (LNAs), peptide Nucleic Acids (PNAs), and morpholino nucleic acids. As used herein, "oligonucleotide" refers to a short synthetic polynucleotide, typically but not necessarily less than about 200 nucleotides in length. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description of polynucleotides applies equally and entirely to oligonucleotides. Unless otherwise indicated, the left hand end of any single stranded polynucleotide sequence disclosed herein is the 5 'end, and the left hand direction of a double stranded polynucleotide sequence is referred to as the 5' direction. The 5 'to 3' addition direction of the nascent RNA transcript is referred to as the transcription direction, the region of the DNA strand having the same sequence as the RNA transcript and located 5 'relative to the 5' end of the RNA transcript is referred to as the "upstream sequence", and the region of the DNA strand having the same sequence as the RNA transcript and located 3 'relative to the 3' end of the RNA transcript is referred to as the "downstream sequence".

As used herein, the term "non-naturally occurring" when used in reference to a nucleic acid molecule as described herein is intended to mean that the nucleic acid molecule is not present in nature. Non-naturally occurring nucleic acids encoding viral peptides or proteins contain at least one genetic alteration or chemical modification that is not normally present in a naturally occurring strain of a virus, including a wild-type strain of a virus. Genetic alterations include, for example, modifications that introduce expressible nucleic acid sequences encoding heterologous peptides or polypeptides of the virus, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, and/or other functional disruption of the genetic material of the virus. Such modifications include, for example, modifications to coding regions of heterologous, homologous, or heterologous and homologous polypeptides of a viral species, as well as functional fragments thereof. Additional modifications include, for example, modifications to non-coding regulatory regions, wherein the modifications alter expression of a gene or an operon. Additional modifications also include, for example, incorporation of the nucleic acid sequence into a vector such as a plasmid or artificial chromosome. Chemical modifications include, for example, one or more functional nucleotide analogs as described herein.

"Isolated nucleic acid" refers to nucleic acids, such as RNA, DNA, or mixed nucleic acids, that are substantially isolated from other genomic DNA sequences that naturally accompany the native sequence, as well as from proteins or complexes such as ribosomes and polymerases. An "isolated" nucleic acid molecule is a nucleic acid molecule that is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid molecule. Furthermore, an "isolated" nucleic acid molecule, such as an mRNA molecule, may be substantially free of other cellular material or culture medium when produced by recombinant techniques, or it may be substantially free of chemical precursors or other chemicals when chemically synthesized. In certain embodiments, one or more nucleic acid molecules encoding an antigen as described herein are isolated or purified. The term includes nucleic acid sequences that have been removed from their naturally occurring environment, and includes recombinant or cloned DNA or RNA isolates as well as chemically synthesized analogs or analogs biosynthesized by heterologous systems. Substantially pure molecules may include isolated forms of the molecule.

The term "encoding nucleic acid" or grammatical equivalents thereof when used in reference to a nucleic acid molecule includes (a) nucleic acid molecules that, when in a native state or manipulated by methods well known to those of skill in the art, can be transcribed to produce mRNA and then translated into peptide and/or polypeptide, and (b) mRNA molecules themselves. The antisense strand is the complement of such a nucleic acid molecule and from which the coding sequence can be deduced. The term "coding region" refers to the portion of a coding nucleic acid sequence that is translated into a peptide or polypeptide. The term "untranslated region" or "UTR" refers to that portion of a coding nucleic acid that is not translated into a peptide or polypeptide. Depending on the orientation of the UTR relative to the coding region of the nucleic acid molecule, the UTR is referred to as a 5'-UTR if it is located at the 5' end of the coding region and the UTR is referred to as a 3'-UTR if it is located at the 3' end of the coding region.

As used herein, the term "mRNA" refers to a messenger RNA molecule comprising one or more Open Reading Frames (ORFs) that can be translated by a cell or organism having the mRNA to produce one or more peptide or protein products. The region containing one or more ORFs is referred to as the coding region of the mRNA molecule. In certain embodiments, the mRNA molecule further comprises one or more untranslated regions (UTRs).

In certain embodiments, the mRNA is a monocistronic mRNA comprising only one ORF. In certain embodiments, the monocistronic mRNA encodes a peptide or protein comprising at least one epitope of a selected antigen (e.g., a pathogenic antigen or a tumor-associated antigen). In other embodiments, the mRNA is a polycistronic mRNA comprising two or more ORFs. In certain embodiments, polycistronic mRNA encodes two or more peptides or proteins that may be the same or different from each other. In certain embodiments, each peptide or protein encoded by the polycistronic mRNA comprises at least one epitope of the selected antigen. In certain embodiments, the different peptides or proteins encoded by the polycistronic mRNA each comprise at least one epitope of a different antigen. In any of the embodiments described herein, the at least one epitope may be at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 epitopes of the antigen.

The term "nucleobase" encompasses purines and pyrimidines, including the natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural or synthetic analogs or derivatives thereof.

As used herein, the term "functional nucleotide analog" refers to a modified version of a classical nucleotide A, G, C, U or T that (a) retains the base pairing properties of the corresponding classical nucleotide and (b) contains at least one chemical modification to (i) a nucleobase, (ii) a glycosyl, (iii) a phosphate group, or (iv) any combination of (i) to (iii) of the corresponding natural nucleotide. As used herein, base pairing encompasses not only classical Watson-Crick (Watson-Crick) adenine-thymine, adenine-uracil, or guanine-cytosine base pairs, but also base pairs formed between a classical nucleotide and a functional nucleotide analogue or between a pair of functional nucleotide analogues, wherein the arrangement of the hydrogen bond donor and the hydrogen bond acceptor allows hydrogen bonding to be formed between a modified nucleobase and a classical nucleobase or between two complementary modified nucleobase structures. For example, functional analogs of guanosine (G) retain the ability to base pair with cytosine (C) or functional analogs of cytosine. An example of such non-classical base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine or uracil. As described herein, functional nucleotide analogs can be naturally occurring or non-naturally occurring. Thus, a nucleic acid molecule containing a functional nucleotide analog may have at least one modified nucleobase, sugar group, and/or internucleoside linkage. Exemplary chemical modifications to nucleobases, glycosyls, or internucleoside linkages of nucleic acid molecules are provided herein.

As used herein, the terms "translational enhancer element," "TEE," and "translational enhancer" refer to a region in a nucleic acid molecule that is used to facilitate translation of a coding sequence of a nucleic acid into a protein or peptide product, such as into a protein or peptide product via cap-dependent or non-cap-dependent translation. TEE is typically located in the UTR region of a nucleic acid molecule (e.g., mRNA) and enhances the level of translation of coding sequences located upstream or downstream. For example, a TEE in the 5' -UTR of a nucleic acid molecule may be located between the promoter and the start codon of the nucleic acid molecule. Various TEE sequences are known in the art (WELLENSIEK et al Genome-wide profiling of human cap-INDEPENDENT TRANSLATION-ENHANCING ELEMENTS, nature Methods, month 8 of 2013; 10 (8): 747-750; chappell et al PNAS, 6, 29, 101 (26) 9590-9594). Some TEEs are known to be conserved across species (P nek et al Nucleic ACIDS RESEARCH, vol.41, 16, 2013, 9, 1, pages 7625-7634).

As used herein, the term "stem-loop sequence" refers to a single stranded polynucleotide sequence having at least two regions that are complementary or substantially complementary to each other when read in opposite directions, and thus are capable of base pairing with each other to form at least one duplex and unpaired loop. The resulting structure is known as a stem-loop structure, hairpin, or hairpin loop, which is a secondary structure found in many RNA molecules.

As used herein, the term "peptide" refers to a polymer containing from two to fifty (2-50) amino acid residues linked by one or more covalent peptide bonds. The term applies to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs or non-natural amino acids).

The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer having more than fifty (50) amino acid residues joined by covalent peptide bonds. That is, the description for polypeptides applies equally to the description for proteins and vice versa. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues are non-naturally occurring amino acids (e.g., amino acid analogs). As used herein, the term encompasses amino acid chains of any length, including full-length proteins (e.g., antigens).

In the case of a peptide or polypeptide, the term "derivative" as used herein refers to a peptide or polypeptide comprising the amino acid sequence of a viral peptide or protein or a fragment of a viral peptide or protein that has been altered by the introduction of amino acid residue substitutions, deletions or additions. As used herein, the term "derivative" also refers to a viral peptide or protein, or a fragment of a viral peptide or protein, which has been chemically modified, for example, by covalently linking any type of molecule to a polypeptide. For example, but not by way of limitation, a viral peptide or protein or fragment of a viral peptide or protein may be chemically modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, chemical cleavage, formulation, metabolic synthesis of tunicamycin, attachment to a cellular ligand or other protein, and the like. Derivatives are modified in a manner that differs from the naturally occurring or starting peptide or polypeptide in the type or position of the attached molecule. Derivatives also include the absence of one or more chemical groups naturally present on the viral peptide or protein. In addition, the viral peptide or protein or a derivative of a fragment of the viral peptide or protein may contain one or more non-classical amino acids. In particular embodiments, a derivative is a functional derivative of a native or unmodified peptide or polypeptide from which the derivative is derived.

The term "functional derivative" refers to a derivative that retains one or more functions or activities of a naturally occurring or starting peptide or polypeptide from which the derivative is derived. For example, a functional derivative of the VZV S protein may retain the ability to bind to one or more of its receptors on a host cell. For example, functional derivatives of VZV N proteins may retain the ability to bind RNA or package viral genomes.

The term "identity" refers to the relationship between sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by aligning and comparing the sequences. "percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to amino acid residues in the reference polypeptide sequence after aligning the sequences and, if necessary, introducing gaps (achieving the maximum percent sequence identity) and not considering any conservative substitutions as part of the sequence identity. The alignment for purposes of determining the percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or megasign (DNAStar, inc.) software. One skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the compared sequences.

"Modification" of an amino acid residue/position refers to a change in the primary amino acid sequence as compared to the starting amino acid sequence, wherein the change is caused by a sequence change involving the amino acid residue/position. For example, typical modifications include substitution of a residue with another amino acid (e.g., conservative or non-conservative substitutions), insertion of one or more (e.g., typically less than 5, 4, or 3) amino acids immediately adjacent to the residue/position, and/or deletion of the residue/position.

In the case of peptides or polypeptides, the term "fragment" as used herein refers to a peptide or polypeptide comprising less than the full length amino acid sequence. Such fragments may, for example, result from amino-terminal truncations, carboxy-terminal truncations and/or internal deletions of residues in the amino acid sequence. Fragments may be produced, for example, by alternative RNA splicing or by protease activity in vivo. In certain embodiments, a fragment refers to a polypeptide comprising an amino acid sequence of at least 5 consecutive amino acid residues, at least 10 consecutive amino acid residues, at least 15 consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 40 consecutive amino acid residues, at least 50 consecutive amino acid residues, at least 60 consecutive amino acid residues, at least 70 consecutive amino acid residues, at least 80 consecutive amino acid residues, at least 90 consecutive amino acid residues, at least 100 consecutive amino acid residues, at least 125 consecutive amino acid residues, at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, at least 650, at least 700, at least 750, at least 850, at least 900 or at least 950 consecutive amino acid residues of the amino acid sequence of the polypeptide. In particular embodiments, fragments of a polypeptide retain at least 1, at least 2, at least 3, or more functions of the polypeptide.

As used herein in the context of a peptide or polypeptide (e.g., a protein), the term "immunogenic fragment" refers to a fragment of a peptide or polypeptide that retains the ability of the peptide or polypeptide to elicit an immune response (including an innate immune response and/or an adaptive immune response) upon contact with the mammalian immune system. In some embodiments, the immunogenic fragment of a peptide or polypeptide may be an epitope.

The term "antigen" refers to a substance that is capable of being recognized by the immune system of a subject (including the adaptive immune system) and is capable of triggering an immune response (including an antigen-specific immune response) upon contacting the subject with the antigen. In certain embodiments, the antigen is a protein (e.g., a tumor-associated antigen (TAA)) associated with a diseased cell, such as a pathogen-infected cell, or a neoplastic cell.

An "epitope" is a site on the surface of an antigen molecule that binds to a single antibody molecule, such as a localized region on the surface of an antigen that is capable of binding to one or more antigen binding regions of an antibody and has antigenic or immunogenic activity in an animal, such as a mammal (e.g., a human being), capable of eliciting an immune response. An epitope with immunogenic activity is a portion of a polypeptide that elicits an antibody response in an animal. Epitopes having antigenic activity are part of the polypeptide to which the antibody binds, as determined by any method well known in the art, including, for example, by an immunoassay. An antigenic epitope is not necessarily immunogenic. Epitopes are generally composed of chemically active surface groups of molecules, such as amino acids or sugar side chains, and have specific three-dimensional structural features as well as specific charge characteristics. The antibody epitope may be a linear epitope or a conformational epitope. Linear epitopes are formed by contiguous amino acid sequences in proteins. Conformational epitopes are formed by amino acids that are discontinuous in the protein sequence, but which group together when the protein folds into its three-dimensional structure. An inductive epitope is formed when the three-dimensional structure of a protein is in an altered conformation, such as after activation or binding of another protein or ligand. In certain embodiments, the epitope is a three-dimensional surface feature of the polypeptide. In other embodiments, the epitope is a linear characteristic of the polypeptide. Typically, an antigen has several or many different epitopes and can react with many different antibodies.

The term "heterologous" refers to an entity that is not found in nature in association with (e.g., encoded and/or expressed by the genome of) a naturally occurring VZV. The term "homologous" refers to an entity found in nature that is associated with (e.g., encoded and/or expressed by the genome of) a naturally occurring VZV.

As used herein, the term "genetic vaccine" refers to a therapeutic or prophylactic composition comprising at least one nucleic acid molecule encoding an antigen associated with a disease of interest (e.g., an infectious disease or neoplastic disease). Administration of a vaccine to a subject ("vaccination") allows for the production of the encoded peptide or protein, thereby eliciting an immune response against the disease of interest in the subject. In certain embodiments, the immune response includes an adaptive immune response, such as the production of antibodies to the encoded antigen, and/or the activation and proliferation of immune cells capable of specifically eliminating diseased cells expressing the antigen. In certain embodiments, the immune response further comprises an innate immune response. According to the present disclosure, the vaccine may be administered to the subject either before or after the onset of clinical symptoms of the disease of interest. In some embodiments, vaccinating healthy or asymptomatic subjects renders the vaccinated subjects immune or less susceptible to the development of the disease of interest. In some embodiments, vaccinating a subject exhibiting symptoms of a disease improves the disease condition or treats the disease in the vaccinated subject.

The term "vector" refers to a substance used to carry or contain a nucleic acid sequence, including, for example, a nucleic acid sequence encoding a viral peptide or protein as described herein, in order to introduce the nucleic acid sequence into a host cell, or to serve as a transcription template to perform an in vitro transcription reaction in a cell-free system to produce mRNA. Vectors suitable for use include, for example, expression vectors, plasmids, phage vectors, viral vectors, episomes, and artificial chromosomes, which may include selection sequences or markers operable for stable integration into the chromosomes of a host cell. In addition, the vector may include one or more selectable marker genes and appropriate transcriptional or translational control sequences. For example, selectable marker genes may be included to provide resistance to antibiotics or toxins, to supplement auxotrophs for deficiency, or to provide key nutrients that are not in the medium. Transcriptional or translational control sequences may include constitutive and inducible promoters, transcriptional enhancers, transcriptional terminators, and the like, as are well known in the art. When two or more nucleic acid molecules (e.g., nucleic acid molecules encoding two or more different viral peptides or proteins) are co-transcribed or co-translated, the two nucleic acid molecules may be inserted, for example, into the same expression vector or into separate expression vectors. For single vector transcription and/or translation, the coding nucleic acids may be operably linked to one common transcriptional or translational control sequence, or to different transcriptional or translational control sequences, such as an inducible promoter and a constitutive promoter. The introduction of a nucleic acid molecule into a host cell can be confirmed using methods well known in the art. Such methods include, for example, nucleic acid analysis, such as Northern blot (Northern blot) or Polymerase Chain Reaction (PCR) amplification of mRNA, immunoblotting for expression of gene products, or other suitable analytical methods for testing the expression of the introduced nucleic acid sequences or their corresponding gene products. Those of skill in the art will understand that a nucleic acid molecule is expressed in sufficient amounts to produce a desired product (e.g., an mRNA transcript of a nucleic acid as described herein), and will further understand that the expression level can be optimized to obtain sufficient expression using methods well known in the art.

The terms "innate immune response" and "innate immunity" are art-recognized and refer to the non-specific defense mechanisms that the body's immune system initiates upon recognition of pathogen-associated molecular patterns that involve different forms of cellular activity, including cytokine production and cell death through various pathways. As used herein, an innate immune response includes, but is not limited to, increased production of inflammatory cytokines (e.g., type I interferon or IL-10 production), activation of the nfkb pathway, increased proliferation, maturation, differentiation and/or survival of immune cells, and in some cases induction of apoptosis. Activation of innate immunity can be detected using methods known in the art, such as measuring (NF) - κb activation.

The terms "adaptive immune response" and "adaptive immunity" are art-recognized and refer to antigen-specific defense mechanisms initiated by the body's immune system upon recognition of a particular antigen, including humoral and cell-mediated responses. As used herein, an adaptive immune response includes a cellular response triggered and/or enhanced by a vaccine composition, such as the genetic compositions described herein. In some embodiments, the vaccine composition comprises an antigen that is a target of an antigen-specific adaptive immune response. In other embodiments, the vaccine composition allows for the production of an antigen in the immunized subject after administration, which antigen is a target of an antigen-specific adaptive immune response. Activation of the adaptive immune response can be detected using methods known in the art, such as measuring the production of antigen-specific antibodies or the level of antigen-specific cell-mediated cytotoxicity.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted immunoglobulins that bind to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., natural Killer (NK) cells, neutrophils, and macrophages) enable these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are absolutely required for such killing. NK cells (the primary cells used to mediate ADCC) express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is known (see, e.g., ravetch and Kinet,1991, annu. Rev. Immunol. 9:457-92). To assess ADCC activity of a target molecule, an in vitro ADCC assay may be performed (see, e.g., U.S. Pat. nos. 5,500,362 and 5,821,337). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule may be assessed in vivo, e.g., in animal models (see, e.g., clynes et al 1998,Proc.Natl.Acad.Sci.USA 95:652-56). Antibodies with little or no ADCC activity may be selected for use.

"Antibody-dependent cellular phagocytosis" or "ADCP" refers to the destruction of target cells via monocyte or macrophage-mediated phagocytosis when immunoglobulins bind to Fc receptors (fcrs) present on certain phagocytes (e.g., neutrophils, monocytes and macrophages) so that these phagocytes can specifically bind to antigen-bearing target cells and subsequently kill the target cells. To assess the ADCP activity of a target molecule, an in vitro ADCP assay may be performed (see, e.g., bracher et al, 2007,J.Immunol.Methods 323:160-71). Useful phagocytes for such assays include Peripheral Blood Mononuclear Cells (PBMCs), purified monocytes from PBMCs, or U937 cells differentiated into a mononuclear type. Alternatively or additionally, ADCP activity of the target molecule may be assessed in vivo, e.g., in animal models (see, e.g., wallace et al 2001,J.Immunol.Methods 248:167-82). Antibodies with little or no ADCP activity may be selected for use.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. An exemplary FcR is a native sequence human FcR. Furthermore, exemplary fcrs are receptors that bind IgG antibodies (e.g., gamma receptors), and include receptors of fcγri, fcγrii, and fcγriii subclasses, including allelic variants and alternatively spliced forms of these receptors. Fcγrii receptors include fcγriia ("activating receptor") and fcγriib ("inhibiting receptor") which have similar amino acid sequences differing primarily in their cytoplasmic domains (see, e.g.1997, Annu. Rev. Immunol. 15:203-34). Various FcRs are known (see, e.g., ravetch and Kinet,1991, annu. Rev. Immunol.9:457-92; capel et al, 1994,Immunomethods 4:25-34; and de Haas et al, 1995, J. Lab. Clin. Med. 126:330-41). The term "FcR" herein encompasses other fcrs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (see, e.g., guyer et al, 1976, J.Immunol.117:587-93; and Kim et al, 1994, eu.J.Immunol.24:2429-34). Antibody variants with improved or reduced binding to FcR have been described (see, e.g., WO 2000/42072; U.S. Pat. No. 7,183,387;7,332,581; and 7.335,742; shields et al, 2001, J.biol. Chem.9 (2): 6591-604).

"Complement-dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (C1 q) to antibodies (of the appropriate subclass) that bind to their cognate antigens. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, 1996,J.Immunol.Methods 202:163). Polypeptide variants having altered amino acid sequences of the Fc region (polypeptides having variant Fc regions) and increased or decreased C1q binding capacity have been described (see, e.g., U.S. Pat. No. 6,194,551;WO 1999/51642; idusogie et al, 2000, J. Immunol. 164:4178-84). Antibodies with little or no CDC activity may be selected for use.

The term "antibody" is intended to include polypeptide products of B cells within polypeptides of the immunoglobulin class that are capable of binding to a particular molecular antigen and are composed of two pairs of identical polypeptide chains, wherein each pair has one heavy chain (about 50-70 kDa) and one light chain (about 25 kDa), each amino-terminal portion of each chain comprises a variable region of about 100 to about 130 amino acids or more, and each carboxy-terminal portion of each chain comprises a constant region. See, e.g., antibody Engineering (Borrebaeck, 2 nd edition, 1995), and Kuby, immunology (3 rd edition, 1997). In particular embodiments, a particular molecular antigen may be bound by an antibody provided herein, including a polypeptide, fragment or epitope thereof. Antibodies also include, but are not limited to, synthetic antibodies, recombinantly produced antibodies, camelized antibodies, intracellular antibodies, anti-idiotype (anti-Id) antibodies, and functional fragments of any of the foregoing, which refers to a portion of an antibody heavy or light chain polypeptide that retains some or all of the binding activity of the antibody from which the fragment is derived. Non-limiting examples of functional fragments include single chain Fv (scFv) (e.g., including monospecific, bispecific, etc.), fab fragments, F (ab ') fragments, F (ab) ₂ fragments, F (ab') ₂ fragments, disulfide-linked Fv (dsFv), fd fragments, fv fragments, diabodies, triabodies, tetrabodies, and minibodies. In particular, antibodies provided herein include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, such as antigen binding domains or molecules that contain an antigen binding site (e.g., one or more CDRs of an antibody). Such antibody fragments can be found, for example, in Harlow and Lane,Antibodies:A LaboratoryManual(1989);Mol.Biology and Biotechnology:A ComprehensiveDeskReference(Myers editors, 1995), huston et al, 1993,Cell Biophysics 22:189-224, pluckthun and Skerra,1989, meth. Enzymol.178:497-515, and Day, advancedImmunochemistry (2 nd edition, 1990). Antibodies provided herein can have any class (e.g., igG, igE, igM, igD and IgA) or any subclass (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2) of immunoglobulin molecules.

The term "administering" (administer/administeration) "refers to the operation of injecting or otherwise physically delivering a substance present in vitro (e.g., a lipid nanoparticle composition as described herein) into a patient, such as transmucosal, intradermal, intravenous, intramuscular delivery, and/or any other physical delivery method described herein or known in the art. When treating a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed after onset of the disease, disorder, condition, or symptom thereof. When preventing a disease, disorder, condition, or symptom thereof, administration of the substance is typically performed prior to onset of the disease, disorder, condition, or symptom thereof.

"Chronic" administration is in contrast to acute mode, meaning that one or more agents are administered in a continuous mode (e.g., for a period of time, such as days, weeks, months, or years), thereby maintaining an initial therapeutic effect (activity) over a longer period of time. By "intermittent" administration is meant that the treatment is not carried out continuously without interruption, but rather is periodic in nature.

As used herein, the term "targeted delivery" or verb form "targeted" refers to a process that facilitates the delivery of an agent (e.g., a therapeutic payload molecule in a lipid nanoparticle composition as described herein) to a particular organ, tissue, cell, and/or intracellular compartment (referred to as a target site) as compared to delivery to any other organ, tissue, cell, or intracellular compartment (referred to as a non-target site). Targeted delivery can be detected using methods known in the art, for example, by comparing the concentration of the delivered agent in the target cell population to the concentration of the delivered agent at the non-target cell population after systemic administration. In certain embodiments, targeted delivery results in a concentration at the target location that is at least 2 times higher than the concentration at the non-target location.

An "effective amount" is generally an amount sufficient to reduce the severity and/or frequency of symptoms, eliminate symptoms and/or underlying causes, prevent the occurrence of symptoms and/or underlying causes thereof, and/or ameliorate or remedy a lesion caused by or associated with a disease, disorder, or condition, including, for example, infection and neoplasia. In some embodiments, the effective amount is a therapeutically effective amount or a prophylactically effective amount.

As used herein, the term "therapeutically effective amount" refers to an amount of an agent (e.g., a vaccine composition) sufficient to reduce and/or ameliorate the severity and/or duration of a given disease, disorder or condition, and/or symptoms associated therewith (e.g., an infectious disease, such as an infectious disease caused by a viral infection, or a neoplastic disease, such as cancer). The "therapeutically effective amount" of a substance/molecule/agent of the present disclosure (e.g., a lipid nanoparticle composition described herein) can vary depending on a number of factors, such as the disease state, age, sex, and weight of the individual, as well as the ability of the substance/molecule/agent to elicit a desired response in the individual. A therapeutically effective amount comprises an amount of the therapeutically beneficial effect of the substance/molecule/agent that outweighs any toxic or detrimental effect thereof. In certain embodiments, the term "therapeutically effective amount" refers to an amount of a lipid nanoparticle composition as described herein or a therapeutic or prophylactic agent (e.g., therapeutic mRNA) contained therein that is effective to "treat" a disease, disorder, or condition in a subject or mammal.

A "prophylactically effective amount" is an amount of an agent or pharmaceutical composition (e.g., a vaccine composition) that, when administered to a subject, will have the intended prophylactic effect, e.g., prevent a disease, disorder, condition, or related symptom (e.g., an infectious disease, such as an infectious disease caused by a viral infection, or a neoplastic disease, such as cancer), delay the onset (or recurrence) thereof, or reduce the likelihood of onset (or recurrence) thereof. In certain embodiments, the term "prophylactically effective amount" refers to an amount of a lipid nanoparticle composition described herein or a prophylactic agent (e.g., a nucleic acid such as, for example, a nucleic acid described in section 5.4 or 6, including mRNA) contained therein that is effective to "prevent" a disease, disorder, or condition in a subject or mammal. Typically, but not necessarily, since the prophylactic dose is for the subject prior to or at an early stage of the disease, disorder or condition, the prophylactically effective amount may be less than the therapeutically effective amount. Complete therapeutic or prophylactic action does not necessarily occur through administration of one dose, but may occur only after administration of a series of doses. Thus, a therapeutically or prophylactically effective amount can be administered in one or more administrations.

The term "preventing" refers to reducing the likelihood of onset (or recurrence) of a disease, disorder, condition, or associated symptom (e.g., an infectious disease, such as an infectious disease caused by a viral infection, or a neoplastic disease, such as cancer).

The terms "control (manage)", "control (managing)" and "management" refer to beneficial effects that a subject obtains from therapy (e.g., prophylactic or therapeutic agents) that do not cause cure of the disease. In certain embodiments, one or more therapies (e.g., prophylactic or therapeutic agents, such as lipid nanoparticle compositions as described herein) are administered to a subject to "control" an infectious or neoplastic disease, one or more symptoms thereof, thereby preventing progression or worsening of the disease.

The term "prophylactic agent" refers to any agent that can inhibit, in whole or in part, the development, recurrence, onset, or spread of a disease and/or symptoms associated therewith in a subject. In some embodiments, the prophylactic agent comprises or consists of a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or chapter 6). In some embodiments, the prophylactic agent comprises or consists of a protein described herein (e.g., a protein described in section 5.3). In some embodiments, the prophylactic agent comprises or consists of a vector comprising a nucleic acid as described herein.

The term "therapeutic agent" refers to any agent that can be used to treat, prevent, or ameliorate a disease, disorder, or condition, including one or more symptoms used to treat, prevent, or ameliorate a disease, disorder, or condition and/or symptoms associated therewith. In some embodiments, the therapeutic agent is a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or chapter 6). In some embodiments, the therapeutic agent is a protein described herein (e.g., a protein described in section 5.3). In some embodiments, the prophylactic agent comprises or consists of a vector comprising a nucleic acid as described herein.

The term "therapy" refers to any regimen, method and/or agent that may be used to prevent, control, treat and/or ameliorate a disease, disorder or condition. In certain embodiments, the term "therapy (therapies/therapy)" refers to biological, supportive, and/or other therapies known to those skilled in the art as useful for preventing, controlling, treating, and/or ameliorating a disease, disorder, or condition by medical personnel.

As used herein, a "prophylactically effective serum titer" is a serum titer of an antibody in a subject (e.g., a human) that completely or partially inhibits the development, recurrence, onset, or spread of a disease, disorder, or condition and/or symptoms associated therewith in the subject.

In certain embodiments, a "therapeutically effective serum titer" is a serum titer of an antibody in a subject (e.g., a human) that reduces the severity, duration, and/or symptoms associated with a disease, disorder, or condition in the subject.

The term "serum titer" refers to the average serum titer in a subject from multiple samples (e.g., at multiple time points) or in a population of at least 10, at least 20, at least 40 subjects, up to about 100,1000 or more subjects.

The term "side effects" encompasses unwanted and/or adverse effects of a therapy (e.g., a prophylactic or therapeutic agent). The unwanted effect is not necessarily bad. Adverse effects of therapies (e.g., prophylactic or therapeutic agents) can be detrimental, uncomfortable, or risky. Examples of side effects include diarrhea, cough, gastroenteritis, wheezing, nausea, vomiting, anorexia, abdominal cramps, fever, pain, weight loss, dehydration, alopecia, dyspnea, insomnia, dizziness, mucositis, nerve and muscle effects, fatigue, dry mouth, loss of appetite, rash or swelling at the site of administration, flu-like symptoms such as fever, chill and fatigue, digestive tract problems and allergic reactions. Other undesirable effects experienced by patients are numerous and known in the art. There are many roles described in Physician's desk reference (68 th edition, 2014).

The term "subject" is used interchangeably with "patient". As used herein, in certain embodiments, the subject is a mammal, such as a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) or a primate (e.g., monkey and human). In particular embodiments, the subject is a human. In one embodiment, the subject is a mammal (e.g., a human) having an infectious disease or neoplastic disease. In another embodiment, the subject is a mammal (e.g., a human) at risk of developing an infectious disease or neoplastic disease.

The term "elderly" refers to people over 65 years old. The term "human adult" refers to a person over 18 years of age. The term "human child" refers to a person aged 1 to 18 years. The term "human infant" refers to a person aged 1 to 3 years. The term "human infant" refers to a newborn to a person of 1 year old.

The term "detectable probe" refers to a composition that provides a detectable signal. The term includes, but is not limited to, any fluorophore, chromophore, radiolabel, enzyme, antibody or antibody fragment, etc. that provides a detectable signal by activity.

The term "detectable agent" refers to a substance that can be used to determine the presence of a desired molecule, such as an antigen encoded by an mRNA molecule described herein, in a sample or subject. The detectable agent may be a substance that can be visualized or a substance that can be otherwise determined and/or measured (e.g., by quantification).

"Substantially all" means at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or about 100%.

As used herein and unless otherwise indicated, the term "about" or "approximately" means an acceptable error for a particular value determined by one of ordinary skill in the art, which depends in part on the manner in which the value is measured or determined. In certain embodiments, the term "about" or "approximately" means within 1, 2, 3, or 4 standard deviations. In certain embodiments, the terms "about" and "approximately" mean within 20%, within 15%, within 10%, within 9%, within 8%, within 7%, within 6%, within 5%, within 4%, within 3%, within 2%, within 1%, within 0.5%, within 0.05% or less of a given value or range.

As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

All publications, patent applications, accession numbers, and other references cited in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the application is not entitled to antedate such publication by virtue of prior application. In addition, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

Various embodiments of the present invention have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the description in the experimental section and examples is intended to illustrate and not limit the scope of the invention as described in the claims.

5.3 Proteins

VZV glycoproteins include glycoprotein E (gE), glycoprotein B (gB), glycoprotein H (gH), and glycoprotein L (gL), where gE is the most abundant VSV glycoprotein expressed by VZV-infected cells. VZV gE is typically 623 amino acid residues in length and comprises a signal peptide (typically amino acid residues 1-30 of full length gE; e.g., the signal peptide of GenBank accession No. AAG32558.1 consists of amino acid residues 1-30 of GenBank accession No. AAG 32558.1) and a transmembrane domain. Mature gE lacks the signal peptide. VZV gE forms heterodimers with gI and heterodimers gE/gI are necessary for intercellular transmission of the virus. Furthermore, VZV gE/gI heterodimers interact with the Fc region of IgG. VZV gE binds to Insulin Degrading Enzymes (IDEs). VZV gE is phosphorylated by a viral kinase encoded by ORF 47. Exemplary VZV ges can be found in GenBank accession nos. AAG32558.1、AAF61669.1、AAK19946.1、AAK01056.1、AAK19955.1、ABF21641.1、ABF22006.1、ABF22152.1、ABF22152.1、ABF22225.1、ABF22298.1、AEW88044.1、AEW88116.1、AAY57677.1、AAY57748.1、Q9J3M8.1、CAA27951.1、QCA47220.1、AEW88980.1、WWU03079.1 and AEW88548.1 and Uniprot nos. Q9J3M8 and P09259. In some embodiments, the VZV gE is the gE of the Dumas strain. In some embodiments, the VZV gE is the gE of strains KPZ-287. In some embodiments, the VZV gE is that of the VZVi/Munich. GER/30.07/Z3 strain. In some embodiments, VZV gE is gE of strain NSYY. In some embodiments, the VZV gE is that of strain 1002/2008. In some embodiments, VZV gE is gE of strain NSYY. In some embodiments, VZV gE is gE of the Oka strain.

In some embodiments, provided herein are fragments of mature VZV gE, wherein the fragments comprise a truncation of at least one and up to 50, 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of up to 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 14 or 37 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of 37 amino acid residues from the C-terminal end of the mature gE. In some embodiments, the truncation is a truncation of up to 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of up to 30 or 29 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of up to 28 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of up to 27 or 26 amino acid residues from the C-terminus of the mature gE. In some embodiments, the truncation is a truncation of up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues or up to 1 amino acid residue from the C-terminus of the mature gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1.

In some embodiments, the fragment of the mature gE further comprises one or more amino acid substitutions. In some embodiments, the fragment of the mature gE further comprises one, two, three, four, five, or all of the amino acid substitutions selected from Y569A, Y582G, S A, S595A, T596A and T598A, wherein amino acid residue positions 569, 582, 593, 595, 596, and 598 are numbered according to the amino acid residue positions of the full-length VZV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55.

In some embodiments, the fragment of mature gE further comprises an amino acid residue substitution Y569A, wherein amino acid residue position number 569 is according to the amino acid residue position number of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitution Y582G, wherein amino acid residue position 582 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A and Y582G, wherein amino acid residue position numbers 569 and 582 are according to the amino acid residue position numbers of full-length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, Y582G and S593A, wherein amino acid residue position numbers 569, 582 and 593 are amino acid residue position numbers according to full length VZV gE. In some embodiments, the fragment of mature gE further comprises the amino acid residue substitution Y569A, Y582G, S595A, wherein amino acid residue position numbers 569, 582 and 595 are according to the amino acid residue position numbers of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, Y582G and T596A, wherein amino acid residue position numbers 569, 582 and 596 are amino acid residue position numbers according to full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, Y582G and T598A, wherein amino acid residue position numbers 569, 582 and 598 are amino acid residue position numbers according to full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, Y582G, S a and S595A, wherein amino acid residue position numbers 569, 582, 593 and 595 are amino acid residue position numbers according to full length VZV gE. In some embodiments, the fragment of the mature gE further comprises the amino acid residue substitution Y569A, Y582G, S593A, S595A, T596A, wherein amino acid residue position numbers 569, 582, 593, 595, and 596 are according to the amino acid residue position numbers of the full length VZV gE. In some embodiments, the fragment of the mature gE further comprises amino acid residue substitutions Y569A, Y582G, S593A, S595A, T596A and T598A, wherein the amino acid residue position numbers 569, 582, 593, 595, 596, and 598 are according to the amino acid residue position numbers of the full length VZV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55.

In some embodiments, the fragment of mature gE further comprises amino acid residue substitution S593A, wherein amino acid residue position 593 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitution S595A, wherein amino acid residue position 595 is numbered according to the amino acid residue position of full-length VZV gE. In some embodiments, the fragment of mature gE further comprises an amino acid residue substitution T596A, wherein amino acid residue position 596 is numbered according to the amino acid residue position of full length VZV gE. In some embodiments, the fragment of mature gE further comprises an amino acid residue substitution T598A, wherein amino acid residue position 598 is numbered according to the amino acid residue position of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S593A and S595A, wherein amino acid residue positions 593 and 595 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S593A and T596A, wherein amino acid residue positions 593 and 596 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S593A and T598A, wherein amino acid residue positions 593 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S595A and T596A, wherein amino acid residue positions 595 and 596 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S595A and T598A, wherein amino acid residue positions 595 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions T596A and T598A, wherein amino acid residue positions 596 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of the mature gE further comprises amino acid residue substitutions S593A, S595A and T596A, wherein amino acid residue positions 593, 595 and 596 are numbered according to the amino acid residue positions of the full length VZV gE. In some embodiments, the fragment of the mature gE further comprises amino acid residue substitutions S593A, S595A and T598A, wherein amino acid residue positions 593, 595 and 598 are numbered according to the amino acid residue positions of the full length VZV gE. In some embodiments, the fragment of the mature gE further comprises amino acid residue substitution S595A, T596A, T598A, wherein amino acid residue positions 595, 596, and 598 are numbered according to the amino acid residue positions of the full-length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions S593A, S595A, T596A, T598A, wherein amino acid residue positions 593, 595, 596 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of the mature gE further comprises amino acid residue substitutions Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue position numbers 582, 593, 595, 596 and 598 are amino acid residue position numbers according to full length VZV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55.

In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A and S593A, wherein amino acid residue positions 569 and 593 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A and S595A, wherein amino acid residue positions 569 and 595 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A and T596A, wherein amino acid residue positions 569 and 596 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A and T598A, wherein amino acid residue positions 569 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S593A and S595A, wherein amino acid residue positions 569, 593 and 595 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S593A and T596A, wherein amino acid residue positions 569, 593 and 596 are numbered according to the amino acid residue positions of full length VZV gE. in some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S593A and T598A, wherein amino acid residue positions 569, 593 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S595A and T596A, wherein amino acid residue positions 569, 595 and 596 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S595A and T598A, wherein amino acid residue positions 569, 595 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, T596A and T598A, wherein amino acid residue positions 569, 596 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S593A, S595A and T596A, wherein amino acid residue positions 569, 593, 595 and 596 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises amino acid residue substitutions Y569A, S593A, S595A and T598A, wherein amino acid residue positions 569, 593, 595 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of mature gE further comprises the amino acid residue substitution Y569A, S595A, T596A, T598A, wherein amino acid residue positions 569, 595, 596 and 598 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the fragment of the mature gE further comprises the amino acid residue substitution Y569A, S593A, S595A, T596A, T598A, wherein amino acid residue positions 569, 593, 595, 596 and 598 are numbered according to the amino acid residue positions of the full length VZV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a heterologous signal peptide, wherein the N-terminus of the fragment is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the mature gE comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the heterologous signal peptide is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the fragment of the mature gE is a fragment described herein (e.g., a fragment of the mature gE described in the examples in this section or section 6). In some embodiments, the fragment of mature gE comprises the amino acid sequence of SEQ ID NO 3, 6, 8, 10 or 12. In a specific embodiment, the fragment comprises the amino acid sequence of SEQ ID NO. 6. In some embodiments, the fragment of mature gE comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83% or at least 84% identical to SEQ ID NO 3, 6, 8, 10 or 12. In some embodiments, the fragment of mature gE comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to SEQ ID NO 3, 6, 8, 10, or 12. In some embodiments, the fragment of mature gE comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93% or at least 94% identical to SEQ ID NO 3, 6, 8, 10 or 12. In some embodiments, the fragment of mature gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO 3, 6, 8, 10 or 12.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of mature gE, and the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide. In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of mature gE and amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are numbered according to the amino acid residue positions of full-length VZV gE, and the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of mature gE and amino acid residue substitutions Y569A and Y582G, wherein amino acid residues 569 and 582 are numbered according to the amino acid residue positions of full length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the fragment comprises the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO 59. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence of SEQ ID NO. 59. In some embodiments, the fusion protein comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO 59.

In some embodiments, provided herein is a fusion protein comprising a mutant of mature gE of VZV and a human IgE signal peptide, wherein the mutant comprises amino acid residue substitutions Y569A, Y582G, S593A, S595A, T596A and T598A, wherein amino acid residues 569, 582, 593, 595, 596 and 598 are numbered according to amino acid residue positions of full length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the full length VZV gE comprises the amino acid sequence of SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence of SEQ ID NO. 10. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23.

In some embodiments, provided herein is a fusion protein comprising a mutant of mature gE of VZV and a human IgE signal peptide, wherein the mutant comprises amino acid residue substitutions Y582G, S593A, S595A, T596A and T598A, wherein amino acid residues 582, 593, 595, 596 and 598 are numbered according to amino acid residue positions of full length VZV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23.

In some embodiments, provided herein is a fusion protein comprising a fragment of mature gE of VZV and a human IgE signal peptide, wherein the fragment comprises a truncation of 50 amino acid residues from the C-terminal end of mature gE and an amino acid residue substitution Y569A, wherein amino acid residues 569 are numbered according to the amino acid residue positions of full-length VZV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the fragment comprises the amino acid sequence of SEQ ID NO. 3. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23.

In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A and Y582G relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G and S593A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G and S595A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G and T596A relative to full length VSV gE.

In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise the amino acid residue substitutions Y569A, Y G and T598A relative to full length VSVgE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S593A and S595A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S593A and T596A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S593A and T598A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S595A and T596A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S595A and T598A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, T596A and T598A relative to full length VSV gE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y582G, S593A, S595A, T596A and T598A relative to full length VSVgE. In some embodiments, provided herein are mutants of full length gE of VZV, wherein the mutants comprise amino acid residue substitutions Y569A, Y582G, S A, S595A, T596A and T598A relative to full length VSV gE. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV, wherein the mutant comprises (i) a truncation of 37 amino acid residues from the C-terminus of mature gE, and (ii) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence shown in SEQ ID NO. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the mutant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 6. In some embodiments, the protein further comprises a signal peptide of VZV gE. In some embodiments, the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 6 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the protein comprises the amino acid sequence set forth in SEQ ID NO. 59. In some embodiments, the amino acid sequence of the protein consists of the amino acid sequence shown in SEQ ID NO. 59. In some embodiments, the heterologous signal peptide of the protein is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV, wherein the mutant comprises amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full-length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence of SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the mutant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the mutant comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the mutant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 8. In some embodiments, the protein further comprises a signal peptide of VZV gE. In some embodiments, the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. in some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 8 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the heterologous signal peptide of the protein is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV, wherein the mutant comprises amino acid residue substitutions Y569A, Y582G, S A, S595A, T596A and T598A, wherein amino acid residue positions 569, 582, 593, 595, 596 and 598 are amino acid residue position numbers of full length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 10. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence shown in SEQ ID NO. 10. In some embodiments, the mutant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the amino acid sequence set forth in SEQ ID NO. 10. In some embodiments, the mutant comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO. 10. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 10. In some embodiments, the mutant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 10. In some embodiments, the protein further comprises a signal peptide of VZV gE. In some embodiments, the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 10 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the heterologous signal peptide of the protein is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV, wherein the mutant comprises amino acid residue substitutions Y582G, S593A, S595A, T a and T598A, wherein amino acid residue positions 582, 593, 595, 596 and 598 are amino acid residue position numbers of full length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence shown in SEQ ID NO. 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the mutant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 12. In some embodiments, the protein further comprises a signal peptide of VZV gE. In some embodiments, the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 12 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the heterologous signal peptide of the protein is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, provided herein is a protein comprising a mutant of mature gE of VZV, wherein the mutant comprises (i) a truncation of 50 amino acid residues from the C-terminus of the mature gE protein, and (ii) an amino acid residue substitution Y569A, wherein amino acid residue position 569 is the amino acid residue position of full-length VSV gE. In some embodiments, the mature gE comprises the amino acid sequence set forth in SEQ ID NO. 1. In some embodiments, the full-length VZV gE comprises the amino acid sequence set forth in SEQ ID NO: 55. In some embodiments, the mutant comprises the amino acid sequence set forth in SEQ ID NO. 3. in some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence shown in SEQ ID NO. 3. In some embodiments, the mutant comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the mutant comprises an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the mutant comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 3. In some embodiments, the protein further comprises a signal peptide of VZV gE comprising the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 18. In some embodiments, the protein further comprises a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide. In some embodiments, the heterologous signal peptide is a human IgE signal peptide. In some embodiments, the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the human IgE signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23. In some embodiments, the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 3 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23. In some embodiments, the heterologous signal peptide of the protein is a human tPA signal peptide. In some embodiments, the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27. In some embodiments, the human tPA signal peptide comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 28.

In some embodiments, provided herein is a protein comprising the amino acid sequence of the VZV gE muteins disclosed in table 1. In some embodiments, provided herein is a protein comprising the amino acid sequence of VZV gE mutein-1 disclosed in table 1. In some embodiments, provided herein is a protein comprising the amino acid sequence of VZV gE mutein-2 disclosed in table 1. In some embodiments, provided herein is a protein comprising the amino acid sequence of VZV gE mutein-3 disclosed in table 1. In some embodiments, provided herein is a protein comprising the amino acid sequence of VZV gE mutein-4 disclosed in table 1. In some embodiments, provided herein is a protein comprising the amino acid sequence of VZV gE mutein-5 disclosed in table 1.

In some embodiments, provided herein are mutants of VZV gE protein described in article section 6 below. In some embodiments, provided herein are mutants of VZV gE described in section 6 below, except for controls. In some embodiments, provided herein are mutants of VZV gE encoded by nucleic acids other than controls described in section 6 below.

In some embodiments, the fragments described herein, mutants described herein, or fusion proteins described herein have a structure similar to wild-type VZV gE, as assessed by techniques known to those of skill in the art (such as, for example, X-ray crystallography, nuclear magnetic resonance) or binding to antibodies specific for conformational epitopes of wild-type VZV gE. In some embodiments, the proteins described herein comprising mutants of mature gE have a structure similar to wild-type VZV gE, as assessed by techniques known to those of skill in the art (such as, for example, X-ray crystallography, nuclear magnetic resonance) or binding to antibodies specific for conformational epitopes of wild-type VZV gE.

In some embodiments, a fragment described herein, a mutant described herein, or a fusion protein described herein retains at least one activity or function of mature VZV gE. In some embodiments, a protein comprising a mutant of mature gE described herein retains at least one activity or function of mature VZV gE. For example, in some embodiments, the ability to form heterodimers with gI is preserved. In another example, in some embodiments, the ability to bind to Fc receptors is preserved. In another example, in some embodiments, the ability to bind to Insulin Degrading Enzymes (IDE) is retained. In another example, in some embodiments, the ability to phosphorylate by a viral kinase encoded by ORF47 is retained.

5.4 Therapeutic nucleic acids

In one aspect, provided herein are therapeutic nucleic acid molecules for controlling, preventing, and treating VZV infection. In some embodiments, the therapeutic nucleic acid encodes a peptide or polypeptide that, when administered to a subject in need thereof, is expressed by cells in the subject to produce the encoded peptide or polypeptide. In some embodiments, the therapeutic nucleic acid molecule is a DNA molecule. In other embodiments, the therapeutic nucleic acid molecule is an RNA molecule. In particular embodiments, the therapeutic nucleic acid molecule is an mRNA molecule.

In some embodiments, the therapeutic nucleic acid molecule is formulated in a vaccine composition. In some embodiments, the vaccine composition is a genetic vaccine as described herein. In some embodiments, the vaccine composition comprises an mRNA molecule as described herein.

In some embodiments, the mRNA molecules of the present disclosure encode a peptide or polypeptide of interest, including any naturally or non-naturally occurring or otherwise modified polypeptide. The peptide or polypeptide encoded by the mRNA may be of any size and may have any secondary structure or activity. In some embodiments, the polypeptide encoded by the mRNA payload may have a therapeutic effect when expressed in a cell.

In some embodiments, the mRNA molecules of the present disclosure comprise at least one coding region (e.g., an Open Reading Frame (ORF)) encoding a peptide or polypeptide of interest. In some embodiments, the nucleic acid molecule further comprises at least one untranslated region (UTR). In certain embodiments, the untranslated region (UTR) is located upstream (5 'to) the coding region, and is referred to herein as the 5' -UTR. In certain embodiments, the untranslated region (UTR) is located downstream (3 'end) of the coding region, and is referred to herein as the 3' -UTR. In particular embodiments, the nucleic acid molecule comprises both a 5'-UTR and a 3' -UTR. In some embodiments, the 5'-UTR comprises a 5' -cap structure. In some embodiments, the nucleic acid molecule comprises a Kozak sequence (e.g., in the 5' -UTR). In some embodiments, the nucleic acid molecule comprises a poly-A region (e.g., in the 3' -UTR). In some embodiments, the nucleic acid molecule comprises a polyadenylation signal (e.g., in the 3' -UTR). In some embodiments, the nucleic acid molecule comprises a stabilizing region (e.g., in the 3' -UTR). In some embodiments, the nucleic acid molecule comprises a secondary structure. In some embodiments, the secondary structure is a stem-loop. In some embodiments, the nucleic acid molecule comprises a stem-loop sequence (e.g., in the 5'-UTR and/or 3' -UTR). In some embodiments, the nucleic acid molecule comprises one or more intron regions capable of excision during splicing. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from the group consisting of 5' -UTR and coding region. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from the group consisting of coding regions and 3' -UTRs. In specific embodiments, the nucleic acid molecule comprises one or more regions selected from the group consisting of 5'-UTR, coding region and 3' -UTR.

5.4.1 Coding region

In some embodiments, the nucleic acid molecules of the present disclosure comprise at least one coding region. In some embodiments, the coding region is an Open Reading Frame (ORF) encoding a single peptide or protein. In some embodiments, the coding region comprises at least two ORFs, each ORF encoding a peptide or protein. In embodiments where the coding region comprises more than one ORF, the peptides and/or proteins encoded may be the same or different from each other. In some embodiments, the multiple ORFs in the coding region are separated by a non-coding sequence. In a specific embodiment, the non-coding sequence separating the two ORFs comprises an Internal Ribosome Entry Site (IRES).

Without being bound by theory, it is contemplated that an Internal Ribosome Entry Site (IRES) can be used as the sole ribosome binding site, or as one of a plurality of ribosome binding sites of an mRNA. mRNA molecules containing more than one functional ribosome binding site can encode several peptides or proteins that are independently translated by the ribosome (e.g., polycistronic mRNA). Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises one or more Internal Ribosome Entry Sites (IRES). Examples of IRES sequences that may be used in connection with the present disclosure include, but are not limited to, those from picornaviruses (e.g., FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot and Mouth Disease Viruses (FMDV), hepatitis C Viruses (HCV), swine fever viruses (CSFV), murine Leukemia Viruses (MLV), monkey immunodeficiency viruses (SIV), or cricket paralysis viruses (CrPV).

In various embodiments, the nucleic acid molecules of the present disclosure encode at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 peptides or proteins. The peptide and protein encoded by the nucleic acid molecule may be the same or different. In some embodiments, the nucleic acid molecules of the present disclosure encode dipeptides (e.g., carnosine and anserine). In some embodiments, the nucleic acid molecule encodes a tripeptide. In some embodiments, the nucleic acid molecule encodes a tetrapeptide. In some embodiments, the nucleic acid molecule encodes a pentapeptide. In some embodiments, the nucleic acid molecule encodes a hexapeptide. In some embodiments, the nucleic acid molecule encodes a heptapeptide. In some embodiments, the nucleic acid molecule encodes an octapeptide. In some embodiments, the nucleic acid molecule encodes a nonapeptide. In some embodiments, the nucleic acid molecule encodes a decapeptide. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 15 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 50 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 100 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 150 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 300 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 500 amino acids. In some embodiments, the nucleic acid molecule encodes a peptide or polypeptide having at least about 1000 amino acids.

In some embodiments, the nucleic acid molecules of the present disclosure are at least about 30 nucleotides (nt) in length. In some embodiments, the nucleic acid molecule is at least about 35nt in length. In some embodiments, the nucleic acid molecule is at least about 40nt in length. In some embodiments, the nucleic acid molecule is at least about 45nt in length. In some embodiments, the nucleic acid molecule is at least about 50nt in length. In some embodiments, the nucleic acid molecule is at least about 55nt in length. In some embodiments, the nucleic acid molecule is at least about 60nt in length. In some embodiments, the nucleic acid molecule is at least about 65nt in length. In some embodiments, the nucleic acid molecule is at least about 70nt in length. In some embodiments, the nucleic acid molecule is at least about 75nt in length. In some embodiments, the nucleic acid molecule is at least about 80nt in length. In some embodiments, the nucleic acid molecule is at least about 85nt in length. In some embodiments, the nucleic acid molecule is at least about 90nt in length. In some embodiments, the nucleic acid molecule is at least about 95nt in length. In some embodiments, the nucleic acid molecule is at least about 100nt in length. In some embodiments, the nucleic acid molecule is at least about 120nt in length. In some embodiments, the nucleic acid molecule is at least about 140nt in length. In some embodiments, the nucleic acid molecule is at least about 160nt in length. In some embodiments, the nucleic acid molecule is at least about 180nt in length. In some embodiments, the nucleic acid molecule is at least about 200nt in length. In some embodiments, the nucleic acid molecule is at least about 250nt in length. In some embodiments, the nucleic acid molecule is at least about 300nt in length. In some embodiments, the nucleic acid molecule is at least about 400nt in length. In some embodiments, the nucleic acid molecule is at least about 500nt in length. In some embodiments, the nucleic acid molecule is at least about 600nt in length. In some embodiments, the nucleic acid molecule is at least about 700nt in length. In some embodiments, the nucleic acid molecule is at least about 800nt in length. In some embodiments, the nucleic acid molecule is at least about 900nt in length. In some embodiments, the nucleic acid molecule is at least about 1000nt in length. In some embodiments, the nucleic acid molecule is at least about 1100nt in length. In some embodiments, the nucleic acid molecule is at least about 1200nt in length. In some embodiments, the nucleic acid molecule is at least about 1300nt in length. In some embodiments, the nucleic acid molecule is at least about 1400nt in length. In some embodiments, the nucleic acid molecule is at least about 1500nt in length. In some embodiments, the nucleic acid molecule is at least about 1600nt in length. In some embodiments, the nucleic acid molecule is at least about 1700nt in length. In some embodiments, the nucleic acid molecule is at least about 1800nt in length. In some embodiments, the nucleic acid molecule is at least about 1900nt in length. In some embodiments, the nucleic acid molecule is at least about 2000nt in length. In some embodiments, the nucleic acid molecule is at least about 2500nt in length. In some embodiments, the nucleic acid molecule is at least about 3000nt in length. In some embodiments, the nucleic acid molecule is at least about 3500nt in length. In some embodiments, the nucleic acid molecule is at least about 4000nt in length. In some embodiments, the nucleic acid molecule is at least about 4500nt in length. In some embodiments, the nucleic acid molecule is at least about 5000nt in length.

In particular embodiments, the therapeutic nucleic acids of the present disclosure are formulated into vaccine compositions (e.g., genetic vaccines) as described herein. In some embodiments, the therapeutic nucleic acid encodes a peptide or protein capable of eliciting an immunity against one or more conditions or diseases of interest. In some embodiments, the condition of interest is associated with or caused by a pathogen infection, such as VZV. In some embodiments, the therapeutic nucleic acid sequence (e.g., mRNA) encodes a pathogenic protein characteristic of a pathogen or an immunogenic fragment (e.g., epitope) or derivative thereof. The vaccine, upon administration to a vaccinated subject, allows expression of the encoded pathogenic protein (or immunogenic fragment or derivative thereof), thereby eliciting immunity against the pathogen in the subject.

In particular embodiments, provided herein are therapeutic compositions (e.g., vaccine compositions) for controlling, preventing, and treating diseases or conditions caused by VZV or by infection with VZV.

Without being bound by theory, it is contemplated that VZV, varicella zoster virus, also known as human herpesvirus type 3, is a double stranded DNA virus that belongs to the alpha herpesvirus. VZV has only one serotype. VZV has a genome comprising 71 genes and encoding 67 proteins, including 6 glycoproteins, now designated gE, gB, gH, gI, gC and gL. Glycoproteins gE, gB and gH are very abundant in infected cells and are also present in the envelope of the virion. Antibodies induced by the three major glycoproteins can neutralize the virus. Specific humoral and cellular immunity, and cytokines such as interferons, play a major role in limiting the spread and recovery of VZV, with specific cellular immunity being particularly important.

Thus, in some embodiments, provided herein are therapeutic nucleic acids encoding viral peptides or proteins derived from VZV. In some embodiments, the nucleic acid encodes a viral peptide or protein derived from VZV, wherein the viral peptide or protein is one or more selected from the group consisting of (a) gE protein, (b) gB protein, (c) gH protein, (d) gL protein, (e) gC protein, (f) gL protein, (g) an immunogenic fragment of any one of (a) to (f), and (h) a functional derivative of any one of (a) to (g).

Thus, in some embodiments, the therapeutic nucleic acids of the present disclosure encode a VZV gE protein, or an immunogenic fragment of a gE protein, or a functional derivative of a gE protein or immunogenic fragment thereof. Table 1 shows, inter alia, exemplary VZV natural antigen sequences.

Table 1 exemplary sequences of VZV antigens.

Note that the italicized sequence (in the case of full length proteins) is a signal peptide.

In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mature gE protein of VZV, wherein the mature gE protein has the amino acid sequence of SEQ ID NO: 1. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mature gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO: 2. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mature gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID NO. 2. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a full length gE protein of VZV, wherein the full length gE protein has the amino acid sequence of SEQ ID No. 55. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a full length gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO: 56. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a full length gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID NO. 56. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

In certain embodiments, the therapeutic nucleic acids of the present disclosure encode an immunogenic fragment of the gE protein of VZV. In certain embodiments, the therapeutic nucleic acids of the present disclosure encode functional derivatives of the gE protein of VZV. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes an immunogenic fragment of a gE protein of VZV, wherein the immunogenic fragment of the gE protein comprises a truncation of at least 1 amino acid residue and up to 49 amino acid residues from the C-terminus compared to the mature gE protein. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes an immunogenic fragment of a gE protein of VZV, wherein the immunogenic fragment of the gE protein comprises a truncation of 49、48、47、46、45、44、43、42、41、40、39、38、37、36、35、34、33、32、31、30、29、28、27、26、25、24、23、22、21、20、19、18、17、16、15、14、13、12、11、10、9、8、7、6、5、4、3、2 or 1 amino acid residue from the C-terminus compared to the mature gE protein. In particular embodiments, the therapeutic nucleic acids of the present disclosure encode an immunogenic fragment of a gE protein of VZV, wherein the immunogenic fragment of the gE protein comprises a truncation of from C-terminal 11-18 (e.g., 11, 12, 13, 14, 15, 16, 17, or 18) or 34-44 (e.g., 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44) amino acid residues as compared to the mature gE protein. In particular embodiments, the therapeutic nucleic acids of the present disclosure encode an immunogenic fragment of a gE protein of VZV, wherein the immunogenic fragment of the gE protein comprises a truncation of from C-terminal 12-16 (e.g., 12, 13, 14, 15, or 16) or 35-39 (e.g., 35, 36, 37, 38, or 39) amino acid residues as compared to the mature gE protein. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes an immunogenic fragment of a gE protein of VZV, wherein the immunogenic fragment of the gE protein comprises a truncation of 14 or 37 amino acid residues from the C-terminus compared to the mature gE protein. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

In certain embodiments, the therapeutic nucleic acids of the present disclosure encode mutants of the gE protein of VZV. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution Y569A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution Y582G. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises substitutions Y569A and Y582G. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution S593A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution S595A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution T596A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises the substitution T598A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises substitutions S593A, S595A, T596A and T598A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant of the gE protein of VZV, wherein the mutant comprises substitutions Y569A, Y582G, S593A, S595A, T596A and T598A. In such embodiments, the amino acid positions are numbered based on the full length gE protein. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

In certain embodiments, the therapeutic nucleic acids of the present disclosure encode mutant fragments of the gE protein of VZV. In certain embodiments, the therapeutic nucleic acids of the present disclosure encode a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 49, 48, 47, 46, 45, 44, 43, or 42 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution Y569A. In certain embodiments, the therapeutic nucleic acids of the present disclosure encode a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution Y582G, with or without the substitution Y569A. In such embodiments, the mutant fragment comprises the substitutions Y569A and Y582G. In certain embodiments, the therapeutic nucleic acids of the present disclosure encode a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 30 or 29 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution S593A, with or without the substitution Y569A and/or Y582G. In such embodiments, the mutant fragment comprises the substitutions Y569A, Y582G and S593A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 28 amino acid residues from the C-terminus compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution S595A, with or without the substitution Y569A and/or Y582G and/or S593A. In such embodiments, the mutant fragment comprises the substitutions Y569A, Y582G, S a and S595A. In certain embodiments, the therapeutic nucleic acids of the present disclosure encode a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 27 or 26 amino acid residues from the C-terminus as compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution T596A, with or without the substitution Y569A and/or Y582G and/or S593A and/or S595A. In such embodiments, the mutant fragment comprises the substitutions Y569A, Y582G, S A, S595A and T596A. In particular embodiments, the therapeutic nucleic acids of the present disclosure encode a mutant fragment of the gE protein of VZV, wherein the mutant fragment comprises a truncation of 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues or 1 amino acid residue from the C-terminus compared to the mature gE protein. In such embodiments, the mutant fragment optionally comprises the substitution T598A, with or without the substitution Y569A and/or Y582G and/or S593A and/or S595A and/or T596A. In such embodiments, the mutant fragment comprises the substitutions Y569A, Y582G, S A, S595A, T596A and T598A. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment has the amino acid sequence of SEQ ID NO: 3. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID No. 4 or 5. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID No. 4 or 5. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment has the amino acid sequence of SEQ ID No. 6. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID No. 7. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID No. 7. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment has the amino acid sequence of SEQ ID No. 8. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO: 9. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID No. 9. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment has the amino acid sequence of SEQ ID No. 10. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO: 11. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID NO: 11. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the mutant fragment has the amino acid sequence of SEQ ID NO: 12. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO: 13. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a mutant fragment of the gE protein of VZV, wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID No. 13. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

Without being bound by theory, it is contemplated that in some embodiments, the therapeutic nucleic acids of the present disclosure encode a fusion protein comprising a VZV gE protein or fragment thereof fused to a trimerization peptide such that the fusion protein is capable of forming a trimeric complex comprising three copies of the gE protein or fragment thereof. In some embodiments, the gE protein or fragment thereof is fused to the trimerized peptide via a peptide linker.

Table 2 shows the sequences of exemplary trimeric and linker peptides, as well as fusion proteins, that can be used in connection with the present disclosure.

Table 2 shows exemplary sequences of linker and trimerized peptides.

In some embodiments, the therapeutic nucleic acid encodes a fusion protein comprising the gE protein of VZV or a functional derivative thereof fused to a trimerizing peptide. In some embodiments, the fusion between the gE protein and the trimerized peptide is via a peptide linker. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO. 14. In some embodiments, the trimerized peptide comprises the amino acid sequence of SEQ ID NO. 16.

In a particular embodiment, the therapeutic nucleic acid encodes a fusion protein comprising a gE protein of VZV fused to a trimerized peptide, wherein the nucleic acid comprises a DNA coding sequence. In a particular embodiment, the therapeutic nucleic acid encodes a fusion protein comprising a gE protein of VZV fused to a trimerized peptide, wherein the nucleic acid comprises an RNA sequence transcribed from a DNA coding sequence. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

Without being bound by theory, it is contemplated that fusion proteins comprising a viral peptide or polypeptide fused to an immunoglobulin Fc region may enhance the immunogenicity of the viral peptide or polypeptide. Thus, in some embodiments, the therapeutic nucleic acid molecules of the present disclosure encode fusion proteins comprising a VZV-derived viral peptide or protein fused to the Fc region of an immunoglobulin. In particular embodiments, the viral peptide or protein is one or more selected from the group consisting of (a) a gE protein, (b) a gB protein, (c) a gH protein, (d) a gI protein, (e) a gC protein, (f) a gL protein, (g) an immunogenic fragment of any one of (a) to (f), and (h) a functional derivative of any one of (a) to (g). In a particular embodiment, the immunoglobulin is a human immunoglobulin (Ig). In a particular embodiment, the immunoglobulin is human IgG, igA, igD, igE or IgM. In particular embodiments, the immunoglobulin is human IgG1, igG2, igG3, or IgG4. In some embodiments, the immunoglobulin Fc is fused to the N-terminus of a viral peptide or polypeptide. In other embodiments, the immunoglobulin Fc is fused to the C-terminus of a viral peptide or polypeptide.

Without being bound by theory, it is contemplated that the signal peptide may mediate transport of the polypeptide to which it is fused to a specific location of the cell. Thus, in some embodiments, the therapeutic nucleic acid molecules of the present disclosure encode fusion proteins comprising a viral peptide or protein fused to a signal peptide. In particular embodiments, the viral peptide or protein is one or more selected from the group consisting of (a) a gE protein, (b) a gB protein, (c) a gH protein, (d) a gI protein, (e) a gC protein, (f) a gL protein, (g) an immunogenic fragment of any one of (a) to (f), and (h) a functional derivative of any one of (a) to (g). In some embodiments, the signal peptide is fused to the N-terminus of the viral peptide or polypeptide. In other embodiments, the signal peptide is fused to the C-terminus of the viral peptide or polypeptide. Table 3 shows exemplary sequences of signal peptides that can be used in conjunction with the present disclosure, as well as exemplary VZV antigen sequences comprising the signal peptides.

TABLE 3 exemplary sequences of signal peptides.

In certain embodiments, the signal peptide is encoded by a gene of VZV from which the viral peptide or polypeptide is derived. In certain embodiments, the signal peptide encoded by a gene of VZV is fused to a viral peptide or polypeptide encoded by a different gene of VZV. In other embodiments, the signal peptide encoded by the gene of VZV is fused to a viral peptide or polypeptide encoded by the same gene of VZV. For example, in some embodiments, a signal peptide having the amino acid sequence of MGTVNKPVVGVLMGFGIITGTLRITNPVRA (SEQ ID NO: 18) is fused to a viral peptide or polypeptide encoded by a nucleic acid molecule of the present disclosure. In various embodiments, the viral peptide or protein is one or more selected from the group consisting of (a) an E protein, (B) a B protein, (C) an H protein, (d) an I protein, (E) a C protein, (f) an L protein, (g) an immunogenic fragment of any one of (a) to (f), and (H) a functional derivative of any one of (a) to (g).

In certain embodiments, the therapeutic nucleic acids of the present disclosure encode the gE protein or fragment of VZV without a natural signal peptide. In a particular embodiment, the encoded gE protein or fragment comprises a signal peptide having the amino acid sequence of SEQ ID NO. 23 or 27. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a gE protein or fragment of VZV having a signal peptide, and wherein the therapeutic nucleic acid comprises the DNA coding sequence of SEQ ID NO:24, 25, 26 or 28. In a particular embodiment, the therapeutic nucleic acid of the present disclosure encodes a gE protein or fragment of VZV having a signal peptide, and wherein the therapeutic nucleic acid comprises an RNA sequence transcribed from the DNA coding sequence of SEQ ID NO:24, 25, 26 or 28. In some embodiments, the RNA sequence is transcribed in vitro. In particular embodiments, the nucleic acid molecule is an mRNA molecule.

In other embodiments, the signal peptide is encoded by a foreign gene sequence that is not present in the VZV from which the viral peptide or polypeptide is derived. In some embodiments, the heterologous signal peptide replaces a homologous signal peptide in a fusion protein encoded by a nucleic acid molecule of the present disclosure. In particular embodiments, the signal peptide is encoded by a mammalian gene. In a specific embodiment, the signal peptide is encoded by a human immunoglobulin gene. In a particular embodiment, the signal peptide is encoded by the human IgE gene. For example, in some embodiments, a signal peptide having the amino acid sequence of MDWTWILFLVAAATRVHS (SEQ ID NO: 23) is fused to a viral peptide or polypeptide encoded by a nucleic acid molecule of the present disclosure. In various embodiments, the viral peptide or protein is one or more selected from the group consisting of (a) an E protein, (B) a B protein, (C) an H protein, (d) an I protein, (E) a C protein, (f) an L protein, (g) an immunogenic fragment of any one of (a) to (f), and (H) a functional derivative of any one of (a) to (g).

In some embodiments, provided herein are nucleic acids encoding fragments of mature gE described in section 5.3. In some embodiments, provided herein are nucleic acids encoding the fusion proteins described in section 5.3. In some embodiments, provided herein are nucleic acids encoding mutants of full length VZV gE described in section 5.3. In some embodiments, provided herein are nucleic acids encoding the proteins described in section 5.3. In some embodiments, provided herein is a nucleic acid comprising the nucleotide sequence set forth in SEQ ID NO 7, 9, 11, 13, 4 or 5. In some embodiments, provided herein is a nucleic acid comprising a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to a nucleotide sequence set forth in SEQ ID NOs 7, 9, 11, 13, 4 or 5. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids encoding a protein or fusion protein described in section 5.3, comprising a mutant or fragment of mature gE and a human IgE signal peptide, wherein the nucleotide sequence encoding the IgE signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, provided herein are nucleic acids encoding a protein or fusion protein described in section 5.3, comprising a mutant or fragment of mature gE and a human IgE signal peptide, wherein the nucleotide sequence encoding the IgE signal peptide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids encoding the proteins described in section 5.3, comprising mutants of mature gE and a signal peptide, wherein the nucleotide sequence encoding the signal peptide comprises the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22. In some embodiments, provided herein are nucleic acids encoding a protein described in section 5.3, comprising a mutant of mature gE and a signal peptide, wherein the nucleotide sequence encoding the signal peptide is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids encoding a protein or fusion protein described in section 5.3, comprising a mutant or fragment of mature gE and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises the nucleotide sequence shown in SEQ ID No. 28. In some embodiments, provided herein is a nucleic acid encoding a protein or fusion protein described in section 5.3, comprising a mutant or fragment of mature gE and a human tPA signal peptide, wherein the nucleotide sequence encoding the human tPA signal peptide comprises a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID No. 28. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. in some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids comprising the nucleotide sequences set forth in SEQ ID NOs 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, provided herein are nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising the nucleotide sequences set forth in SEQ ID NOs 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting essentially of, or comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identity to a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, provided herein is a nucleic acid consisting essentially of, or comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity to a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, provided herein is a nucleic acid consisting essentially of, or comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identity to a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, provided herein is a nucleic acid consisting essentially of, or comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. in some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids encoding the amino acid sequences set forth in SEQ ID NO. 6. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 7. In some embodiments, provided herein are nucleic acids encoding the amino acid sequences set forth in SEQ ID NO. 8. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 9. In some embodiments, provided herein are nucleic acids encoding the amino acid sequences set forth in SEQ ID NO. 10. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 11. In some embodiments, provided herein are nucleic acids encoding the amino acid sequences set forth in SEQ ID NO. 12. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 13. In some embodiments, provided herein are nucleic acids encoding the amino acid sequences set forth in SEQ ID NO. 3. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 4 or 5. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. in some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a 3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids encoding the amino acid sequence of SEQ ID NO. 59. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63 or 64. In some embodiments, the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63 or 64. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identical to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identical to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identical to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, the nucleic acid comprises a nucleotide sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids comprising the nucleotide sequences set forth in SEQ ID NOs 51, 60, 61, 62, 63 or 64. In some embodiments, provided herein are nucleic acids consisting of the nucleotide sequences set forth in SEQ ID NOs 51, 60, 61, 62, 63 or 64. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identity to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identity to the nucleotide sequence set forth in SEQ ID NO. 51, 60, 61, 62, 63, or 64. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO 51, 60, 61, 62, 63, or 64. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids comprising the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting of the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identity to the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity to the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% identity to the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO. 63. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. in some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are nucleic acids comprising the nucleotide sequences set forth in SEQ ID NOs 49, 50, 52, 53 or 54. In some embodiments, provided herein are nucleic acids consisting of the nucleotide sequences set forth in SEQ ID NOs 49, 50, 52, 53 or 54. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 80%, at least 81%, at least 82%, at least 83%, or at least 84% identity to the nucleotide sequence set forth in SEQ ID NO 49, 50, 52, 53, or 54. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 85%, at least 86%, at least 87%, at least 88%, or at least 89% identity to the nucleotide sequence set forth in SEQ ID NO 49, 50, 52, 53, or 54. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence of at least 90%, at least 91%, at least 92%, at least 93% with 49, 50, 52, 53, or 5460, 61, 62, 63, or 64. In some embodiments, provided herein are nucleic acids consisting of, consisting essentially of, or comprising a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the nucleotide sequence set forth in SEQ ID NO 49, 50, 52, 53, or 54. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is DNA. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In some embodiments, provided herein are the nucleic acids shown in table 1 or the mRNA of the nucleic acids shown in table 1. In some embodiments, provided herein are the nucleic acids shown in Table 1 other than SEQ ID NOS 2 and 56, or the mRNA of the nucleic acids shown in Table 1 other than SEQ ID NOS 2 and 56. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein coding sequence shown in table 1, or a nucleic acid of a VZV gE mutein coding sequence shown in table 1. In some embodiments, provided herein is a nucleic acid consisting of a VZV gE mutein coding sequence shown in table 1, or an mRNA of a nucleic acid of a VZV gE mutein coding sequence shown in table 1. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein-1 coding sequence set forth in table 1, or a nucleic acid of a VZV gE mutein-1 coding sequence set forth in table 1. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein-2 coding sequence set forth in table 1, or a nucleic acid of a VZV gE mutein-2 coding sequence set forth in table 1. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein-3 coding sequence shown in table 1, or a nucleic acid of a VZV gE mutein-3 coding sequence shown in table 1. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein-4 coding sequence set forth in table 1, or a nucleic acid of a VZV gE mutein-4 coding sequence set forth in table 1. In some embodiments, provided herein are mRNA comprising a nucleic acid of a VZV gE mutein-5 coding sequence set forth in table 1, or a nucleic acid of a VZV gE mutein-5 coding sequence set forth in table 1. In some embodiments, the nucleic acid comprises one or more functional nucleotide analogs (e.g., one or more functional nucleotide analogs described in chapter 5.4.6, 5.4.7, and/or 5.4.8). In some embodiments, the nucleic acid is an mRNA and the nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine. In some embodiments, the nucleic acid comprises one or more modified internucleoside linkages (e.g., one or more modified internucleoside linkages described in section 5.4.9). In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5' -cap structure as described, for example, in chapter 5.4.2. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the nucleic acid further comprises a 5 'untranslated region (5' -UTR) as described, for example, in chapter 5.4.3 and a3 'untranslated region (3' -UTR) as described, for example, in chapter 5.4.3. In some embodiments, the 5' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOs 29-38. In some embodiments, the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the nucleic acid further comprises a 5'-UTR and a 3' -UTR, wherein the 5'-UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 29-38 and the 3' -UTR comprises the nucleotide sequence set forth in any one of SEQ ID NOS: 39-46. In some embodiments, the 3' -UTR further comprises a poly-a tail or polyadenylation signal as described, for example, in chapter 5.4.4.

In particular embodiments, the coding nucleotide sequences of the nucleic acids described herein (e.g., naturally occurring nucleic acids) have been codon optimized for expression in cells of a subject, optionally, wherein the subject is a non-human mammal or human.

In particular embodiments, a nucleic acid described herein (e.g., a non-naturally occurring nucleic acid) is an mRNA, and wherein thymine (e.g., in a corresponding DNA sequence) is replaced by uracil or a functional analog thereof in the nucleic acid.

In a specific embodiment, provided herein are the nucleic acids described in section 6 below. In a specific embodiment, provided herein is a codon optimized nucleic acid described in article section 6 below. In particular embodiments, provided herein are non-naturally occurring nucleic acids described in section 6 below. In some embodiments, provided herein are nucleic acids encoding mutants of VZV gE other than the control described in section 6 below.

5.4.25' -Cap structure

Without being bound by theory, it is expected that the 5' -cap structure of the polynucleotide participates in nuclear export and increases polynucleotide stability, and binds to mRNA Cap Binding Protein (CBP), which is responsible for polynucleotide stability in cells, and induces translational capacity by associating CBP with poly-a binding protein to form mature circular mRNA species. The 5 '-cap structure further facilitates removal of the 5' -proximal intron during mRNA splicing. Thus, in some embodiments, the nucleic acid molecules of the present disclosure comprise a 5' -cap structure.

The nucleic acid molecule may be capped at the 5 'end by a cellular endogenous transcription machinery, thereby creating a 5' -ppp-5 '-triphosphate linkage between the terminal guanosine cap residue of the polynucleotide and the 5' end transcribed sense nucleotide. This 5' -guanylate cap may then be methylated to produce an N7-methyl-guanylate residue. The ribose of the nucleotide transcribed at the 5 'end and/or before the end (ANTETERMINAL) of the polynucleotide may also optionally be 2' -O-methylated. 5' -uncapping via hydrolysis and cleavage of guanylate cap structures can target nucleic acid molecules, e.g., mRNA molecules, for degradation.

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more alterations to the native 5' -cap structure produced by endogenous processes. Without being bound by theory, modification of the 5' -cap may increase the stability of the polynucleotide, increase the half-life of the polynucleotide, and may increase the translational efficiency of the polynucleotide.

Exemplary alterations to the native 5' -cap structure include the creation of a non-hydrolyzable cap structure to prevent uncapping and thereby increase the half-life of the polynucleotide. In some embodiments, because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphodiester linkage, in some embodiments, modified nucleotides may be used during the capping reaction. For example, in some embodiments, vaccinia virus capping Enzyme (VACCINIA CAPPING Enzyme) from NEW ENGLAND Biolabs (Ipswich, mass.) can be used for α -thioguanosine nucleotides to generate phosphorothioate linkages in the 5' -ppp-5' cap according to the manufacturer's instructions. Additional modified guanosine nucleotides such as alpha-methylphosphonic acid and selenophosphate nucleotides may be used.

Additional exemplary alterations to the native 5' -cap structure also include modifications at the 2' and/or 3' positions of the capped Guanosine Triphosphate (GTP), substitution of sugar epoxy (oxygen yielding a carbocyclic ring) for a methylene moiety (CH 2), modifications at the triphosphate bridge moiety of the cap structure, or modifications at the nucleobase (G) moiety.

Additional exemplary alterations to the native 5' -cap structure include, but are not limited to, 2' -O-methylation of ribose of the 5' -end and/or 5' -end pre-nucleotides of the polynucleotide at the sugar 2' -hydroxyl (as described above). Multiple different 5 '-cap structures can be used to create a 5' -cap of a polynucleotide (such as an mRNA molecule). Additional exemplary 5' -cap structures that may be used in conjunction with the present disclosure also include those described in international patent publications WO 2008/127688, WO 2008/016473, and WO 2011/015347, the entire contents of each of which are incorporated herein by reference.

In various embodiments, the 5' -end cap can comprise a cap analog. Cap analogs are also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs that differ in chemical structure from the natural (i.e., endogenous, wild-type, or physiological) 5' -cap while retaining cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and/or linked to a polynucleotide.

For example, an anti-reverse cap analogue (ARCA) cap contains two guanosine groups linked by a 5'-5' -triphosphate group, wherein one guanosine contains an N7-methyl group as well as a3 '-O-methyl group (i.e., N7,3' -O-dimethyl-guanosine-5 '-triphosphate-5' -guanosine, i.e., m7G-3'mppp-G, which may equivalently be referred to as 3' O-Me-m7G (5 ') ppp (5') G). The 3'-O atom of the other unchanged guanosine is attached to the 5' -terminal nucleotide of a capped polynucleotide (e.g.mRNA). N7-and 3' -O-methylated guanines provide the terminal portion of a capped polynucleotide (e.g., mRNA). Another exemplary cap structure is a mCAP, which is similar to ARCA, but has a2 '-O-methyl group on guanosine (i.e., N7,2' -O-dimethyl-guanosine-5 '-triphosphate-5' -guanosine, i.e., m7 Gm-ppp-G).

In some embodiments, the cap analog can be a dinucleotide cap analog. As non-limiting examples, dinucleotide cap analogs can be modified with a borane phosphate group (borophosphate) or a selenophosphate group (phophoroselenoate) at different phosphate positions, such as the dinucleotide cap analogs described in U.S. patent No. 8,519,110, the entire contents of which are incorporated herein by reference in their entirety.

In some embodiments, the cap analog can be an N7- (4-chlorophenoxyethyl) -substituted dinucleotide cap analog known in the art and/or described herein. Non-limiting examples of N7- (4-chlorophenoxyethyl) -substituted dinucleotide cap analogs include N7- (4-chlorophenoxyethyl) -G (5 ') ppp (5 ') G and N7- (4-chlorophenoxyethyl) -m3' -OG (5 ') ppp (5 ') G cap analogs (see, e.g., the various cap analogs and methods of synthesizing cap analogs described in Kore et al Bioorganic & MEDICINAL CHEMISTRY 201321:4570-4574; the entire contents of which are incorporated herein by reference). In other embodiments, the cap analogs that can be used in conjunction with the nucleic acid molecules of the present disclosure are 4-chloro/bromophenoxyethyl analogs.

In various embodiments, the cap analog can include a guanosine analog. Useful guanosine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2' -fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

Without being bound by theory, it is expected that although cap analogs allow for simultaneous capping of polynucleotides in an in vitro transcription reaction, up to 20% of transcripts remain uncapped. This and the structural differences in the native 5' -cap structure of the cap analogue and the polynucleotide produced by the endogenous transcriptional machinery of the cell may lead to reduced translational capacity and reduced cell stability.

Thus, in some embodiments, the nucleic acid molecules of the present disclosure may also be capped post-transcriptionally using enzymes in order to produce a more authentic (authentic) 5' -cap structure. As used herein, the phrase "more realistic" refers to a feature that closely reflects or mimics an endogenous or wild-type feature in structure or function. That is, a "more authentic" feature better represents an endogenous, wild-type, natural, or physiological cell function and/or structure, or it outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects, as compared to the synthetic feature or analog of the prior art. Non-limiting examples of more realistic 5' -cap structures that can be used in conjunction with the nucleic acid molecules of the present disclosure are synthetic 5' -cap structures (or compared to wild-type, natural or physiological 5' -cap structures) as known in the art, particularly structures with enhanced binding to cap binding proteins, increased half-life, reduced sensitivity to 5' -endonucleases, and/or reduced 5' -uncapping. For example, in some embodiments, the recombinant vaccinia virus capping enzyme and the recombinant 2 '-O-methyltransferase can create a classical 5' -5 '-triphosphate linkage between a 5' -terminal nucleotide of a polynucleotide and a guanosine cap nucleotide, wherein the guanosine cap contains N7-methylation and the 5 '-terminal nucleotide of the polynucleotide contains a 2' -O-methyl group. This structure is referred to as the cap 1 structure. Such caps result in higher translational capacity, cell stability, and reduced activation of cellular pro-inflammatory cytokines compared to other 5' cap analog structures known in the art, for example. Other exemplary cap structures include 7mG (5 ') ppp (5 ') N, pN2p (cap 0), 7mG (5 ') ppp (5 ') NlmpNp (cap 1), 7mG (5 ') -ppp (5 ') NlmpN mp (cap 2), and m (7) Gpppm (3) (6,6,2 ') Apm (2 ') Cpm (2) (3, 2 ') Up (cap 4). In some embodiments, the cap structure comprises m ⁷ GpppAmpU.

Without being bound by theory, it is contemplated that the nucleic acid molecules of the present disclosure may be capped post-transcriptionally, and since this approach is more efficient, nearly 100% of the nucleic acid molecules may be capped.

In some embodiments, provided herein are non-naturally occurring nucleic acids consisting of, consisting essentially of, or comprising (1) the nucleotide sequence set forth in SEQ ID NO:63, or (2) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising (1) the nucleotide sequence set forth in SEQ ID NO:63, or (2) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU. In some embodiments, provided herein is a nucleic acid consisting of, consisting essentially of, or comprising the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with N1-methyl pseudouridine and the first nucleotide G is replaced with m ⁷ GpppAmpU.

5.4.3 Untranslated regions (UTR)

In some embodiments, the nucleic acid molecules of the present disclosure comprise one or more untranslated regions (UTRs). In some embodiments, the UTR is located upstream of the coding region in the nucleic acid molecule and is referred to as a 5' -UTR. In some embodiments, the UTR is located downstream of the coding region in the nucleic acid molecule and is referred to as a 3' -UTR. The sequence of the UTR may be homologous or heterologous to the sequence of the coding region found in the nucleic acid molecule. Multiple UTRs may be included in a nucleic acid molecule and may have the same or different sequences and/or genetic origins. According to the present disclosure, any portion (including none) of the UTRs in a nucleic acid molecule may be codon optimized, and any portion may independently contain one or more different structural or chemical modifications before and/or after codon optimization.

In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises UTR and coding regions that are homologous with respect to each other. In other embodiments, the nucleic acid molecules (e.g., mRNA) of the present disclosure comprise UTR and coding regions that are heterologous with respect to each other. In some embodiments, to monitor the activity of a UTR sequence, a nucleic acid molecule comprising a coding sequence of a UTR and a detectable probe may be administered in vitro (e.g., a cell or tissue culture) or in vivo (e.g., to a subject), and the effect of the UTR sequence (e.g., modulating expression levels, cellular localization of the encoded product, or half-life of the encoded product) may be measured using methods known in the art.

In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one Translational Enhancer Element (TEE) that functions to increase the amount of polypeptide or protein produced by the nucleic acid molecule. In some embodiments, the TEE is located in the 5' -UTR of the nucleic acid molecule. In other embodiments, the TEE is located at the 3' -UTR of the nucleic acid molecule. In other embodiments, at least two TEEs are located at the 5'-UTR and 3' -UTR, respectively, of a nucleic acid molecule. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure may comprise one or more copies of a TEE sequence or comprise more than one different TEE sequence. In some embodiments, the different TEE sequences present in the nucleic acid molecules of the disclosure may be homologous or heterologous with respect to each other.

Various TEE sequences are known in the art and may be used in connection with the present disclosure. For example, in some embodiments, the TEE may be an Internal Ribosome Entry Site (IRES), HCV-IRES, or IRES element. Chappell et al Proc. Natl. Acad. Sci. USA 101:9590-9594,2004; zhou et al Proc. Natl. Acad. Sci.102:6273-6278,2005. Additional Internal Ribosome Entry Sites (IRES) that can be used in conjunction with the present disclosure include, but are not limited to, IRES described in U.S. patent No. 7,468,275, U.S. patent publication No. 2007/0048776, and U.S. patent publication No. 2011/0123410, and international patent publication nos. WO 2007/025008 and WO 2001/055369, the respective contents of which are incorporated herein by reference in their entirety. In some embodiments, the TEE may be WELLENSIEK et al Genome-wide profiling of human cap-INDEPENDENT TRANSLATION-ENHANCING ELEMENTS, nature Methods, month 8 of 2013, 10 (8): 747-750, complemented with the TEE described in Table 1 and complemented with Table 2, the contents of which are incorporated herein by reference in their entirety.

Additional exemplary TEEs that may be used in conjunction with the present disclosure include, but are not limited to, TEE sequences described in U.S. patent No. 6,310,197, U.S. patent No. 6,849,405, U.S. patent No. 7,456,273, U.S. patent No. 7,183,395, U.S. patent publication No. 2009/0226470, U.S. patent publication No. 2013/0177581, U.S. patent publication No. 2007/0048776, U.S. patent publication No. 2011/0127410, U.S. patent publication No. 2009/0093049, international patent publication No. WO 2009/075886, international patent publication No. WO 2012/009644 and international patent publication No. WO 1999/02455, international patent publication No. WO 2007/025008, international patent publication No. WO 2001/055371, european patent No. 2610341, european patent No. 2610340, the respective contents of which are incorporated herein by reference in their entirety.

In various embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one UTR comprising at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, or more than 60 TEE sequences. In some embodiments, the TEE sequence in the UTR of the nucleic acid molecule is a copy of the same TEE sequence. In other embodiments, at least two TEE sequences in the UTR of the nucleic acid molecule have different TEE sequences. In some embodiments, a plurality of different TEE sequences are arranged in one or more repeating patterns in the UTR region of the nucleic acid molecule. For illustration purposes only, the repeating pattern may be, for example ABABAB, AABBAABBAABB, ABCABCABC, etc., where in these exemplary patterns each capital letter (A, B or C) represents a different TEE sequence. In some embodiments, at least two TEE sequences are contiguous with each other (i.e., without a spacer sequence therebetween) in the UTR of a nucleic acid molecule. In other embodiments, at least two TEE sequences are separated by a spacer sequence. In some embodiments, UTRs may comprise TEE sequence-spacer sequence modules that are repeated at least once, at least twice, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, or more than 9 times in UTRs. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, the 3' -UTR, or both the 5'-UTR and the 3' -UTR of the nucleic acid molecule.

In some embodiments, the UTR of a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one translational inhibiting element that functions to reduce the amount of polypeptide or protein produced by the nucleic acid molecule. In some embodiments, the UTR of the nucleic acid molecule comprises one or more miR sequences or fragments thereof (e.g., miR seed sequences) that are recognized by one or more micrornas. In some embodiments, the UTR of the nucleic acid molecule comprises one or more stem-loop structures that down-regulate the translational activity of the nucleic acid molecule. Other mechanisms for inhibiting translational activity associated with nucleic acid molecules are known in the art. In any of the embodiments described in this paragraph, the UTR can be the 5'-UTR, the 3' -UTR, or both the 5'-UTR and the 3' -UTR of the nucleic acid molecule. Table 4 shows exemplary 5'-UTR and 3' -UTR sequences that may be used in connection with the present disclosure.

Table 4 illustrates an exemplary untranslated region (UTR) sequence.

In a specific embodiment, the nucleic acid molecules of the present disclosure comprise a 5' -UTR selected from any one of SEQ ID NOS: 29-38. In a specific embodiment, the nucleic acid molecules of the present disclosure comprise a 3' -UTR selected from any one of SEQ ID NOS: 39-46. In a specific embodiment, the nucleic acid molecules of the present disclosure comprise a 5'-UTR selected from any one of SEQ ID NOS: 29-38 and a 3' -UTR selected from any one of SEQ ID NOS: 39-46. In any of the embodiments described in this paragraph, the nucleic acid molecule may further comprise a coding region having a sequence as described herein, such as any of the DNA coding sequences in tables 1 to 4 or an equivalent RNA sequence thereof. In particular embodiments, the nucleic acid molecule described in this paragraph may be an in vitro transcribed RNA molecule.

TABLE 5 exemplary DNA constructs

A _n = 120 mer of a in the sequence in table 5. More generally, for example, a _n = 30 to 3000 polymers.

5.4.4 Polyadenylation (Poly-A) region

Long-chain adenosine nucleotides (poly-a regions) are typically added to messenger RNA (mRNA) molecules during natural RNA processing to increase the stability of the molecules. Immediately after transcription, the 3 '-end of the transcript is cleaved to release the 3' -hydroxyl group. Next, a poly-A polymerase adds a series of adenosine nucleotides to the RNA. This process is known as polyadenylation and the addition of a poly-A region between 100 and 250 residues in length. Without being bound by theory, it is contemplated that the poly-a region may confer a number of advantages to the nucleic acid molecules of the present disclosure.

Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a polyadenylation signal. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises one or more polyadenylation (poly-A) regions. In some embodiments, the poly-A region consists entirely of adenine nucleotides or functional analogs thereof. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 3' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5' end. In some embodiments, the nucleic acid molecule comprises at least one poly-A region at its 5 'end and at least one poly-A region at its 3' end.

In accordance with the present disclosure, the poly-A regions may have different lengths in different embodiments. In particular, in some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 30 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 35 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 40 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 45 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 50 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 55 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 60 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 65 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 70 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 75 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 80 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 85 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 90 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 95 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 110 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 120 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 130 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 140 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 150 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 160 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 170 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 180 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 190 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 200 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 225 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 275 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 300 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 350 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 400 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 450 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 600 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 800 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 900 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1000 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1100 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1200 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 1300 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1400 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1500 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 1600 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 1700 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 1800 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 1900 nucleotides in length. in some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2000 nucleotides in length. In some embodiments, the poly-a region of a nucleic acid molecule of the present disclosure is at least 2250 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2500 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 2750 nucleotides in length. In some embodiments, the poly-A region of a nucleic acid molecule of the present disclosure is at least 3000 nucleotides in length.

In some embodiments, the length of the poly-a region in a nucleic acid molecule can be selected based on the total length of the nucleic acid molecule or a portion thereof (such as the length of the coding region or the length of the open reading frame of the nucleic acid molecule, etc.). For example, in some embodiments, the poly-a region comprises about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the total length of the nucleic acid molecule comprising the poly-a region.

Without being bound by theory, it is contemplated that certain RNA binding proteins may bind to the poly-A region located at the 3' end of the mRNA molecule. These poly-A binding proteins (PABP) may regulate mRNA expression, for example, by interacting with translation initiation mechanisms in cells and/or protecting the 3' -poly-A tail from degradation. Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises at least one binding site for a poly-a binding protein (PABP). In other embodiments, the nucleic acid molecule is allowed to form a conjugate or complex with the PABP prior to loading into a delivery vehicle (e.g., a lipid nanoparticle).

In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a poly-A-G quadruplex. G quadruplets are circular arrays of four guanosine nucleotides that can hydrogen bond formed by G-rich sequences in DNA and RNA. In this embodiment, the G-quadruplet is incorporated at the end of the poly-A region. The stability, protein yield and other parameters of the resulting polynucleotide (e.g., mRNA) can be determined, including half-life at various time points. It has been found that the protein yield of the poly-A-G quadruplex structure is equal to at least 75% of the protein yield observed with the poly-A region containing only 120 nucleotides.

In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure can include a poly-a region and can be stabilized by the addition of a 3' -stabilizing region. In some embodiments, the 3' -stabilizing region useful for stabilizing a nucleic acid molecule (e.g., mRNA) includes the poly-a or poly-a-G tetrad structure described in international patent publication No. WO 2013/103659, the disclosure of which is incorporated herein by reference in its entirety.

In other embodiments, the 3' -stabilizing region that can be used in conjunction with the nucleic acid molecules of the present disclosure includes a chain terminating nucleoside, such as, but not limited to, 3' -deoxyadenosine (cordycepin (cordycepin)), 3' -deoxyuridine, 3' -deoxycytosine, 3' -deoxyguanosine, 3' -deoxythymine, 2',3' -dideoxynucleoside, such as 2',3' -dideoxyadenosine, 2',3' -dideoxyuridine, 2',3' -dideoxyguanosine, 2',3' -dideoxythymine, 2' -deoxynucleoside, or O-methyl nucleoside, 3' -deoxynucleoside, 2',3' -dideoxynucleoside, 3' -O-methyl nucleoside, 3' -O-ethyl nucleoside, 3' -arabinoside, and/or other alternative nucleosides known in the art and/or described herein.

5.4.5 Secondary Structure

Without being bound by theory, it is contemplated that the stem-loop structure may guide RNA folding, preserve the structural stability of the nucleic acid molecule (e.g., mRNA), provide recognition sites for RNA binding proteins, and serve as substrates for enzymatic reactions. For example, the incorporation of miR sequences and/or TEE sequences will alter the shape of the stem-loop region, whereby translation can be increased and/or decreased (Kedde et al, APumilio-induced RNA structure switch in p27-3'UTR controls miR-221and miR-222accessibility.Nat Cell Biol.,2010, month 10; 12 (10): 1014-20, the contents of which are incorporated herein by reference in their entirety).

Thus, in some embodiments, a nucleic acid molecule (e.g., mRNA) described herein, or a portion thereof, may be in a stem-loop structure, such as, but not limited to, a histone stem-loop. In some embodiments, the stem-loop structure is formed from a stem-loop sequence of about 25 or about 26 nucleotides in length, such as, but not limited to, the structure described in international patent publication No. WO 2013/103659, the contents of which are incorporated herein by reference in their entirety. Additional examples of stem-loop sequences include those described in international patent publication No. WO 2012/019780 and international patent publication No. WO 2015/02667, the contents of which are incorporated herein by reference. In some embodiments, the stem-loop sequence comprises a TEE as described herein. In some embodiments, the stem-loop sequence comprises a miR sequence as described herein. In particular embodiments, the stem-loop sequence can include a miR-122 seed sequence. In a specific embodiment, the nucleic acid molecule comprises a stem-loop sequence CAAAGGCTCTTTTCAGAGCCACCA (SEQ ID NO: 47). In other embodiments, the nucleic acid molecule comprises a stem-loop sequence CAAAGGCUCUUUUCAGAGCCACCA (SEQ ID NO: 48).

In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located upstream (at the 5' end) of the coding region in the nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 5' -UTR of the nucleic acid molecule. In some embodiments, a nucleic acid molecule (e.g., mRNA) of the present disclosure comprises a stem-loop sequence located downstream (at the 3' end) of the coding region in the nucleic acid molecule. In some embodiments, the stem-loop sequence is located within the 3' -UTR of the nucleic acid molecule. In some cases, the nucleic acid molecule may contain more than one stem-loop sequence. In some embodiments, the nucleic acid molecule comprises at least one stem-loop sequence in the 5'-UTR and at least one stem-loop sequence in the 3' -UTR.

In some embodiments, the nucleic acid molecule comprising a stem-loop structure further comprises a stabilizing region. In some embodiments, the stabilizing region comprises at least one chain terminating nucleoside that acts to slow degradation and thereby increase the half-life of the nucleic acid molecule. Exemplary chain terminating nucleosides that can be used in conjunction with the nucleic acid molecules of the present disclosure include, but are not limited to, 3 '-deoxyadenosine (cordycepin), 3' -deoxyuridine, 3 '-deoxycytosine, 3' -deoxyguanosine, 3 '-deoxythymine, 2',3 '-dideoxynucleosides, such as 2',3 '-dideoxyadenosine, 2',3 '-dideoxyuridine, 2',3 '-dideoxycytidine, 2',3 '-dideoxythymine, 2' -deoxynucleosides, or O-methylnucleosides, 3 '-deoxynucleosides, 2',3 '-dideoxynucleosides, 3' -O-methylnucleosides, 3 '-O-ethylnucleosides, 3' -arabinoside, as well as other alternative nucleosides known in the art and/or described herein. In other embodiments, the stem-loop structure may be stabilized by altering the 3' -region of the polynucleotide, which may prevent and/or inhibit the addition of oligo (U) (international patent publication No. WO 2013/103659, which is incorporated herein by reference in its entirety).

In some embodiments, the nucleic acid molecules of the present disclosure comprise at least one stem-loop sequence and a poly-A region or polyadenylation signal. Non-limiting examples of polynucleotide sequences comprising at least one stem-loop sequence and a poly-a region or polyadenylation signal include the sequences described in international patent publication No. WO 2013/120497, international patent publication No. WO 2013/120629, international patent publication No. WO 2013/120500, international patent publication No. WO 2013/120627, international patent publication No. WO 2013/120498, international patent publication No. WO 2013/120626, international patent publication No. WO 2013/120499, and international patent publication No. WO 2013/120628, the respective contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-a region or polyadenylation signal may encode a pathogen antigen or fragment thereof, such as the polynucleotide sequences described in international patent publication No. WO 2013/120499 and international patent publication No. WO 2013/120628, the respective contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-a region or polyadenylation signal may encode a therapeutic protein, such as the polynucleotide sequences described in international patent publication No. WO 2013/120497 and international patent publication No. WO 2013/120629, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-a region or polyadenylation signal may encode a tumor antigen or fragment thereof, such as the polynucleotide sequences described in international patent publication No. WO 2013/120500 and international patent publication No. WO 2013/120627, the respective contents of which are incorporated herein by reference in their entirety.

In some embodiments, a nucleic acid molecule comprising a stem-loop sequence and a poly-a region or polyadenylation signal may encode a sensitising antigen or an autoimmune autoantigen, such as the polynucleotide sequences described in international patent publication No. WO 2013/120498 and international patent publication No. WO 2013/120626, the contents of each of which are incorporated herein by reference in their entirety.

5.4.6 Functional nucleotide analogues

In some embodiments, the payload nucleic acid molecules described herein contain only classical nucleotides selected from a (adenosine), G (guanosine), C (cytosine), U (uridine), and T (thymidine). Without being bound by theory, it is expected that certain functional nucleotide analogs may confer useful properties to a nucleic acid molecule. In the context of the present disclosure, examples of such useful properties include, but are not limited to, increased stability of the nucleic acid molecule, reduced immunogenicity of the nucleic acid molecule in inducing an innate immune response, increased production of proteins encoded by the nucleic acid molecule, increased intracellular delivery and/or retention of the nucleic acid molecule, and/or reduced cytotoxicity of the nucleic acid molecule, among others.

Thus, in some embodiments, the payload nucleic acid molecule comprises at least one functional nucleotide analog as described herein. In some embodiments, the functional nucleotide analog contains at least one chemical modification to a nucleobase, a sugar group, and/or a phosphate group. Thus, a payload nucleic acid molecule comprising at least one functional nucleotide analogue contains at least one chemical modification directed to nucleobases, sugar groups and/or internucleoside linkages. Exemplary chemical modifications to nucleobases, glycosyls, or internucleoside linkages of nucleic acid molecules are provided herein.

As described herein, nucleotides ranging from 0% to 100% of all nucleotides in a payload nucleic acid molecule can be functional nucleotide analogs as described herein. For example, in various embodiments, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 1% to about 95%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, from about 10% to about 95%, from about 10% to about 100%, from about 20% to about 25%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 95%, from about 20% to about 100%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 50% to about 95%, from about 50% to about 100%, from about 70%, from about 50% to about 80%, from about 95% to about 95%, from about 95% to about 100%, from about 80%, from about 95% to about 100% of the nucleotide in all nucleotides in a nucleic acid molecule. In any of these embodiments, the functional nucleotide analog may be present at any position of the nucleic acid molecule, including the 5 '-terminus, the 3' -terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule may contain different sugar modifications, different nucleobase modifications, and/or different types of internucleoside linkages (e.g., backbone structures).

As described herein, from 0% to 100% of the nucleotides in one type of all nucleotides in a payload nucleic acid molecule (e.g., as all purine-containing nucleotides of one type, or as all pyrimidine-containing nucleotides of one type, or as all A, G, C, T or U of one type) can be functional nucleotide analogs described herein. For example, in various embodiments, from about 1% to about 20%, from about 1% to about 25%, from about 1% to about 50%, from about 1% to about 60%, from about 1% to about 70%, from about 1% to about 80%, from about 1% to about 90%, from about 1% to about 95%, from about 10% to about 20%, from about 10% to about 25%, from about 10% to about 50%, from about 10% to about 60%, from about 10% to about 70%, from about 10% to about 80%, from about 10% to about 90%, from about 10% to about 95%, from about 10% to about 100%, from about 20% to about 25%, from about 20% to about 50%, from about 20% to about 60%, from about 20% to about 70%, from about 20% to about 80%, from about 20% to about 95%, from about 20% to about 100%, from about 50% to about 70%, from about 50% to about 80%, from about 50% to about 90%, from about 50% to about 95%, from about 50% to about 100%, from about 50% to about 70%, from about 80%, from about 95% to about 100%, from about 80% to about 95%, from about 95% to about 100% of the nucleotide in one type of nucleotide in the nucleic acid molecule. In any of these embodiments, the functional nucleotide analog may be present at any position of the nucleic acid molecule, including the 5 '-terminus, the 3' -terminus, and/or one or more internal positions. In some embodiments, a single nucleic acid molecule may contain different sugar modifications, different nucleobase modifications, and/or different types of internucleoside linkages (e.g., backbone structures).

5.4.7 Modification of nucleobases

In some embodiments, the functional nucleotide analog contains a non-classical nucleobase. In some embodiments, classical nucleobases (e.g., adenine, guanine, uracil, thymine, and cytosine) in a nucleotide may be modified or substituted to provide one or more functional analogs of the nucleotide. Exemplary modifications of nucleobases include, but are not limited to, one or more substitutions or modifications including, but not limited to, alkyl, aryl, halo, oxo, hydroxy, alkoxy, and/or thio substitutions, one or more condensed or open rings, oxidation, and/or reduction.

In some embodiments, the non-classical nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracils include pseudouridine (ψ), pyridin-4-ketoribonucleoside, 5-azauracil, 6-azauracil, 2-thio-5-azauracil, 2-thiouracil (s ² U), 4-thio-uracil (s ⁴ U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uracil (ho ⁵ U), 5-aminoallyl-uracil, 5-halouracil (e.g., 5-iodouracil or 5-bromouracil), 3-methyluracil (m ³ U), 5-methoxyuracil (mo ⁵ U), uracil 5-oxyacetic acid (cmo ⁵ U), Uracil 5-oxoacetic acid methyl ester (mcmo ⁵ U), 5-carboxymethyl-uracil (cm ⁵ U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uracil (chm ⁵ U), 5-carboxyhydroxymethyl-uracil methyl ester (mchm ⁵ U), 5-Methoxycarbonylmethyl-uracil (mcm ⁵ U), 5-Methoxycarbonylmethyl-2-thiouracil (mcm ⁵s² U), 5-aminomethyl-2-thiouracil (nm ⁵s² U), 5-methylaminomethyl uracil (mcm ⁵ U), 5-methylaminomethyl-2-thiouracil (mna ⁵s² U), 5-methylaminomethyl-2-selenouracil (mna ⁵se² U), 5-carbamoylmethyluracil (ncm ⁵ U), 5-carboxymethylaminomethyl-uracil (cmnm ⁵ U), 5-carboxymethylaminomethyl-2-thiouracil (cmnm ⁵s² U), 5-propynyl-uracil, 1-propynyl-pseudouracil, 5-taurine methyl-uracil (τm ⁵ U), 1-taurine methyl-pseudouridine, 5-taurine methyl-2-thiouracil (τm ⁵5s² U), 1-taurine methyl-4-thio-pseudouridine, 5-methyl-uracil (m ⁵ U, i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m ¹ ψ), 1-ethyl-pseudouridine (Et ¹ ψ), a, 5-methyl-2-thio-uracil (m ⁵s² U), 1-methyl-4-thio-pseudouridine (m ¹s⁴. Phi.), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m ³. Phi.), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouracil (D), dihydropseudouridine, 5, 6-dihydrouracil, 5-methyl-dihydrouracil (m ⁵ D), 2-thio-dihydrouracil, 2-thio-dihydropseudouridine, 2-methoxy-uracil, 2-methoxy-4-thio-uracil, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine, 3- (3-amino-3-carboxypropyl) uracil (acp ³ U), 1-methyl-3- (3-amino-3-carboxypropyl) pseudouridine (acp ³. Phi.), 5- (isopentenylaminomethyl) uracil (m ⁵ U), 5- (isopentenylaminomethyl) -2-thio-uracil (m ⁵s² U), 5,2 '-O-dimethyl-uridine (m ⁵ Um), 2-thio-2' -O-methyl-uridine (s ² Um), 5-methoxycarbonylmethyl-2 '-O-methyl-uridine (mcm ⁵ Um), 5-carbamoylmethyl-2' -O-methyl-uridine (ncm ⁵ Um), 5-carboxymethylaminomethyl-2 ' -O-methyl-uridine (cmnm ⁵ Um), 3,2' -O-dimethyl-uridine (m ³ Um) and 5- (isopentenylaminomethyl) -2' -O-methyl-uridine (inm ⁵ Um), 1-thio-uracil, deoxythymidine, 5- (2-methoxycarbonylvinyl) -uracil, 5- (carbamoyl hydroxymethyl) -uracil, 5-carbamoylmethyl-2-thio-uracil, 5-carboxymethyl-2-thio-uracil, 5-cyanomethyl-uracil, 5-methoxy-2-thio-uracil and 5- [3- (1-E-propenyl amino) ] uracil.

In some embodiments, the non-classical nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-azacytosine, 6-azacytosine, pseudoisocytosine, 3-methylcytosine (m 3C), N4-acetylcytosine (ac 4C), 5-formylcytosine (f 5C), N4-methyl-cytosine (m 4C), 5-methyl-cytosine (m 5C), 5-halo-cytosine (e.g., 5-iodo-cytosine), 5-hydroxymethyl-cytosine (hm 5C), 1-methyl-pseudoisocytosine, pyrrolo-cytosine, pyrrolo-pseudoisocytosine, 2-thiocytosine (s 2C), 2-thio-5-methylcytosine, 4-thio-pseudoisocytosine, 4-thio-1-methyl-1-deaza-pseudoisocytosine, zepine (zepine), 5-aza-bunyamine, 5-methyl-cytidine, 2-thioisocytosine, 2-thiocytidine, 4-thiocyline (s 2C), 2-thio-5-methyl-5-cytaroline, 2-thiocytidine, 4-methyl-39-5-thiocytidine, 4-methyl-5-thiocytidine, 4-methyl-3-methyl-thiocytidine, 4-methyl-3-methyl-C, 5,2' -O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2 ' -O-methyl-cytidine (ac 4 Cm), N4,2' -O-dimethyl-cytidine (m 4 Cm), 5-formyl-2 ' -O-methyl-cytidine (fSCm), N4,2' -O-trimethyl-cytidine (m 42 Cm), 1-thio-cytosine, 5-hydroxy-cytosine, 5- (3-azidopropyl) -cytosine, and 5- (2-azidoethyl) -cytosine.

In some embodiments, the non-canonical nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having substituted adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyl-adenine (m 1A), 2-methyl-adenine (m 2A), N6-methyl-adenine (m 6A), 2-methylthio-N6-methyl-adenine (ms 2m 6A), N6-isopentenyl-adenine (i 6A), 2-methylthio-N6-isopentenyl-adenine (m 6A), 2-hydroxy-isopentenyl adenine (m 6A), 2- (hydroxy-3-hydroxy-5-adenine (m 6A) N6-glycylcarbamoyl-adenine (g 6A), N6-threonyl carbamoyl-adenine (t 6A), N6-methyl-N6-threonyl carbamoyl-adenine (m 6t 6A), 2-methylsulfanyl-N6-threonyl carbamoyl-adenine (ms 2g 6A), N6-dimethyl-adenine (m 62A), N6-hydroxy-N-valyl carbamoyl-adenine (hn 6A), 2-methylsulfanyl-N6-hydroxy-N-valyl carbamoyl-adenine (ms 2hn 6A), N6-acetyl-adenine (ac 6A), 7-methyl-adenine, 2-methylsulfanyl-adenine, N6,2 '-O-dimethyl-adenine (m 6 Am), N6,2' -O-trimethyl-adenine (m 62A), 1,2 '-O-dimethyl-adenine (m 6A), 1,2' -N6-hydroxy-N-valyl-adenine (ms 2hn 6A), N6-acetyl-adenine (ac 6A), 7-methyl-adenine, 2-methoxy-adenine, N6,2 '-O-dimethyl-adenine (m 6 Am), N6,2' -O-trimethyl-adenine (m 6A), 1, 2-hydroxy-N-adenine, 8-methyl-adenine, N8-amino-N-azaadenine and nona-methyl-adenine.

In some embodiments, the non-canonical nucleobase is a modified guanine. Exemplary nucleobases and nucleosides with modified guanines include inosine (I), 1-methyl-inosine (m 1I), bosyl (wyosine) (imG), methyl bosyl (mimG), 4-demethyl-bosyl (imG-14), isobornyl (imG), huai Dinggan (wybutosine) (yW), peroxy Huai Dinggan (o 2 yW), hydroxy Huai Dinggan (OHyW), hydroxy Huai Dinggan (OHyW) with modification deficiency (undermodified), 7-deaza-guanine, pigtail (queuosine) (Q), epoxy pigtail (oQ), galactosyl-pigtail (galQ), mannosyl-pigtail (manQ), 7-cyano-7-deaza-guanine (preQO), 7-aminomethyl-7-deaza-guanine (preQ 1), gulin (archaeosine) (G+), 7-deaza-8-aza-guanine, 6-thioguanine, 6-thioguanosine (queuosine) (Q), epoxy pigtail (oQ), galactosyl-pigtail (327-deaza-guanosine) (preQO), 7-aminomethyl-7-deaza-guanosine (preQ), 7-amino methyl-7-deaza-guanine (G+), 6-thioguanosine (6-thioguanosine), 6-thioguanosine (6-methyl-7-thioguanosine (3-6-methyl-7-deaza-guanosine (3) N2-methyl-guanine (m 2G), N2-dimethyl-guanine (m 22G), N2, 7-dimethyl-guanine (m 2, 7G), N2, 7-dimethyl-guanine (m 2,2,7G), 8-oxo-guanine, 7-methyl-8-oxo-guanine, 1-methyl-6-thioguanine, N2-dimethyl-6-thioguanine, N2-methyl-2 ' -O-methyl-guanosine (m 2 Gm), N2-dimethyl-2 ' -O-methyl-guanosine (m 22 Gm), 1-methyl-2 ' -O-methyl-guanosine (m 1 Gm), N2, 7-dimethyl-2 ' -O-methyl-guanosine (m 2,7 Gm), 2' -O-methyl-inosine (Im), 1,2' -O-dimethyl-2 ' -O-guanosine (m 2, m) and 1-thioguanosine (Im).

In some embodiments, the non-classical nucleobases of the functional nucleotide analogs can independently be purines, pyrimidines, purine analogs, or pyrimidine analogs. For example, in some embodiments, the non-canonical nucleobase can be a modified adenine, cytosine, guanine, uracil, or hypoxanthine. In other embodiments, non-classical nucleobases may also include naturally occurring and synthetic derivatives of, for example, bases, including pyrazolo [3,4-d ] pyrimidines; 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazapine, 7-deazaguanine, 3-deazaguanine, 7-deazaadenine, 3-deazaadenine, pyrazolo [3,4-d ] pyrimidine, imidazo [1,5-a ]1,3, 5-triazinone, 9-deazapurine, imidazo [4,5-d ] pyrazine, thiazolo [4,5-d ] pyrimidine, pyrazin-2-one, 1,2, 4-triazine, pyridazine, or 1,3, 5-triazine.

Modification of 5.4.8 sugar

In some embodiments, the functional nucleotide analog contains a non-canonical glycosyl. In various embodiments, the non-classical sugar group may be a 5-carbon or 6-carbon sugar (such as pentose, ribose, arabinose, xylose, glucose, galactose, or deoxy derivatives thereof) having one or more substitutions such as halogen, hydroxy, thiol, alkyl, alkoxy, alkenyloxy, alkynyloxy, cycloalkyl, aminoalkoxy, alkoxyalkoxy, hydroxyalkoxy, amino, azido, aryl, aminoalkyl, aminoalkenyl, aminoalkyl, and the like.

In general, RNA molecules contain ribosyl groups that are oxygen-containing 5-membered rings. Exemplary non-limiting alternative nucleotides include oxygen substitution in ribose (e.g., substitution with S, se or alkylene groups, such as methylene or ethylene), addition of double bonds (e.g., substitution of ribose with cyclopentenyl or cyclohexenyl groups), ring contraction of ribose (e.g., a 4-membered ring for forming cyclobutane or oxetane), ring expansion of ribose (e.g., for forming a 6-or 7-membered ring with additional carbon or heteroatoms, such as for anhydrohexitol, altritol (altritol), mannitol, cyclohexenyl, and N-morpholinyl (which also has an phosphoramidate backbone)), polycyclic forms (e.g., tricyclic and "unlocked" forms, such as a diol nucleic acid (GNA) (e.g., R-GNA or S-GNA in which ribose is substituted with a diol unit attached to a phosphodiester linkage), threose nucleic acid (TNA in which ribose is substituted with α -L-threose- (3 '. Fwdarw.2') and Peptide Nucleic Acid (PNA).

In some embodiments, the glycosyl group contains one or more carbons having a stereochemical configuration opposite to the corresponding carbon in ribose. Thus, a nucleic acid molecule may comprise a nucleotide containing, for example, arabinose or L-ribose as sugar. In some embodiments, the nucleic acid molecule comprises at least one nucleoside wherein the sugar is L-ribose, 2 '-O-methyl ribose, 2' -fluoro ribose, arabinose, hexitol, LNA, or PNA.

Modification of 5.4.9 internucleoside linkages

In some embodiments, the payload nucleic acid molecules of the present disclosure may contain one or more modified internucleoside linkages (e.g., phosphate backbones). The backbone phosphate group may be altered by replacing one or more oxygen atoms with different substituents.

In some embodiments, the functional nucleotide analogs can include substitution of an unaltered phosphate moiety with another internucleoside linkage described herein. Examples of alternative phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenos, boranophosphates, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates and phosphotriesters. Both non-linking oxygens of the dithiophosphate are replaced by sulfur. The phosphate linker can also be altered by replacing the linking oxygen with nitrogen (bridged phosphoramidate), sulfur (bridged phosphorothioate) and carbon (bridged methylphosphonate).

Alternative nucleosides and nucleotides can include one or more non-bridging oxygens replaced with borane moieties (BH ₃), thio (thio), methyl, ethyl and/or methoxy groups. As a non-limiting example, two non-bridging oxygens at the same position (e.g., alpha (α), beta (β), or gamma (γ) positions) can be replaced with a thio (thio) and methoxy group. Replacement of one or more oxygen atoms at the phosphate moiety (e.g., alpha-phosphorothioate) position may confer RNA and DNA stability (such as stability against exonucleases and endonucleases) via non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and therefore have a longer half-life in the cellular environment.

Other internucleoside linkages, including internucleoside linkages that do not contain a phosphorus atom, that can be used in accordance with the present disclosure are described herein.

Additional examples of nucleic acid molecules (e.g., mRNA), compositions, formulations, and/or methods related thereto that can be used in connection with the present disclosure also include those described in WO 2002/098443、WO 2003/051401、WO 2008/052770、WO 2009/127230、WO 2006/122828、WO 2008/083949、WO 2010/088927、WO 2010/037539、WO 2004/004743、WO 2005/016376、WO 2006/024518、WO 2007/095976、WO 2008/014979、WO 2008/077592、WO 2009/030481、WO 2009/095226、WO 2011069586、WO 2011026641、WO 2011/144358、WO 2012019780、WO 2012013326、WO 2012/089338、WO 2012/113513、WO 2012/116811、WO 2012/116810、WO 2013/113502、WO 2013/113501、WO 2013/113736、WO 2013/143698、WO 2013/143699、WO 2013/143700、WO 2013/120626、WO 2013120627、WO 2013/120628、WO 2013/120629、WO 2013/174409、WO 2014/127917、WO 2015/024669、WO 2015/024668、WO 2015/024667、WO 2015/024665、WO 2015/024666、WO 2015/024664、WO 2015/101415、WO 2015/101414、WO 2015/024667、WO 2015/062738、WO 2015/101416, the respective contents of which are incorporated herein in their entirety.

Therapeutic nucleic acid molecules as described herein can be isolated or synthesized by using methods known in the art. In some embodiments, the DNA or RNA molecules used in connection with the present disclosure are chemically synthesized. In other embodiments, the DNA or RNA molecules used in connection with the present disclosure are isolated from a natural source.

In some embodiments, mRNA molecules used in connection with the present disclosure are biosynthesized using host cells. In certain embodiments, the mRNA is produced by transcription of the corresponding DNA sequence using a host cell. In some embodiments, the DNA sequence encoding the mRNA sequence is incorporated into an expression vector using methods known in the art, and then the vector is introduced into a host cell (e.g., e.coli). The host cell is then cultured under suitable conditions to produce mRNA transcripts. Other methods of generating mRNA molecules from coding DNA are known in the art. For example, in some embodiments, mRNA transcripts may be produced using a cell-free (in vitro) transcription system comprising enzymes of the transcription machinery of the host cell. An exemplary cell-free transcription reaction system is described in this disclosure.

5.4.10 Vectors and host cells

In one aspect, provided herein are vectors comprising the nucleic acids described herein (e.g., in chapter 5.4 or 6). In particular embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the vector may be a viral or non-viral vector (e.g., phage, transposon, cosmid, bacmid, or miniplasmid). In a specific embodiment, the vector is an IVT (in vitro transcription) plasmid.

In another aspect, provided herein are host cells (e.g., in vivo or in vitro host cells) comprising a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6) or a vector described herein. The host cell may be in the form of a single cell, a population of similar or different cells, for example in the form of a culture (such as a liquid culture or a culture on a solid substrate), an organism or a part thereof. The term "host cell" is intended to include a particular subject cell transformed, transduced or transfected with a nucleic acid described herein or a vector described herein as well as progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transformed, transduced or transfected with the nucleic acid or vector due to mutations or environmental effects that may occur during passage or integration of the nucleic acid into the cell genome. The term "host cell" may be any type of cell, for example a primary cell, a cell in culture or a cell from a cell line. Any cell that allows the nucleic acid or vector to replicate and/or produce the protein encoded by the nucleic acid or vector and that can be maintained in culture is part of the present disclosure. In some embodiments, the host cell is a cultured cell. In some embodiments, the host cell is an in vitro or ex vivo cell. In particular embodiments, the host cell is suitable for expressing a recombinant gene or protein. The present disclosure is not intended to be limited to any particular cell type. Indeed, it is contemplated that any suitable cell will be used herein. In some embodiments, the cell or host cell is isolated.

The host cell of the present disclosure may be, for example, a bacterial, yeast, insect or mammalian cell or a human cell. Examples of insect cells are High Five, sf9, se301, seIZD2109, seUCR1, sf900+, sf21, BTI-TN-5B1-4, MG-1, tn368, hzAm1, BM-N, ha2302, hz2E5 or Ao38. Examples of mammalian cells are HEK293, heLa, CHO, NS, SP2/0, PER.C6, vero, RD, BHK, HT 1080, A549, cos-7, ARPE-19 or MRC-5 cells. The host cell may be a host cell as described herein. In some embodiments, the host cell is from a mouse. In particular embodiments, the host cell is a non-human mammalian cell. In other embodiments, the host cell is a bacterial, yeast or insect cell. In some embodiments, the host cell is a human cell. In particular embodiments, the human cells are primary cells isolated from a human subject. In some embodiments, the host cell is from a cell line. In some embodiments, the host cell is in vitro or in cell culture (i.e., cultured cells). In other embodiments, the host cell is in vivo. In particular embodiments, the host cell is isolated from tissue.

5.5 Nanoparticle compositions

In one aspect, the nucleic acid molecules described herein are formulated for in vitro and in vivo delivery. In particular, in some embodiments, the nucleic acid molecule is formulated as a lipid-containing composition. In some embodiments, the lipid-containing composition forms a lipid nanoparticle that encapsulates the nucleic acid molecule within a lipid shell. In some embodiments, the lipid shell protects the nucleic acid molecule from degradation. In some embodiments, the lipid nanoparticle also facilitates transport of the encapsulated nucleic acid molecule into an intracellular compartment and/or mechanism to perform a desired therapeutic or prophylactic function. In certain embodiments, the nucleic acid, when present in the lipid nanoparticle, resists degradation by nucleases in aqueous solution. Lipid nanoparticles comprising nucleic acids and methods of making the same are known in the art, such as those disclosed in, for example, U.S. patent publication No. 2004/0142025, U.S. patent publication No. 2007/0042031, PCT publication No. WO 2017/004143, PCT publication No. WO 2015/199952, PCT publication No. WO 2013/016058, and PCT publication No. WO 2013/086373, the complete disclosure of each of which is incorporated herein by reference in its entirety for all purposes.

In some embodiments, the nanoparticle compositions provided herein have a maximum dimension of 1 μm or less (e.g., ,≤1μm、≤900nm、≤800nm、≤700nm、≤600nm、≤500nm、≤400nm、≤300nm、≤200nm、≤175nm、≤150nm、≤125nm、≤100nm、≤75nm、≤50nm or less), such as when measured by Dynamic Light Scattering (DLS), transmission electron microscopy, scanning electron microscopy, or another method. In one embodiment, the lipid nanoparticle provided herein has at least one dimension in the range of about 40nm to about 200 nm. In one embodiment, the at least one dimension is in the range of about 40nm to about 100 nm.

Nanoparticle compositions that can be used in connection with the present disclosure include, for example, lipid Nanoparticles (LNP), nanolipoprotein particles, liposomes, lipid vesicles, and lipid complexes (lipoplex). In some embodiments, the nanoparticle composition is a vesicle comprising one or more lipid bilayers. In some embodiments, the nanoparticle composition comprises two or more concentric bilayers separated by an aqueous compartment. The lipid bilayers may be functionalized and/or crosslinked to each other. The lipid bilayer may include one or more ligands, proteins, or channels.

In some embodiments, the nanoparticle composition comprises one or more lipid components described in section 6 below. In some embodiments, the nanoparticle composition comprises one or more lipid components described in international patent application publication No. WO 2021// 204175. In some embodiments, the nanoparticle composition comprises one or more lipid components described in international patent application publication No. WO 2022/152109. In some embodiments, the nanoparticle composition comprises one or more lipid components described in International patent application publication No. WO 2022/247755. In some embodiments, the nanoparticle composition is a nanoparticle composition as described in article section 6 below. In some embodiments, the nanoparticle composition is produced as described in section 6. In some embodiments, lipids (e.g., compounds according to one of lipid series 01, 02, 03, and 04, such as, for example, compounds according to one of formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-formulae thereof)) are produced as described in section 6.

In some embodiments, the nanoparticle composition comprises a lipid component comprising at least one lipid, such as a compound according to one of the lipid families 01, 02, 03, and 04 as described herein, e.g., a compound according to one of the formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-formulae thereof). For example, in some embodiments, the nanoparticle composition can comprise a lipid component comprising one of the compounds provided herein. The nanoparticle composition may also include one or more other lipid or non-lipid components as described below.

5.5.1 Cationic lipid

Cationic lipids include the following lipid series 01-04 (and its subformulae).

Lipid series 01

In one embodiment, provided herein are compounds of formula (01-I):

Or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof, wherein:

Each of G ¹ and G ² is independently a bond, C ₂-C₁₂ alkylene, or C ₂-C₁₂ alkenylene, wherein one or more of the alkylene or alkenylene groups-CH ₂ -is optionally replaced by-O-;

l ¹ is -OC(＝O)R¹、-C(＝O)OR¹、-OC(＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^c、-NR^aC(＝O)OR¹、-SC(＝S)R¹、-C(＝S)SR¹、-C(＝S)R¹、-CH(OH)R¹、-P(＝O)(OR^b)(OR^c)、-(C₆-C₁₀ arylene) -R ¹, - (6 to 10 membered heteroarylene) -R ¹ or R ¹;

L ² is -OC(＝O)R²、-C(＝O)OR²、-OC(＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^dC(＝O)NR^eR^f、-OC(＝O)NR^eR^f、-NR^dC(＝O)OR²、-SC(＝S)R²、-C(＝S)SR²、-C(＝S)R²、-CH(OH)R²、-P(＝O)(OR^e)(OR^f)、-(C₆-C₁₀ arylene) -R ², - (6 to 10 membered heteroarylene) -R ² or R ²;

r ¹ and R ² are each independently C ₆-C₃₂ alkyl or C ₆-C₃₂ alkenyl;

R ^a、R^b、R^d and R ^e are each independently H, C ₁-C₂₄ alkyl or C ₂-C₂₄ alkenyl;

R ^c and R ^f are each independently C ₁-C₃₂ alkyl or C ₂-C₃₂ alkenyl;

G ³ is C ₂-C₂₄ alkylene, C ₂-C₂₄ alkenylene, C ₃-C₈ cycloalkylene, or C ₃-C₈ cycloalkenyl;

R ³ is-N (R ⁴)R⁵;

R ⁴ is C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, 4 to 8 membered heterocyclyl or C ₆-C₁₀ aryl, or a portion of R ⁴、G³ or G ³ together with the nitrogen to which they are attached form a cyclic moiety;

R ⁵ is C ₁-C₁₂ alkyl or C ₃-C₈ cycloalkyl, or R ⁴、R⁵ together with the nitrogen to which they are attached form a cyclic moiety;

x is 0, 1 or 2, and

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocyclyl, aryl, alkylene, alkenylene, cycloalkylene, cycloalkenylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, provided herein are compounds of formula (01-II):

is a single bond or a double bond;

G ⁴ is a bond, C ₁-C₂₃ alkylene, C ₂-C₂₃ alkenylene, C ₃-C₈ cycloalkylene, or C ₃-C₈ cycloalkenyl;

R ³ is-N (R ⁴)R⁵;

R ⁴ is C ₁-C₁₂ alkyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, 4-to 8-membered heterocyclyl or C ₆-C₁₀ aryl, or R ⁴、G³ or a portion of G ³ together with the nitrogen to which they are attached form a cyclic moiety;

x is 0, 1 or 2, and

In one embodiment, the compound is a compound of table 01-1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 01-1.

Lipid series 02

In one embodiment, provided herein are compounds of formula (02-I):

Each of G ¹ and G ² is independently C ₂-C₁₂ alkylene or C ₂-C₁₂ alkenylene, wherein one or more of G ¹ and G ² -CH ₂ -is optionally replaced by-O-, -C (=o) O-or-OC (=o) -;

each L ¹ is independently -OC(＝O)R¹、-C(＝O)OR¹、-OC(＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^c、-NR^aC(＝O)OR¹、-SC(＝S)R¹、-C(＝S)SR¹、-C(＝S)R¹、-CH(OH)R¹、-P(＝O)(OR^b)(OR^c)、-NR^aP(＝O)(OR^b)(OR^c);

Each L ² is independently -OC(＝O)R²、-C(＝O)OR²、-OC(＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^dC(＝O)NR^eR^f、-OC(＝O)NR^eR^f、-NR^dC(＝O)OR²、-SC(＝S)R²、-C(＝S)SR²、-C(＝S)R²、-CH(OH)R²、-P(＝O)(OR^e)(OR^f)、-NR^dP(＝O)(OR^e)(OR^f);

R ¹ and R ² are each independently C ₆-C₂₄ alkyl or C ₆-C₂₄ alkenyl;

r ^c and R ^f are each independently C ₁-C₂₄ alkyl or C ₂-C₂₄ alkenyl;

G ³ is C ₂-C₁₂ alkylene or C ₂-C₁₂ alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced by C ₃-C₈ cycloalkylene or C ₃-C₈ cycloalkenylene;

R ³ is-N (R ⁴)R⁵、-OR⁶ or-SR ⁶;

R ⁴ is C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, C ₆-C₁₀ aryl, or 4 to 8 membered heterocycloalkyl;

R ⁵ is H, C ₁-C₁₂ alkyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, C ₆-C₁₀ aryl, or 4 to 8 membered heterocycloalkyl;

R ⁶ is hydrogen, C ₁-C₁₂ alkyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, or C ₆-C₁₀ aryl;

x is 0, 1 or 2, and

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, cycloalkylene, and cycloalkenylene is independently optionally substituted.

In one embodiment, the compound is a compound of table 02-1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 02-1.

Lipid series 03

In one embodiment, provided herein are compounds of formula (03-I):

Each of G ¹ and G ² is independently a bond, C ₂-C₁₂ alkylene, or C ₂-C₁₂ alkenylene, wherein one or more of G ¹ and G ² -CH ₂ -is optionally replaced by-O-;

each L ¹ is independently -OC(＝O)R¹、-C(＝O)OR¹、-OC(＝O)OR¹、-C(＝O)R¹、-OR¹、-S(O)_xR¹、-S-SR¹、-C(＝O)SR¹、-SC(＝O)R¹、-NR^aC(＝O)R¹、-C(＝O)NR^bR^c、-NR^aC(＝O)NR^bR^c、-OC(＝O)NR^bR^c、-NR^aC(＝O)OR¹、-SC(＝S)R¹、-C(＝S)SR¹、-C(＝S)R¹、-CH(OH)R¹、-P(＝O)(OR^b)(OR^c)、-NR^aP(＝O)(OR^b)(OR^c)、-(C₆-C₁₀ arylene) -R ¹, - (6-to 10-membered heteroarylene) -R ¹, - (4-to 8-membered heterocyclylene) -R ¹ or R ¹;

Each L ² is independently -OC(＝O)R²、-C(＝O)OR²、-OC(＝O)OR²、-C(＝O)R²、-OR²、-S(O)_xR²、-S-SR²、-C(＝O)SR²、-SC(＝O)R²、-NR^dC(＝O)R²、-C(＝O)NR^eR^f、-NR^dC(＝O)NR^eR^f、-OC(＝O)NR^eR^f、-NR^dC(＝O)OR²、-SC(＝S)R²、-C(＝S)SR²、-C(＝S)R²、-CH(OH)R²、-P(＝O)(OR^e)(OR^f)、-NR^dP(＝O)(OR^e)(OR^f)、-(C₆-C₁₀ arylene) -R ², - (6-to 10-membered heteroarylene) -R ², - (4-to 8-membered heterocyclylene) -R ² or R ²;

G ³ is C ₂-C₁₂ alkylene or C ₂-C₁₂ alkenylene, wherein part or all of the alkylene or alkenylene is optionally replaced with C ₃-C₈ cycloalkylene, C ₃-C₈ cycloalkenylene, C ₃-C₈ cycloalkynylene, 4-to 8-membered heterocyclylene, C ₆-C₁₀ arylene, or 5-to 10-membered heteroarylene;

R ³ is hydrogen, C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₂-C₁₂ alkynyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, C ₃-C₈ cycloalkynyl, 4 to 8 membered heterocyclyl, C ₆-C₁₀ aryl or 5 to 10 membered heteroaryl, or a part of R ³、G¹ or G ¹ together with the nitrogen to which they are attached forms a cyclic moiety, or a part of R ³、G³ or G ³ together with the nitrogen to which they are attached forms a cyclic moiety

R ⁴ is C ₁-C₁₂ alkyl or C ₃-C₈ cycloalkyl;

x is 0, 1 or 2;

n is 1 or 2;

m is 1 or 2, and

Wherein each alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, heterocyclyl, aryl, heteroaryl, alkylene, alkenylene, cycloalkylene, cycloalkenyl, cycloalkynylene, heterocyclylene, arylene, heteroarylene, and cyclic moiety is independently optionally substituted.

In one embodiment, the compound is a compound of table 03-1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 03-1.

Lipid series 04

In one embodiment, provided herein are compounds of formula (04-I):

Each of G ¹ and G ² is independently a bond, C ₂-C₁₂ alkylene, or C ₂-C₁₂ alkenylene;

r ¹ and R ² are each independently C ₅-C₃₂ alkyl or C ₅-C₃₂ alkenyl;

R ⁰ is C ₁-C₁₂ alkyl, C ₂-C₁₂ alkenyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, C ₆-C₁₀ aryl, or 4 to 8 membered heterocycloalkyl;

G ³ is C ₂-C₁₂ alkylene or C ₂-C₁₂ alkenylene;

R ⁵ is C ₁-C₁₂ alkyl, C ₃-C₈ cycloalkyl, C ₃-C₈ cycloalkenyl, C ₆-C₁₀ aryl, or 4 to 8 membered heterocycloalkyl;

x is 0, 1 or 2;

s is 0 or 1, and

Wherein each alkyl, alkenyl, cycloalkyl, cycloalkenyl, heterocycloalkyl, aryl, alkylene, alkenylene, arylene, and heteroarylene is independently optionally substituted.

In one embodiment, the compound is a compound of table 04-1, or a pharmaceutically acceptable salt, prodrug, or stereoisomer thereof.

Table 04-1.

It is to be understood that any embodiment of a compound provided herein as set forth above, and any particular substituent and/or variable of a compound provided herein as set forth above, may be independently combined with other embodiments and/or substituents and/or variables of a compound to form embodiments not specifically set forth above. Furthermore, where a list of substituents and/or variables is listed for any particular group or variable, it is to be understood that each individual substituent and/or variable may be deleted from a particular embodiment and/or claim and that the remaining list of substituents and/or variables is to be considered within the scope of embodiments provided herein.

It is to be understood that in this specification, combinations of the various substituents and/or variables depicted are permissible only if such contributions result in stable compounds.

5.5.2 Other ionizable lipids

As described herein, in some embodiments, nanoparticle compositions provided herein comprise one or more charged or ionizable lipids in addition to compounds according to series 01, 02, 03, and 04, e.g., lipids according to formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-formulas thereof). Without being bound by theory, it is expected that certain charged or zwitterionic lipid components of the nanoparticle composition are similar to the lipid components in the cell membrane, thereby improving cellular uptake of the nanoparticles. Exemplary charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention include, but are not limited to, 3- (didodecylamino) -N1, 4-tris (dodecyl) -1-piperazineethylamine (KL 10), N1- [2- (didodecylamino) -ethyl ] -N1, N4-tris (dodecyl) -1, 4-piperazineethylamine (KL 22), 14, 25-ditridecyl-15,18,21,24-tetraaza-trioctadecane (KL 25), 1, 2-ditolyloxy-N, N-dimethylaminopropane (DLinDMA), 2-ditolyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), Triseventeen-carbon-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butyrate (DLin-MC 3-DMA), 2-diimine-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), 1, 2-dioleyloxy-N, N-dimethylaminopropane (DODMA), 2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA), (2R) -2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9Z, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA (2R)), (2S) -2- ({ 8- [ (3β) -cholest-5-en-3-yloxy ] octyl } oxy) -N, N-dimethyl-3- [ (9Z-, 12Z) -octadeca-9, 12-dien-1-yloxy ] propan-1-amine (octyl-CLinDMA (2S)), (12Z, 15Z) -N, N-dimethyl-2-nonyldi-undec-12, 15-dien-1-amine, N, N-dimethyl-1- { (1S, 2R) -2-octylcyclopropyl } heptadec-8-amine. Additional exemplary charged or ionizable lipids that may form part of the nanoparticle compositions of the present invention include lipids described in Sabnis et al "ANovel Amino Lipid Series formRNA Delivery:Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates",Molecular Therapy, volume 26, phase 6, 2018 (e.g., lipid 5), the entire contents of which are incorporated herein by reference.

In some embodiments, suitable cationic lipids include N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTMA), N- [1- (2, 3-dioleyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTAP), 1, 2-dioleoyl-sn-glycerol-3-ethylphosphocholine (DOEPC), 1, 2-dilauryl-sn-glycerol-3-ethylphosphocholine (DLEPC), 1, 2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1, 2-dimyristoyl-sn-glycerol-3-ethylphosphocholine (14:1), N1- [2- ((1S) -1- [ (3-aminopropyl) amino ] -4- [ bis (3-amino-propyl) amino ] butylformamido) ethyl ] -3, 4-dioleyloxy ] -benzamide (MVL 5), dioctadecylamino-glycyl-sn-glycerol-3-ethylphosphocholine (DMEPC), 1, 2-dimyristoyl-glycerol-3-ethylphosphocholine (14:1). N' -dimethylaminoethyl carbamoyl ] cholesterol (DC-Chol); dioctadecyl Dimethyl Ammonium Bromide (DDAB); SAINT-2, N-methyl-4- (dioleyl) methylpyridinium, 1, 2-dimyristoxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), 1, 2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORIE), 1, 2-dioleoyloxypropyl-3-dimethylhydroxyethylammonium chloride (DORI), dialkylated amino acids (DILA ²) (e.g., C18:1-norArg-C16), dioleyldimethylammonium chloride (DODAC), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine (POEPC), 1, 2-dimyristoyl-sn-glycero-3-ethylphosphocholine (MOEPC), dioleoyl (R) -5- (dimethylamino) pentane-1, 2-diyl ester hydrochloride (DODAPen-Cl), dioleoyl (R) -5-guanidinopyran-1, 2-diyl ester hydrochloride (DOPen-G), and N, N-palmitoyl-2-oleoyl-sn-3-ethylphosphocholine (N62-62). Cationic lipids having a head group charged at physiological pH are also suitable, such as primary amines (e.g., DODAG N ', N' -dioctadecyl-N-4, 8-diaza-10-aminodecanoylglycinamide) and guanidinium head groups (e.g., bis-guanidinium-spermidine-cholesterol (BGSC), bis-guanidinium-tren-cholesterol (BGTC), PONA and dioleate (R) -5-guanidinium-1, 2-diyl ester hydrochloride (DOPen-G)). Another suitable cationic lipid is dioleate (R) -5- (dimethylamino) pentane-1, 2-diyl ester hydrochloride (DODAPen-Cl). In certain embodiments, the cationic lipids are in specific enantiomer or racemic forms, and include various salt forms (e.g., chloride or sulfate) of the cationic lipids described above. For example, in some embodiments, the cationic lipid is N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium chloride (DOTAP-Cl) or N- [1- (2, 3-dioleoyloxy) propyl ] -N, N, N-trimethylammonium sulfate (DOTAP-sulfate). In some embodiments, the cationic lipid is an ionizable cationic lipid, such as Dioctadecyl Dimethyl Ammonium Bromide (DDAB), 1, 2-dioleyloxy-3-dimethylaminopropane (DLinDMA), 2-dioleyloxy-4- (2-dimethylaminoethyl) - [1,3] -dioxolane (DLin-KC 2-DMA), thirty-seventeen carbon 4- (dimethylamino) butyrate-6,9,28,31-tetraen-19-yl ester (DLin-MC 3-DMA), 1, 2-dioleoyloxy-3-dimethylaminopropane (DODAP), 1, 2-dioleyloxy-3-dimethylaminopropane (DODMA), and morpholino cholesterol (Mo-CHOL). In certain embodiments, the lipid nanoparticle comprises a combination of two or more cationic lipids (e.g., two or more of the cationic lipids described above).

Additionally, in some embodiments, the charged or ionizable lipid that may form part of the nanoparticle compositions of the present invention is a lipid comprising a cyclic amine group. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO 2015/199952, WO 2016/1762330, and WO 2015/01633, the respective entireties of which are incorporated herein by reference in their entirety. Additionally, in some embodiments, the charged or ionizable lipid that may form part of the nanoparticle compositions of the present invention is a lipid comprising a cyclic amine group. Additional cationic lipids suitable for the formulations and methods disclosed herein include those described in WO 2015/199952, WO 2016/1762330, and WO 2015/01633, the respective entireties of which are incorporated herein by reference in their entirety.

5.5.3 Polymer-bound lipids

In some embodiments, the lipid component of the nanoparticle composition may include one or more polymer-bound lipids, such as pegylated lipids (PEG lipids). Without being bound by theory, it is expected that the polymer-bound lipid component in the nanoparticle composition may improve colloidal stability and/or reduce protein absorption of the nanoparticle. Exemplary polymer-bound lipids that can be used in conjunction with the present disclosure include, but are not limited to, PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof. For example, the PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, ceramide-PEG 2000, or Chol-PEG2000.

In one embodiment, the polymer-bound lipid is a pegylated lipid. For example, some embodiments include polyethylene glycol diacylglycerols (PEG-DAG), such as 1- (monomethoxy-polyethylene glycol) -2, 3-dimyristoylglycerol (PEG-DMG), polyethylene glycol phosphatidylethanolamine (PEG-PE), PEG succinate diacylglycerols (PEG-S-DAG), such as 4-O- (2 ',3' -di (tetradecyloxy) propyl-1-O- (omega-methoxy (polyethoxy) ethyl) succinate (PEG-S-DMG), polyethylene glycol ceramides (PEG-cer), or PEG dialkoxypropyl carbamates, such as omega-methoxy (polyethoxy) ethyl-N- (2, 3-di (tetradecyloxy) propyl) carbamate or 2, 3-di (tetradecyloxy) propyl-N- (omega-methoxy) (polyethoxy) ethyl) carbamate.

In one embodiment, the polymer-bound lipid is present at a concentration in the range of 1.0 mol% to 2.5 mol%. In one embodiment, the polymer-bound lipid is present at a concentration of about 1.7 mole%. In one embodiment, the polymer-bound lipid is present at a concentration of about 1.5 mole%.

In one embodiment, the molar ratio of cationic lipid to polymer-bound lipid is in the range of about 35:1 to about 25:1. In one embodiment, the molar ratio of cationic lipid to polymer-bound lipid is in the range of about 100:1 to about 20:1.

In one embodiment, the pegylated lipid has the formula:

or a pharmaceutically acceptable salt, tautomer, or stereoisomer thereof, wherein:

R ¹² and R ¹³ are each independently a linear or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds, and

W has an average value in the range of 30 to 60. In one embodiment, R12 and R13 are each independently a straight saturated alkyl chain containing from 12 to 16 carbon atoms. In other embodiments, the w average value is in the range of 42 to 55, e.g., the w average value is 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55. In some embodiments, w average is about 49.

In one embodiment, the pegylated lipid has the formula:

Wherein the average w is about 49.

Lipid series 05

In one embodiment, provided herein are compounds of formula (05-I):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:

L is a lipid;

X is a linker;

Each R ³ is independently H or C ₁-C₆ alkyl;

Each Y ¹ is independently a bond, O, S or NR ^a;

Each G ⁴ is independently a bond or C ₁-C₁₂ alkylene, wherein one or more-CH ₂ -are independently optionally replaced by-O-, -S-or-NR ^a -;

Each G ⁵ is independently a bond or C ₁-C₁₂ alkylene, wherein one or more-CH ₂ -are independently optionally replaced by-O-, -S-or-NR ^a -;

Each R ^a is independently H, C ₁-C₁₂ alkyl or C ₂-C₁₂ alkenyl;

One of Z ¹ and Z ² is a positively charged moiety and the other of Z ¹ and Z ² is a negatively charged moiety;

n is an integer from 2 to 100;

T is hydrogen, halogen, alkyl, alkenyl 、-OR"、-SR"、-COOR"、-OCOR"、-NR"R"、-N⁺(R")₃、-P⁺(R")₃、-S-C(＝S)-S-R"、-S-C(＝S)-O-R"、-S-C(＝S)-NR"R"、-S-C(＝S)- aryl, cyano, azido, aryl, heteroaryl, or a targeting group, wherein R' is independently at each occurrence hydrogen or alkyl, and wherein each alkyl, alkenyl, alkylene, aryl, and heteroaryl is independently optionally substituted.

5.5.4 Structural lipids

In some embodiments, the lipid component of the nanoparticle composition may include one or more structural lipids. Without being bound by theory, it is expected that the structural lipids may stabilize the amphiphilic structure of the nanoparticle, such as, but not limited to, the lipid bilayer structure of the nanoparticle. Exemplary structural lipids that can be used in connection with the present disclosure include, but are not limited to, cholesterol, fecal sterols, sitosterols, ergosterols, campesterols, stigmasterols, brassicasterol, lycorine, lycoside, ursolic acid, alpha-tocopherol, and mixtures thereof. In certain embodiments, the structural lipid is cholesterol. In some embodiments, the structural lipids include cholesterol and corticosteroids such as prednisolone (prednisolone), dexamethasone (dexamethasone), prednisone (prednisone), and hydrocortisone (hydrocortisone), or combinations thereof.

In one embodiment, the lipid nanoparticle provided herein comprises a steroid or steroid analogue. In one embodiment, the steroid or steroid analogue is cholesterol. In one embodiment, the steroid is present at a concentration in the range of 39 mole% to 49 mole%, 40 mole% to 46 mole%, 40 mole% to 44 mole%, 40 mole% to 42 mole%, 42 mole% to 44 mole%, or 44 mole% to 46 mole%. In one embodiment, the steroid is present at a concentration of 40 mole%, 41 mole%, 42 mole%, 43 mole%, 44 mole%, 45 mole%, or 46 mole%.

In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the steroid is present at a concentration in the range of 32 mole% to 40 mole% steroid.

5.5.5 Phospholipids

In some embodiments, the lipid component of the nanoparticle composition may include one or more phospholipids, such as one or more (poly) unsaturated lipids. Without being bound by theory, it is contemplated that phospholipids may assemble into one or more lipid bilayer structures. Exemplary phospholipids that may form part of the nanoparticle compositions of the present invention include, but are not limited to, 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC), 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DLPC), 1, 2-dimyristoyl-sn-glycero-phosphorylcholine (DMPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DOPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (DPPC), 1, 2-di (undecoyl) -sn-glycero-phosphorylcholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine (POPC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (18:6-dioleoyl-sn-glycero-3-phosphorylcholine (dapc), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (p PC), 1, 2-dioleoyl-sn-glycero-3-phosphorylcholine (p), 1, 2-di (undecoyl-sn-glycero-3-phosphorylcholine (p), 1, 2-glycero-3-phosphorylcholine (p), and C-hexadecyl-glycero-3-phosphorylcholine (p), 16-phosphorylcholine (p), 1, 2-di (n-undecoyl) -sn-3-phosphorylcholine (p), and p-3-phosphorylcholine (p) 1, 2-di-arachidonyl-sn-glycero-3-phosphorylcholine, 1, 2-di (docosahexaenoic acid) -sn-glycero-3-phosphorylcholine, 1, 2-di-phytanic acid-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 1, 2-distearoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-oleoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-linolenoyl-sn-glycero-3-phosphoethanolamine, 1, 2-di-arachidonyl-sn-glycero-3-phosphoethanolamine, 1, 2-di (docosahexaenoic acid) -sn-glycero-3-phosphoethanolamine, 1, 2-dioleoyl-sn-glycero-3-phospho-racemic- (1-glycero) sodium salt (DOPG), and sphingomyelin. In certain embodiments, the nanoparticle composition comprises DSPC. In certain embodiments, the nanoparticle composition comprises DOPE. In some embodiments, the nanoparticle composition comprises both DSPC and DOPE.

Additional exemplary neutral lipids include, for example, dipalmitoyl phosphatidylglycerol (DPPG), palmitoyl Oleoyl Phosphatidylethanolamine (POPE), and dioleoyl phosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoyl phosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl phosphatidylethanolamine (SOPE), and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans-DOPE). In one embodiment, the neutral lipid is 1, 2-distearoyl-sn-glycero-3-phosphorylcholine (DSPC). In one embodiment, the neutral lipid is selected from DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM.

In one embodiment, the neutral lipid is Phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic Acid (PA), or Phosphatidylglycerol (PG).

In addition, phospholipids that may form part of the nanoparticle compositions of the present invention also include those described in WO 2017/112865, the entire contents of which are incorporated herein by reference in their entirety.

5.5.6 Formulations

According to the present disclosure, nanoparticle compositions described herein can comprise at least one lipid component and one or more additional components, such as therapeutic and/or prophylactic agents (e.g., therapeutic nucleic acids described herein). In particular embodiments, nanoparticle compositions described herein comprise at least one lipid component (e.g., a lipid component described herein (e.g., in section 5.5 or 6)) and a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6). Nanoparticle compositions can be designed for one or more specific applications or targets. The components of the nanoparticle composition can be selected based on the particular application or goal, and/or based on the efficacy, toxicity, cost, ease of use, availability, or other characteristics of one or more of the components. Similarly, the particular formulation of the nanoparticle composition may be selected for a particular application or goal, depending on, for example, the efficacy and toxicity of a particular combination of each ingredient.

The lipid component of the nanoparticle composition may comprise, for example, a lipid, a phospholipid (such as an unsaturated lipid, e.g., DOPE or DSPC), a polymer-bound lipid (such as a PEG lipid), and a structural lipid (such as a steroid) according to one of formulas 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and subformulae thereof) described herein. The ingredients of the lipid component may be provided at specific fractions.

In one embodiment, provided herein are nanoparticle compositions comprising a cationic or ionizable lipid compound provided herein, a therapeutic agent, and one or more excipients. In one embodiment, the cationic or ionizable lipid compound comprises a compound according to one of formulas 01-I, 01-II, 02-I, 02-II, 03-I, 03-II-A, 03-II-B, 03-II-C, 03-II-D, 04-I, 04-III, 04-IV (and sub-formulae thereof), and optionally one or more other ionizable lipid compounds, as described herein. In one embodiment, the one or more excipients are selected from neutral lipids, phospholipids, steroids, and polymer-bound lipids. In one embodiment, the therapeutic agent is encapsulated within or associated with the lipid nanoparticle.

In one embodiment, provided herein is a nanoparticle composition (lipid nanoparticle) comprising:

i) About 40 to about 50 mole% of a cationic lipid;

ii) neutral lipids;

iii) A steroid;

iv) Polymer-bound lipid, and

V) a therapeutic agent.

i) About 20 mole percent to about 65 mole percent of a cationic lipid;

ii) about 5 mole percent to about 40 mole percent of a phospholipid;

iii) About 20 mole percent to about 50 mole percent of a steroid;

iv) Polymer-bound lipid, and

V) a therapeutic agent.

In some embodiments, the nanoparticle composition (lipid nanoparticle) comprises:

i) About 40 mole% to about 55 mole% of a cationic lipid;

ii) about 5 mole% to about 15 mole% of a phospholipid;

iii) About 35 mole% to about 50 mole% of a steroid, and

Iv) from about 2 mole% to about 10 mole% of polymer-bound lipid.

In one embodiment, the nanoparticle composition (lipid nanoparticle) comprises:

i) About 45 mole% to about 55 mole% of a cationic lipid;

ii) about 6 to about 10 mole% of a phospholipid;

iii) About 40 mole% to about 48 mole% of a steroid, and

Iv) from about 1 mole% to about 2.5 mole% of polymer-bound lipid.

As used herein, "mole%" refers to the mole percentage of one component relative to the total moles of all lipid components in the LNP (i.e., the total moles of cationic lipid, neutral lipid, steroid, and polymer-bound lipid).

In one embodiment, the therapeutic agent is a nucleic acid molecule according to the present disclosure. In some embodiments, the therapeutic agent comprises any one or more of the nucleic acid sequences described in present Wen Zhangjie 5.3.3 (therapeutic nucleic acid).

In one embodiment, the lipid nanoparticle comprises 40 to 50 mole%, 41 to 49 mole%, 41 to 48 mole%, 42 to 48 mole%, 43 to 48 mole%, 44 to 48 mole%, 45 to 48 mole%, 46 to 48 mole%, or 47.2 to 47.8 mole% of the cationic lipid. In one embodiment, the lipid nanoparticle comprises about 47.0 mole%, 47.1 mole%, 47.2 mole%, 47.3 mole%, 47.4 mole%, 47.5 mole%, 47.6 mole%, 47.7 mole%, 47.8 mole%, 47.9 mole%, or 48.0 mole% cationic lipid. In one embodiment, the lipid nanoparticle comprises about 50 mole% cationic lipid.

In one embodiment, the neutral lipid is present at a concentration in the range of 5 to 15 mole%, 7 to 13 mole%, or 9 to 11 mole%. In one embodiment, the neutral lipid is present at a concentration of about 9.5 mole%, 10 mole%, or 10.5 mole%. In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 4.1:1.0 to about 4.9:1.0, about 4.5:1.0 to about 4.8:1.0, or about 4.7:1.0 to 4.8:1.0.

In one embodiment, the steroid is present at a concentration in the range of 38 mole% to 49 mole%, 40 mole% to 46 mole%, 40 mole% to 44 mole%, 40 mole% to 42 mole%, 42 mole% to 44 mole%, or 44 mole% to 46 mole%. In one embodiment, the steroid is present at a concentration of 40 mole%, 41 mole%, 42 mole%, 43 mole%, 44 mole%, 45 mole%, or 46 mole%. In one embodiment, the steroid is present at a concentration of 38.0 mole%, 38.1 mole%, 38.2 mole%, 38.3 mole%, 38.4 mole%, 38.5 mole%, 38.6 mole%, 38.7 mole%, 38.8 mole%, or 38.9 mole%. In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2. In one embodiment, the steroid is cholesterol.

In one embodiment, the lipid nanoparticle comprises about 50 mole% cationic lipid and about 38.5 mole% steroid. In some embodiments, the molar ratio of cationic lipid to steroid is 1.3:1.0. In one embodiment, the steroid is cholesterol.

In one embodiment, the ratio of therapeutic agent to lipid in the LNP (i.e., N/P, where N represents the number of moles of cationic lipid and P represents the number of moles of phosphate ester present as part of the nucleic acid backbone) is in the range of 2:1 to 30:1, e.g., in the range of 3:1 to 22:1. In one embodiment, N/P is in the range of 6:1 to 20:1 or 2:1 to 12:1. Exemplary N/P ranges include about 3:1, about 6:1, about 12:1, and about 22:1.

In one embodiment, provided herein is a lipid nanoparticle comprising i) a cationic lipid having an effective pKa greater than 6.0 ii) 5 to 15 mole% of a neutral lipid;

iii) 1 to 15 mole% of an anionic lipid;

iv) 30 to 45 mole% of a steroid;

v) Polymer-bound lipid, and

Vi) a therapeutic agent or a pharmaceutically acceptable salt or prodrug thereof, wherein the mole% is determined based on the total moles of lipids present in the lipid nanoparticle.

In one embodiment, the cationic lipid may be any of a variety of lipid species that carry a net positive charge at a selected pH, e.g., physiological pH. Exemplary cationic lipids are described below. In one embodiment, the cationic lipid has a pKa value greater than 6.25. In one embodiment, the cationic lipid has a pKa greater than 6.5. In one embodiment, the cationic lipid has a pKa value greater than 6.1, greater than 6.2, greater than 6.3, greater than 6.35, greater than 6.4, greater than 6.45, greater than 6.55, greater than 6.6, greater than 6.65, or greater than 6.7.

In one embodiment, the lipid nanoparticle comprises 40 to 45 mole% cationic lipid. In one embodiment, the lipid nanoparticle comprises 45 to 50 mole% cationic lipid.

In one embodiment, the molar ratio of cationic lipid to neutral lipid is in the range of about 2:1 to about 8:1. In one embodiment, the lipid nanoparticle comprises 5 to 10 mole% neutral lipid.

Exemplary anionic lipids include, but are not limited to, phosphatidylglycerol, dioleoyl phosphatidylglycerol (DOPG), dipalmitoyl phosphatidylglycerol (DPPG), or 1, 2-distearoyl-sn-glycerol-3-phosphate- (1' -rac-glycerol) (DSPG).

In one embodiment, the lipid nanoparticle comprises 1 to 10 mole% anionic lipid. In one embodiment, the lipid nanoparticle comprises 1 to 5 mole% anionic lipid. In one embodiment, the lipid nanoparticle comprises 1 to 9 mole%, 1 to 8 mole%, 1 to 7 mole%, or 1 to 6 mole% of an anionic lipid. In one embodiment, the molar ratio of anionic lipid to neutral lipid is in the range of 1:1 to 1:10.

In one embodiment, the steroid is cholesterol. In one embodiment, the molar ratio of cationic lipid to cholesterol is in the range of about 5:1 to 1:1. In one embodiment, the lipid nanoparticle comprises 32 mole% to 40 mole% of a steroid.

In one embodiment, the sum of the mole% of neutral lipids and the mole% of anionic lipids is in the range of 5 mole% to 15 mole%. In one embodiment, wherein the sum of the mole% of neutral lipids and the mole% of anionic lipids is in the range of 7 mole% to 12 mole%.

In one embodiment, the molar ratio of anionic lipid to neutral lipid is in the range of 1:1 to 1:10. In one embodiment, the sum of the mole% of neutral lipids and the mole% of steroids is in the range of 35 mole% to 45 mole%.

In one embodiment, the lipid nanoparticle comprises:

i) 45 to 55 mole% of a cationic lipid;

ii) 5 to 10 mole% neutral lipid;

iii) 1 to 5 mole% of an anionic lipid, and

Iv) 32 to 40 mole% of a steroid.

In one embodiment, the lipid nanoparticle comprises 1.0 mol% to 2.5 mol% of bound lipid. In one embodiment, the polymer-bound lipid is present at a concentration of about 1.5 mole%.

In one embodiment, the steroid is cholesterol. In some embodiments, the steroid is present at a concentration in the range of 39 mole% to 49 mole%, 40 mole% to 46 mole%, 40 mole% to 44 mole%, 40 mole% to 42 mole%, 42 mole% to 44 mole%, or 44 mole% to 46 mole%. In one embodiment, the steroid is present at a concentration of 40 mole%, 41 mole%, 42 mole%, 43 mole%, 44 mole%, 45 mole%, or 46 mole%. In certain embodiments, the molar ratio of cationic lipid to steroid is in the range of 1.0:0.9 to 1.0:1.2, or 1.0:1.0 to 1.0:1.2.

In one embodiment, the molar ratio of cationic lipid to steroid is in the range of 5:1 to 1:1.

In one embodiment, the molar ratio of cationic lipid to polymer-bound lipid is in the range of about 100:1 to about 20:1. In one embodiment, the molar ratio of cationic lipid to polymer-bound lipid is in the range of about 35:1 to about 25:1.

In one embodiment, the lipid nanoparticle has an average diameter in the range of 50nm to 100nm or 60nm to 85 nm.

In one embodiment, the composition comprises a cationic lipid, DSPC, cholesterol, and PEG-lipid as provided herein, as well as mRNA. In one embodiment, the cationic lipid, DSPC, cholesterol, and PEG-lipid provided herein are in a molar ratio of about 50:10:38.5:1.5.

Nanoparticle compositions can be designed for one or more specific applications or targets. For example, nanoparticle compositions can be designed for delivery of therapeutic and/or prophylactic agents, such as RNA, to a particular cell, tissue, organ or system or group thereof in a mammal. The physicochemical properties of the nanoparticle composition can be altered to increase selectivity for a particular bodily target. For example, granularity may be adjusted based on the fenestration size of different organs. The therapeutic and/or prophylactic agents included in the nanoparticle composition may also be selected based on one or more desired delivery objectives. For example, a therapeutic and/or prophylactic agent may be selected for a particular indication, disorder, disease, or condition and/or for delivery to a particular cell, tissue, organ, or system or group thereof (e.g., local or specific delivery). In certain embodiments, nanoparticle compositions can comprise an mRNA encoding a polypeptide of interest that is capable of translation within a cell to produce the polypeptide of interest. Such compositions may be designed to specifically deliver to a particular organ. In certain embodiments, the composition may be designed for specific delivery to the liver of a mammal.

The amount of therapeutic and/or prophylactic agent in the nanoparticle composition can depend on the size, composition, desired target and/or application, or other characteristics of the nanoparticle composition, as well as the characteristics of the therapeutic and/or prophylactic agent. For example, the amount of RNA that can be used in the nanoparticle composition can depend on the size, sequence, and other characteristics of the RNA. The relative amounts of therapeutic and/or prophylactic agents and other ingredients (e.g., lipids) in the nanoparticle composition can also vary. In some embodiments, the weight/weight ratio of lipid component to therapeutic and/or prophylactic agent in the nanoparticle composition can be about 5:1 to about 60:1, such as about 5:1、6:1、7:1、8:1、9:1、10:1、11:1、12:1、13:1、14:1、15:1、16:1、17:1、18:1、19:1、20:1、22:1、25:1、30:1、35:1、40:1、45:1、50:1 and 60:1. For example, the wt/wt ratio of lipid component to therapeutic and/or prophylactic agent may be about 10:1 to about 40:1. In certain embodiments, the wt/wt ratio is about 20:1. The amount of therapeutic and/or prophylactic agent in the nanoparticle composition can be measured, for example, using absorption spectroscopy (e.g., ultraviolet-visible spectroscopy).

In some embodiments, the nanoparticle composition comprises one or more RNAs, and the one or more RNAs, lipids, and amounts thereof can be selected to provide a particular N: P ratio. The N: P ratio of the composition refers to the molar ratio of nitrogen atoms in one or more lipids to the number of phosphate groups in the RNA. In some embodiments, a lower N to P ratio is selected. The one or more RNAs, lipids, and amounts thereof may be selected to provide an N to P ratio of about 2:1 to about 30:1, e.g., 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 12:1, 14:1, 16:1, 18:1, 20:1, 22:1, 24:1, 26:1, 28:1, or 30:1. In certain embodiments, the N to P ratio may be from about 2:1 to about 8:1. In other embodiments, the N to P ratio is from about 5:1 to about 8:1. For example, the N to P ratio may be about 5.0:1, about 5.5:1, about 5.67:1, about 6.0:1, about 6.5:1, or about 7.0:1. For example, the N to P ratio may be about 5.67:1.

The physical properties of the nanoparticle composition may depend on its components. For example, nanoparticle compositions comprising cholesterol as a structural lipid may have different characteristics than nanoparticle compositions comprising a different structural lipid. Similarly, the characteristics of a nanoparticle composition may depend on the absolute or relative amounts of its components. For example, nanoparticle compositions comprising higher mole fractions of phospholipids may have different characteristics than nanoparticle compositions comprising lower mole fractions of phospholipids. The characteristics may also vary depending on the method and conditions of preparation of the nanoparticle composition.

Nanoparticle compositions can be characterized by a variety of methods. For example, microscopy (e.g., transmission electron microscopy or scanning electron microscopy) can be used to examine the morphology and size distribution of the nanoparticle composition. Zeta potential can be measured using dynamic light scattering or potentiometry (e.g., potentiometry). Dynamic light scattering can also be used to determine particle size. The various characteristics of the nanoparticle composition, such as particle size, polydispersity index, and zeta potential, can also be measured using an instrument, such as Zetasizer Nano ZS (Malvem Instruments Ltd, malvem, worcestershire, UK).

In various embodiments, the average size of the nanoparticle composition may be between tens of nanometers and hundreds of nanometers. For example, the average size may be about 40nm to about 150nm, such as about 40nm、45nm、50nm、55nm、60nm、65nm、70nm、75nm、80nm、85nm、90nm、95nm、100nm、105nm、110nm、115nm、120nm、125nm、130nm、135nm、140nm、145nm or 150nm. In some embodiments, the nanoparticle composition can have an average size of about 50nm to about 100nm, about 50nm to about 90nm, about 50nm to about 80nm, about 50nm to about 70nm, about 50nm to about 60nm, about 60nm to about 100nm, about 60nm to about 90nm, about 60nm to about 80nm, about 60nm to about 70nm, about 70nm to about 100nm, about 70nm to about 90nm, about 70nm to about 80nm, about 80nm to about 100nm, about 80nm to about 90nm, or about 90nm to about 100nm. In certain embodiments, the nanoparticle composition can have an average size of about 70nm to about 100nm. In some embodiments, the average size may be about 80nm. In other embodiments, the average size may be about 100nm.

The nanoparticle composition can be relatively homogeneous. The polydispersity index may be used to indicate the uniformity of the nanoparticle composition, such as the particle size distribution of the nanoparticle composition. A small (e.g., less than 0.3) polydispersity index generally indicates a narrow particle size distribution. The nanoparticle composition can have a polydispersity index of about 0 to about 0.25, such as 0.01、0.02、0.03、0.04、0.05、0.06、0.07、0.08、0.09、0.10、0.11、0.12、0.13、0.14、0.15、0.16、0.17、0.18、0.19、0.20、0.21、0.22、0.23、0.24 or 0.25. In some embodiments, the nanoparticle composition can have a polydispersity index of about 0.10 to about 0.20.

The zeta potential of the nanoparticle composition can be used to indicate the zeta potential of the composition. For example, the zeta potential may describe the surface charge of the nanoparticle composition. Nanoparticle compositions having relatively low positive or negative charges are generally desirable because higher charged species can undesirably interact with cells, tissues and other components in the body. In some embodiments, the zeta potential of the nanoparticle composition may be from about-10 to about +20mV, from about-10 to about +15mV, from about-10 to about +10mV, from about-10 to about +5mV, from about-10 to about 0mV, from about-10 to about-5 mV, from about-5 to about +20mV, from about-5 to about +15mV, from about-5 to about +10mV, from about-5 to about +5mV, from about-5 to about 0mV, from about 0 to about +20mV, from about 0 to about +15mV, from about 0 to about +10mV, from about 0 to about +5mV, from about +5 to about +20mV, from about +5 to about +15mV, or from about +5 to about +10mV.

Encapsulation efficiency of a therapeutic and/or prophylactic agent (e.g., a nucleic acid as described herein) describes the amount of therapeutic and/or prophylactic agent that is encapsulated or otherwise associated with a nanoparticle composition after preparation relative to the initial amount provided. High encapsulation efficiency (e.g., near 100%) is desirable. Encapsulation efficiency may be measured, for example, by comparing the amount of therapeutic and/or prophylactic agent in a solution containing the nanoparticle composition before and after disruption of the nanoparticle composition with one or more organic solvents or detergents. Fluorescence can be used to measure the amount of free therapeutic and/or prophylactic agent (e.g., RNA) in a solution. For nanoparticle compositions described herein, the encapsulation efficiency of the therapeutic and/or prophylactic agent can be at least 50%, e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 80%. In certain embodiments, the encapsulation efficiency may be at least 90%.

The nanoparticle composition may optionally comprise one or more coatings. For example, the nanoparticle composition can be formulated as a capsule, film or tablet with a coating. Capsules, films or tablets comprising the compositions described herein may be of any useful size, tensile strength, hardness or density.

5.5.7 Pharmaceutical compositions

In one aspect, provided herein are compositions comprising a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6), a vector comprising a nucleic acid described herein (e.g., a vector described in chapter 5.4 or 6), or a protein, fusion protein, fragment, mutant described herein (e.g., a protein, fusion protein, fragment, mutant described in chapter 5.3 or 6). In some embodiments, provided herein are pharmaceutical compositions comprising a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6), a vector comprising a nucleic acid described herein (e.g., a vector described in chapter 5.4 or 6), or a protein, fusion protein, fragment, mutant described herein (e.g., a protein, fusion protein, fragment, mutant described in chapter 5.3 or 6), and a pharmaceutically acceptable carrier, diluent, or excipient.

Nanoparticle compositions according to the present disclosure may be formulated in whole or in part as pharmaceutical compositions. The pharmaceutical composition may comprise one or more nanoparticle compositions disclosed herein. For example, a pharmaceutical composition can comprise one or more nanoparticle compositions comprising one or more different therapeutic and/or prophylactic agents (e.g., a nucleic acid as described herein (such as, for example, in section 5.4 or 6)). The pharmaceutical composition may also comprise one or more pharmaceutically acceptable excipients or auxiliary ingredients, such as those described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a nanoparticle composition described herein (e.g., a nanoparticle composition described in section 5.5 or 6) and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, provided herein are pharmaceutical compositions comprising a nanoparticle composition described herein comprising a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6), and a pharmaceutically acceptable carrier, diluent, or excipient. General guidelines for the formulation and manufacture of pharmaceutical compositions and agents can be found, for example, in Remington' S THE SCIENCE AND PRACTICE of Pharmacy, 21 st edition, a.r. gennaro; lippincott, williams & Wilkins, baltimore, md.,2006. Conventional excipients and adjunct ingredients can be used in any pharmaceutical composition unless any conventional excipient or adjunct ingredient is incompatible with one or more components of the nanoparticle composition. The excipient or adjunct ingredient is incompatible with the components of the nanoparticle composition if the combination of the excipient or adjunct ingredient and the components of the nanoparticle composition can result in any undesirable biological or other deleterious effects.

In some embodiments, the one or more excipients or adjunct ingredients can comprise more than 50% of the total mass or volume of the pharmaceutical composition comprising the nanoparticle composition. For example, the one or more excipients or auxiliary ingredients may constitute 50%, 60%, 70%, 80%, 90% or higher percent of the pharmaceutical convention (pharmaceutical convention). In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% pure. In some embodiments, the excipient is approved for human and veterinary use. In some embodiments, the excipient is approved by the U.S. food and drug administration. In some embodiments, the excipient is pharmaceutical grade. In some embodiments, the excipient meets the standards of the United States Pharmacopeia (USP), the European Pharmacopeia (EP), the british pharmacopeia, and/or the international pharmacopeia.

The relative amounts of one or more nanoparticle compositions, one or more pharmaceutically acceptable excipients, and/or any additional ingredients in the pharmaceutical compositions according to the present disclosure will vary depending on the identity, build, and/or condition of the subject being treated and further depending on the route of administration of the composition. For example, the pharmaceutical composition may comprise between 0.1% and 100% (wt/wt) of one or more nanoparticle compositions.

In certain embodiments, nanoparticle compositions and/or pharmaceutical compositions of the present disclosure are stored and/or transported (e.g., stored at a temperature of 4 ℃ or less, such as between about-150 ℃ and about 0 ℃ or between about-80 ℃ and about-20 ℃ (e.g., about-5 ℃, -10 ℃, -15 ℃, -20 ℃, -25 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃, -130 ℃, or-150 ℃)). For example, a pharmaceutical composition comprising a compound according to series 01, 02, 03 and 04, e.g. a compound of any of formulae (01-I), (01-II), (02-I), (03-I) and (04-I) (and sub-formulae thereof), is a solution that is stored and/or transported refrigerated at, e.g., about-20 ℃, 30 ℃,40 ℃,50 ℃, 60 ℃, 70 ℃ or-80 ℃. In certain embodiments, the present disclosure also relates to a method of increasing the stability of nanoparticle and/or pharmaceutical compositions comprising a compound according to series 01, 02, 03, and 04, e.g., any of formulas (01-I), (01-II), (02-I), (03-I), and (04-I) (and sub-formulas thereof), by storing the nanoparticle and/or pharmaceutical composition at a temperature of 4 ℃ or less, such as between about-150 ℃ and about 0 ℃ or between about-80 ℃ and about-20 ℃ (e.g., about-5 ℃, -10 ℃, -15 ℃, -20 ℃, -25 ℃, -30 ℃, -40 ℃, -50 ℃, -60 ℃, -70 ℃, -80 ℃, -90 ℃, -130 ℃, or-150 ℃). For example, nanoparticle compositions and/or pharmaceutical compositions disclosed herein are stable for about at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 1 month, at least 2 months, at least 4 months, at least 6 months, at least 8 months, at least 10 months, at least 12 months, at least 14 months, at least 16 months, at least 18 months, at least 20 months, at least 22 months, or at least 24 months at a temperature of, for example, 4 ℃ or less (e.g., between about 4 ℃ and-20 ℃). In one embodiment, the formulation is stable for at least 4 weeks at about 4 ℃.

In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition disclosed herein and a pharmaceutically acceptable carrier selected from one or more of Tris, acetate (e.g., sodium acetate), citrate (e.g., sodium citrate), saline, PBS, and sucrose. In certain embodiments, the pharmaceutical compositions of the present disclosure have a pH of between about 7 and 8 (e.g., 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0, or between 7.5 and 8, or between 7 and 7.8). In certain embodiments, the pharmaceutical compositions of the present disclosure comprise a nanoparticle composition as disclosed herein, tris, saline, and sucrose, and have a pH of about 7.5-8, which is suitable for storage and/or transport at, for example, about-20 ℃. For example, the pharmaceutical compositions of the present disclosure comprise the nanoparticle compositions disclosed herein and PBS, and have a pH of about 7-7.8, suitable for storage or transport at, for example, about 4 ℃ or less. In the context of the present disclosure, "stability," "stabilized," and "stable" refer to nanoparticle compositions and/or pharmaceutical compositions disclosed herein that are resistant to chemical or physical changes (e.g., degradation, particle size change, aggregation, change in encapsulation, etc.) under given manufacturing, transportation, storage, and/or use conditions, such as when pressure is applied, such as shear forces, freeze/thaw pressures, and the like.

In certain embodiments, the pharmaceutical compositions (e.g., aqueous compositions or frozen compositions) of the present disclosure comprise the nanoparticle compositions and buffers disclosed herein. Representative buffers include, but are not limited to, acetic acid, adipic acid, ammonia, ammonium phosphate, ammonium sulfate, arginine, asparagine, boric acid, calcium carbonate, calcium lactate, tricalcium phosphate, citric acid monohydrate, dipotassium phosphate, disodium phosphate, diethanolamine, glycine, histidine, hydroxyethylpiperazine ethane sulfonic acid, lysine acetate, lysine hydrochloride, maleic acid, malic acid, meglumine, methionine, sodium dihydrogen phosphate, monoethanolamine, monosodium glutamate, phosphoric acid, potassium citrate, potassium metaphosphate, sodium acetate, sodium bicarbonate, sodium borate, sodium carbonate, sodium citrate, sodium lactate, triethanolamine, tromethamine, phosphate buffers (e.g., sodium dihydrogen phosphate monohydrate, disodium hydrogen phosphate), acetate buffers, citrate buffers, borate buffers, and HBSS (hank's balanced salt solution), tris-aminomethane buffer, tris-aminomethane hydrochloride buffers, tris (hydroxymethyl) -aminomethane (Tris) buffers, borate buffers, diethylamine buffers, and the like.

In certain embodiments, the pharmaceutical compositions (e.g., aqueous compositions or frozen compositions) of the present disclosure comprise the nanoparticle compositions and buffers disclosed herein, and further comprise one or more cryoprotectants, including, but not limited to, sugars, surfactants, polyols, amino acids, peptides, proteins, and/or the like.

Representative sugars include monosaccharides, disaccharides, and polysaccharides. Representative monosaccharides include glucose, fructose, galactose, arabinose, xylose, mannose, lactulose, allose, altrose, gulose, idose, talose, ribose, lyxose, and the like. Representative disaccharides include sucrose, trehalose, lactose, maltose, isomaltose, cellobiose, and the like. Representative polysaccharides include starch and starch derivatives, cellulose and cellulose derivatives (e.g., methylcellulose, ethylcellulose, hydroxycellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose), chitosan and its derivatives, chitin, inulin, chitin, glycogen, pectin, and hyaluronic acid.

Surfactants include nonionic surfactants, specifically polyoxyethylene fatty alcohol ethers (Brij), polysorbates (e.g., tween 80, tween 60, tween 20, tween 40), polyoxyethylene fatty acid esters (OEO), polyoxyethylene castor oil derivatives, polyoxyethylene polypropylene glycol copolymers, sucrose fatty acid esters, polyethylene glycol fatty acid esters, polyoxyethylene sorbitan monofatty acid esters, polyoxyethylene sorbitan di-fatty acid esters, polyoxyethylene monoglycerides, polyoxyethylene diglycerides, polyglycerol fatty acid esters, polypropylene glycol monoesters, arylalkyl polyether alcohols, polyoxyethylene-polyoxypropylene copolymers (poloxamers), polyvinyl alcohol (PVA) derivatives, and polyvinylpyrrolidone (PVP).

Polyols include sugar alcohols, polymeric polyols, low molecular weight polyols, and the like. Representative sugar alcohols include maltitol, sorbitol, xylitol, erythritol, isomalt, maltitol and the like, mannitol, polyglucitol (polyglycitol) and the like. Representative polymeric polyols include polyvinyl alcohol, polyethylene glycol, polyether polyols, polyester polyols, polypropylene glycol, and the like. Representative low molecular weight polyols include ethanol, ethylene glycol, propylene glycol, glycerol, trimethylol propane, butylene glycol, and the like.

In one embodiment, the pharmaceutical composition (e.g., aqueous composition or frozen composition) of the present disclosure comprises propylene glycol. For example, the concentration of propylene glycol in the pharmaceutical composition is in the range of about 0.05% w/v to about 50% w/v, about 0.05% w/v to about 25% w/v, about 0.5% w/v to 10% w/v, or about 1% w/v to about 5% w/v. For example, the concentration of glycerol in the formulation is in the range of about 1.5% w/v to about 3% w/v or about 2% w/v to about 2.5% w/v.

Representative amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

Representative peptides include glycylglycine and triglycine. Proteins include albumin, collagen, casein and gelatin.

The nanoparticle composition and/or pharmaceutical composition comprising one or more nanoparticle compositions can be administered to any patient or subject, including patients or subjects who may benefit from the therapeutic effect provided by delivery of a therapeutic and/or prophylactic agent to one or more specific cells, tissues, organs or systems or groups thereof, such as the renal system. Although the description provided herein of nanoparticle compositions and pharmaceutical compositions comprising nanoparticle compositions is primarily directed to compositions suitable for administration to humans, those skilled in the art will appreciate that such compositions are generally suitable for administration to any other mammal. Improvements to compositions suitable for administration to humans in order to render the compositions suitable for administration to a variety of animals are well known and veterinary pharmacologists of ordinary skill can design and/or make such improvements by mere routine experimentation, if any. It is contemplated that subjects to which the compositions are administered include, but are not limited to, humans, other primates, and other mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and/or rats.

Pharmaceutical compositions comprising one or more nanoparticle compositions may be prepared by any method known in the pharmacological arts or later developed. Generally, such methods of preparation involve combining the active ingredient with excipients and/or one or more other auxiliary ingredients and then, if necessary or desired, dividing, shaping and/or packaging the product into the desired single or multi-dose units.

In some embodiments, provided herein are pharmaceutical compositions comprising a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6). In some embodiments, provided herein are pharmaceutical compositions comprising nucleic acids encoding proteins described herein (e.g., proteins described in section 5.3). In some embodiments, provided herein are pharmaceutical compositions comprising a nucleic acid encoding a fragment as described herein, a nucleic acid encoding a mutant as described herein, a nucleic acid encoding a protein as described herein, or a nucleic acid encoding a fusion protein as described herein. In some embodiments, provided herein are pharmaceutical compositions comprising a vector (e.g., a vector described in chapter 5.4 or 6) comprising a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6). The vector may be a viral vector or a non-viral vector (e.g., a plasmid). In some embodiments, provided herein are pharmaceutical compositions comprising a protein described herein (e.g., a protein described in section 5.3). In some embodiments, provided herein are pharmaceutical compositions comprising a fragment as described herein, a mutant as described herein, a protein as described herein, or a fusion protein as described herein. The pharmaceutical compositions may comprise a buffer (e.g., a buffer as disclosed herein) and/or one or more cryoprotectants (e.g., one or more cryoprotectants as disclosed herein).

In some embodiments, provided herein are pharmaceutical compositions comprising a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6) and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, provided herein are pharmaceutical compositions comprising a nucleic acid encoding a protein described herein (e.g., a protein described in section 5.3) and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, provided herein are pharmaceutical compositions comprising a nucleic acid encoding a fragment as described herein, a nucleic acid encoding a mutant as described herein, a nucleic acid encoding a protein as described herein, or a nucleic acid encoding a fusion protein as described herein, and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, provided herein are pharmaceutical compositions comprising a vector comprising a nucleic acid as described herein.

In some embodiments, provided herein are pharmaceutical compositions comprising a protein described herein (e.g., a protein described in section 5.3) and a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, provided herein are pharmaceutical compositions comprising a fragment as described herein, a mutant as described herein, a protein as described herein or a fusion protein as described herein, and a pharmaceutically acceptable carrier, diluent or excipient.

In particular embodiments, the pharmaceutical compositions described herein are immunogenic compositions. For example, in some embodiments, the pharmaceutical composition induces an immune response in a subject (e.g., a human subject), as described in section 6 (e.g., a gE-specific antibody). In particular embodiments, the pharmaceutical compositions described herein are vaccine compositions.

Pharmaceutical compositions according to the present disclosure may be prepared, packaged and/or sold in bulk, as single unit doses and/or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition comprising a predetermined amount of an active ingredient (e.g., a nanoparticle composition). The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the subject and/or a convenient fraction of such dose, for example half or one third of such dose.

Pharmaceutical compositions may be prepared in a variety of forms suitable for a variety of routes and methods of administration. For example, pharmaceutical compositions may be prepared in liquid dosage forms (e.g., emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and elixirs), injectable forms, solid dosage forms (e.g., capsules, tablets, pills, powders and granules), dosage forms for topical and/or transdermal administration (e.g., ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and patches), suspensions, powders and other forms.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, nanoemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredient, the liquid dosage form may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1, 3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. In addition to inert diluents, the oral compositions can also include additional therapeutic and/or prophylactic agents, additional agents, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and/or perfuming agents. In certain embodiments for parenteral administration, the compositions are mixed with a solubilizing agent, such as Cremophor ^TM, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.

Injectable formulations, such as sterile injectable aqueous or oleaginous suspensions, may be formulated according to the known art using suitable dispersing, wetting and/or suspending agents. The sterile injectable preparation may be a sterile injectable solution, suspension and/or emulsion in a non-toxic parenterally acceptable diluent and/or solvent, for example, as a solution in 1, 3-butanediol. Acceptable vehicles and solvents that may be used include water, ringer's solution, USP, and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono-or diglycerides. Fatty acids such as oleic acid find use in the preparation of injectables.

The injectable formulation may be sterilized, for example, by filtration through a bacterial-retaining filter and/or by incorporating sterilizing agents in the form of sterile solid compositions which may be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.

In some embodiments, the pharmaceutical composition comprises a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6). In some embodiments, the pharmaceutical composition comprises a non-naturally occurring nucleic acid described herein (e.g., a non-naturally occurring nucleic acid described in section 5.4 or 6). In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding a protein described herein (e.g., a protein described in section 5.3). In some embodiments, the pharmaceutical composition comprises a nucleic acid encoding a fragment as described herein, a nucleic acid encoding a mutant as described herein, a nucleic acid encoding a protein as described herein, or a nucleic acid encoding a fusion protein as described herein. In some embodiments, the pharmaceutical composition comprises a protein described herein (e.g., a protein described in section 5.3). In some embodiments, the pharmaceutical composition comprises a fragment as described herein, a mutant as described herein, a protein as described herein, or a fusion protein as described herein. In some embodiments, the pharmaceutical composition comprises a fragment as described herein or a nucleic acid as described herein. In some embodiments, the pharmaceutical composition comprises a non-naturally occurring nucleic acid as described herein.

In particular embodiments, provided herein are pharmaceutical compositions comprising a non-naturally occurring nucleic acid as described herein and at least a first lipid, optionally wherein the first lipid is a compound according to formula 01-I or formula 01-II, or a compound listed in Table 01-1, or a compound according to formula 02-I, or a compound listed in Table 02-1, or a compound according to formula 03-I, or a compound listed in Table 03-1, or a compound according to formula 04-I, or a compound listed in Table 04-1. In specific embodiments, the pharmaceutical composition further comprises a second lipid, optionally wherein the second lipid is a compound according to formula 05-I. In particular embodiments, the pharmaceutical composition is formulated as a lipid nanoparticle encapsulating a nucleic acid in a lipid shell.

The present disclosure features methods of delivering a therapeutic and/or prophylactic agent (i.e., a polypeptide of interest) to a mammalian cell or organ and treating a disease or disorder in a mammal in need thereof, the methods comprising administering the therapeutic and/or prophylactic agent (i.e., a polypeptide of interest) to the mammal and/or contacting the mammalian cell with the therapeutic and/or prophylactic agent.

The present disclosure provides methods of delivering a therapeutic and/or prophylactic agent to a mammalian cell or organ, producing a polypeptide of interest in a mammalian cell, and treating a disease or disorder in a mammal in need thereof, the methods comprising administering to the mammal a nanoparticle composition comprising the therapeutic and/or prophylactic agent and/or contacting the mammalian cell with the nanoparticle composition.

5.6 Method

In one aspect, provided herein are also methods for controlling, preventing, and treating a disease or disorder in a subject (e.g., a human subject) caused by VZV or by infection with VZV. In some embodiments, provided herein is a method for controlling, preventing, and/or treating a disease or disorder caused by VZV or by infection of VZV in a subject (e.g., a human subject), the method comprising administering to the subject a protein described herein (e.g., in chapter 5.3 or 6), a fragment described herein (e.g., in chapter 5.3 or 6), a fusion protein described herein (e.g., in chapter 5.3 or 6), a nucleic acid (including a non-naturally occurring nucleic acid described herein) (e.g., in chapter 5.4 or 6), a vector described herein (e.g., in chapter 5.4 or 6), or a pharmaceutical composition described herein (e.g., in chapter 5.5 or 6). In some embodiments, the disease or disorder controlled, prevented or treated with the methods described herein is caused by VZV or by infection with VZV. In particular embodiments, the methods provided herein are for preventing a disease or disorder caused by VZV. In some embodiments, the method inhibits VZV infection. In some embodiments, the method inhibits reactivation of a latent VZV infection.

In particular embodiments, the disease or condition to be controlled, prevented or treated with the methods described herein is one or more of varicella, shingles, post-herpetic neuralgia (PHN), meningoencephalitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis and pancreatitis.

In some embodiments, the methods of the invention for controlling, preventing, and treating a disease or disorder caused by VZV or by infection of VZV in a subject comprise administering to the subject a therapeutically effective amount of a fragment, nucleic acid, or therapeutic nucleic acid as described herein. In specific embodiments, the therapeutic nucleic acid is an mRNA molecule as described herein.

In some embodiments, the methods of the invention for controlling, preventing, and treating a disease or disorder caused by VZV or by infection of VZV in a subject comprise administering to the subject a therapeutically effective amount of a therapeutic composition comprising a fragment, nucleic acid, or therapeutic nucleic acid as described herein. In specific embodiments, the therapeutic nucleic acid is an mRNA molecule as described herein.

In some embodiments, the methods of the invention for controlling, preventing, and treating a disease or disorder caused by VZV or by infection of VZV in a subject comprise administering to the subject a therapeutically effective amount of a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid as described herein. In specific embodiments, the therapeutic nucleic acid is an mRNA molecule as described herein.

In some embodiments, the methods of the invention for controlling, preventing, and treating a disease or disorder caused by VZV or by infection of VZV in a subject comprise administering to the subject a therapeutically effective amount of a lipid-containing composition comprising a therapeutic nucleic acid as described herein. In specific embodiments, the therapeutic nucleic acid is an mRNA molecule as described herein.

In some embodiments, the methods of the invention for controlling, preventing, and treating a disease or disorder caused by VZV or by infection of VZV in a subject comprise administering to the subject a therapeutically effective amount of a lipid-containing composition comprising a therapeutic nucleic acid as described herein, wherein the lipid-containing composition is formulated as a lipid nanoparticle encapsulating the therapeutic nucleic acid in a lipid shell. In specific embodiments, the therapeutic nucleic acid is an mRNA molecule as described herein. In particular embodiments, cells in a subject are effective to ingest lipid-containing compositions (e.g., lipid nanoparticles) described herein after administration. In particular embodiments, the lipid-containing compositions (e.g., lipid nanoparticles) described herein are endocytosed by a cell of a subject.

In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a protein described herein (e.g., a protein described in chapter 5.3 or 6) or a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6). In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a fragment (e.g., a fragment described in chapter 5.4 or 6), a mutant described herein (e.g., a mutant described in chapter 5.3 or 6), or a fusion protein described herein (e.g., a fusion protein described in chapter 5.3 or 6). In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a vector described herein (e.g., a vector described in section 5.4.10). In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6). In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is mRNA. In particular embodiments, the nucleic acid is a non-naturally occurring mRNA. In specific embodiments, the disease or condition includes one or more of varicella, shingles (herpes zoster) (or shingles), post-herpetic neuralgia (PHN), meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In specific embodiments, the disease or disorder is shingles (herpes zoster) (or shingles).

In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a lipid-containing composition comprising a nucleic acid described herein. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is mRNA. In particular embodiments, the nucleic acid is a non-naturally occurring mRNA. In specific embodiments, the disease or condition includes one or more of varicella, shingles (herpes zoster) (or shingles), post-herpetic neuralgia (PHN), meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In specific embodiments, the disease or disorder is shingles (herpes zoster) (or shingles).

In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a lipid-containing composition comprising a nucleic acid described herein, wherein the lipid-containing composition is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid shell. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is mRNA. In particular embodiments, the nucleic acid is a non-naturally occurring mRNA. In particular embodiments, cells in a subject are effective to ingest lipid-containing compositions (e.g., lipid nanoparticles) described herein after administration. In particular embodiments, the lipid-containing compositions (e.g., lipid nanoparticles) described herein are endocytosed by a cell of a subject. In specific embodiments, the disease or condition includes one or more of varicella, shingles (herpes zoster) (or shingles), post-herpetic neuralgia (PHN), meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In specific embodiments, the disease or disorder is shingles (herpes zoster) (or shingles).

In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), wherein the nanoparticle composition encapsidates a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6). In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is mRNA. In particular embodiments, the nucleic acid is a non-naturally occurring mRNA. In particular embodiments, the cells in the subject are effective to ingest the nanoparticle composition after administration. In particular embodiments, the nanoparticle composition is endocytosed by a cell of the subject. In specific embodiments, the disease or condition includes one or more of varicella, shingles (herpes zoster) (or shingles), post-herpetic neuralgia (PHN), meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In specific embodiments, the disease or disorder is shingles (herpes zoster) (or shingles).

In some embodiments, provided herein is a method for preventing a disease or disorder caused by VZV in a subject (e.g., a human subject), the method comprising administering to the subject a prophylactically effective amount of a pharmaceutical composition described herein (e.g., a pharmaceutical composition described in section 5.5.7). In particular embodiments, the disease or condition includes one or more of varicella, shingles, post-herpetic neuralgia (PHN), meningoepithymitis, myelitis, cranial nerve palsy, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis. In specific embodiments, the disease or disorder is shingles (herpes zoster) (or shingles).

Without being bound by any theory, the nucleic acids described herein administered to a subject (e.g., a human subject) are taken up by cells in the subject, a protein encoded by the nucleic acid is expressed, and the protein induces an immune response in the subject. In some embodiments, the protein induces antibodies (e.g., igG) that are specific for VZV gE. In some embodiments, the antibody specific for VZV gE is a neutralizing antibody. In some embodiments, the immune response induced by the protein comprises one or more of the responses described in section 5.6.1 or 6. Without being bound by any theory, the immune response induced by the protein provides protection against diseases or conditions caused by VZV. Protection may be complete or partial protection against diseases or conditions caused by VZV (e.g., shingles).

In some embodiments, after administration of a therapeutic nucleic acid as described herein, a vaccine composition comprising a therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein to a subject in need thereof, cells in the subject ingest and express the administered therapeutic nucleic acid to produce a peptide or polypeptide encoded by the nucleic acid. In some embodiments, the encoded peptide or polypeptide is derived from VZV that causes a disease or disorder that is controlled, prevented or treated by the method.

5.6.1 Immune response

In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a nucleic acid described herein (e.g., a nucleic acid described in section 5.4 or 6) or a vector described herein (e.g., a vector described in section 5.4.10) to the subject. In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a lipid-containing composition comprising a nucleic acid described herein to the subject, wherein the lipid-containing composition is formulated as a lipid nanoparticle encapsulating the nucleic acid in a lipid shell. In some embodiments, one or more immune responses to VZV are elicited in a subject in need thereof following administration of a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), wherein the nanoparticle composition encapsidates a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6). In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a protein described herein (e.g., a protein described in chapter 5.3 or 6) to the subject. In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a fragment described herein (e.g., a fragment described in chapter 5.3 or 6), a mutant (e.g., a mutant described in chapter 5.3 or 6), or a fusion protein described herein (e.g., a fusion protein described in chapter 5.3 or 6) to the subject. In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a pharmaceutical composition described herein (e.g., a pharmaceutical composition described in section 5.5.7) to the subject. The one or more immune responses may be in the form of, for example, an antibody response (humoral response) or a cellular immune response such as cytokine secretion (e.g., interferon-gamma), helper activity, or cytotoxicity. In some embodiments, expression of an activation marker on an immune cell, expression of a co-stimulatory receptor on an immune cell, expression of a ligand of a co-stimulatory receptor, cytokine secretion, infiltration of an infected cell by an immune cell (e.g., a T lymphocyte, a B lymphocyte, and/or an NK cell), production of antibodies that specifically recognize one or more viral proteins (e.g., viral peptides or proteins encoded by a therapeutic nucleic acid), effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and/or NK cell proliferation. In some embodiments, activation and proliferation of bone Marrow Derived Suppressor Cells (MDSCs) and Treg cells are inhibited. In some embodiments, the one or more immune responses comprise an antibody response. In some embodiments, the antibody reaction comprises generating an antibody that specifically binds to VZV gE (e.g., a VZV gE neutralizing antibody). In some embodiments, the one or more immune responses include production of a cytokine in a lymphocyte (e.g., increased production of a cytokine in a lymphocyte) and/or an increase in the proportion of lymphocytes that express a cytokine. In some embodiments, the immune response includes an increase in production of one or more cytokines (e.g., IFN-gamma, IL-2, and/or TNF-alpha) by one or more lymphocyte populations. in some embodiments, the lymphocyte is a CD4 ⁺ T cell and/or a CD8 ⁺ T cell. In some embodiments, the one or more immune responses are one or more of the immune responses described in section 6.

In some embodiments, one or more immune responses against VZV are elicited in a subject in need thereof following administration of a fragment, nucleic acid, or therapeutic nucleic acid as described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid as described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid as described herein, to the subject. In some embodiments, the immune response elicited comprises one or more adaptive immune responses against VZV. In some embodiments, the immune response elicited comprises one or more innate immune responses against VZV. The one or more immune responses may be in the form of, for example, an antibody response (humoral response) or a cellular immune response such as cytokine secretion (e.g., interferon-gamma), helper activity, or cytotoxicity. In some embodiments, expression of an activation marker on an immune cell, expression of a co-stimulatory receptor on an immune cell, expression of a ligand of a co-stimulatory receptor, cytokine secretion, infiltration of an infected cell by an immune cell (e.g., a T lymphocyte, a B lymphocyte, and/or an NK cell), production of antibodies that specifically recognize one or more viral proteins (e.g., viral peptides or proteins encoded by a therapeutic nucleic acid), effector function, T cell activation, T cell differentiation, T cell proliferation, B cell differentiation, B cell proliferation, and/or NK cell proliferation. In some embodiments, activation and proliferation of bone Marrow Derived Suppressor Cells (MDSCs) and Treg cells are inhibited.

In some embodiments, upon administration of a fragment, nucleic acid, or therapeutic nucleic acid as described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid as described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid as described herein to a subject in need thereof, one or more lymphocyte populations that produce cytokines in the subject are increased. In some embodiments, the lymphocyte is a CD4 ⁺ T cell and/or a CD8 ⁺ T cell. In some embodiments, the cytokine is one or more of IFN-gamma, IL-2, and TNF-alpha. In some embodiments, the proportion of cd4+ cells expressing IFN- γ is increased. In some embodiments, the proportion of CD4+ cells expressing IL-2 is increased. In some embodiments, the proportion of cd4+ cells expressing TNF- α is increased. In some embodiments, the proportion of cd8+ cells expressing IFN- γ is increased. In some embodiments, the proportion of CD8+ cells expressing IL-2 is increased. In some embodiments, the proportion of cd8+ cells expressing TNF- α is increased.

In particular embodiments, neutralizing antibodies specifically bind to one or more epitopes of the VZV gE protein and inhibit or reduce the function or activity of one or more gE proteins.

In particular embodiments, the neutralizing antibodies bind to one or more viral proteins present on the surface of a viral particle or infected cell and label the viral particle or infected cell for destruction by the immune system of the subject. In some embodiments, endocytosis of the viral particle by the white blood cells (e.g., macrophages) is induced or enhanced. In some embodiments, antibody-dependent cell-mediated cytotoxicity (ADCC) against the infected cells in the subject is induced or enhanced. In some embodiments, antibody-dependent cell phagocytosis (ADCP) is induced or enhanced in a subject against an infected cell. In some embodiments, complement Dependent Cytotoxicity (CDC) against the infected cells in the subject is induced or enhanced.

5.6.2 Combination therapies

In some embodiments, the compositions of the present disclosure may further comprise one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an adjuvant capable of enhancing the immunogenicity of the composition (e.g., a genetic vaccine). In some embodiments, the additional therapeutic agent is an immunomodulatory agent that enhances an immune response in the subject. In some embodiments, the adjuvant and therapeutic nucleic acid in the composition may have a synergistic effect in eliciting an immune response in a subject.

In some embodiments, the additional therapeutic agent and the therapeutic nucleic acid of the present disclosure may be co-formulated in one composition. For example, the additional therapeutic agent may be formulated as part of a composition comprising a therapeutic nucleic acid of the present disclosure. Alternatively, in some embodiments, the additional therapeutic agent and therapeutic nucleic acid of the present disclosure may be formulated as separate compositions or dosage units for co-administration to a subject sequentially or simultaneously.

In certain embodiments, the therapeutic nucleic acids of the present disclosure are formulated as part of a lipid-containing composition as described in section 5.5, and the additional therapeutic agent is formulated as a separate composition. In certain embodiments, the therapeutic nucleic acids of the present disclosure are formulated as part of a lipid-containing composition as described in section 5.5, wherein the additional therapeutic agent is also formulated as part of the lipid-containing composition.

In certain embodiments, the therapeutic nucleic acids of the present disclosure are formulated such that the therapeutic nucleic acids are encapsulated in the lipid shell of the lipid nanoparticle as described in section 5.5, and the additional therapeutic agent is formulated as a separate composition. In certain embodiments, the therapeutic nucleic acids of the present disclosure are formulated such that the therapeutic nucleic acids are encapsulated in the lipid shell of a lipid nanoparticle as described in section 5.5, wherein the lipid nanoparticle also encapsulates an additional therapeutic molecule or a nucleic acid encoding an additional therapeutic molecule. In certain embodiments, the therapeutic nucleic acids of the present disclosure are formulated such that the therapeutic nucleic acids are encapsulated in the lipid shell of the lipid nanoparticle as described in section 5.5, wherein the lipid nanoparticle and the additional therapeutic agent are formulated as a single composition.

In particular embodiments, the additional therapeutic agent is an adjuvant. In some embodiments, the adjuvant comprises an agent that promotes Dendritic Cell (DC) maturation in the vaccinated subject, such as, but not limited to, lipopolysaccharide, TNF-a, or CD40 ligand. In some embodiments, the adjuvant is an agent recognized as a "danger signal" by the immune system of the vaccinated subject, such as LPS, GP96, and the like.

In some embodiments, the adjuvant comprises an immunostimulatory cytokine, such as but not limited to IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-12、IL-13、IL-14、IL-15、IL-16、IL-17、IL-18、IL-19、IL-20、IL-21、IL-22、IL-23、IL-24、IL-25、IL-26、IL-27、IL-28、IL-29、IL-30、IL-31、IL-32、IL-33、INF-α、IFN-β、INF-γ、GM-CSF、G-CSF、M-CSF、LT-β or TNF-a, a growth factor such as hGH.

In some embodiments, the adjuvant comprises a compound known to be capable of eliciting an innate immune response. An exemplary class of such compounds are Toll-like receptor ligands, such as those of the human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, and murine Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR 13. Another exemplary class of such compounds are immunostimulatory nucleic acids, such as oligonucleotides containing CpG motifs. The CpG-containing nucleic acid may be a DNA (CpG-DNA) or RNA (CpG-RNA) molecule. The CpG-RNA or CpG-DNA may be single-stranded CpG-DNA (ss CpG-DNA), double-stranded CpG-DNA (dsDNA), single-stranded CpG-RNA (ss CpG-RNA) or double-stranded CpG-RNA (ds CpG-RNA). In some embodiments, the CpG nucleic acid is in the form of CpG-RNA. In particular embodiments, the CpG nucleic acid is in the form of single stranded CpG-RNA (ss CpG-RNA). In some embodiments, the CpG nucleic acid contains at least one or more (mitogenic) cytosine/guanine dinucleotide sequences (CpG motifs). In some embodiments, at least one CpG motif contained in these sequences (i.e., C (cytosine) and/or G (guanine) forming the CpG motif) is unmethylated.

In some embodiments, the additional therapeutic agent is an immunomodulatory agent that activates, boosts, or resumes normal immune function. In particular embodiments, the immunomodulator is an agonist of a costimulatory signal of an immune cell, such as a T lymphocyte, NK cell, or antigen presenting cell (e.g., a dendritic cell or macrophage). In particular embodiments, the immunomodulator is an antagonist of an inhibitory signal of an immune cell, such as a T lymphocyte, NK cell, or antigen presenting cell (e.g., a dendritic cell or macrophage).

Various immune cell stimulators known to those of skill in the art may be used in conjunction with the present disclosure. In certain embodiments, the agonist of the costimulatory signal is an agonist of a costimulatory molecule (e.g., a costimulatory receptor) found on an immune cell such as a T lymphocyte (e.g., a cd4+ or cd8+ T lymphocyte), an NK cell, and/or an antigen-presenting cell (e.g., a dendritic cell or macrophage). Specific examples of co-stimulatory molecules include glucocorticoid-induced tumor necrosis factor receptor (GITR), inducible T-cell co-stimulatory (ICOS or CD 278), OX40 (CD 134), CD27, CD28, 4-IBB (CD 137), CD40, lymphotoxin alpha (lta), LIGHT (lymphotoxoid, which exhibits inducible expression and competes with herpes simplex virus glycoprotein D for HVEM (receptor expressed by T lymphocytes)), CD226, cytotoxic and regulatory T-cell molecules (CRT AM), death receptor 3 (DR 3), lymphotoxin beta receptor (LTBR), transmembrane activator and CAML interactive factor (transmembrane activator and CAML interactor, TACI), B-cell activator receptor (BAFFR) and B-cell maturation protein (BCMA).

In particular embodiments, the agonist of a co-stimulatory receptor is an antibody or antigen binding fragment thereof that specifically binds to the co-stimulatory receptor. Specific examples of co-stimulatory receptors include GITR, ICOS, OX, CD27, CD28, 4-1BB, CD40, LT alpha, LIGHT, CD226, CRT AM, DR3, LTBR, TACI, BAFFR, and BCMA. In certain embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on immune cells. In other embodiments, the bispecific antibody binds to a receptor on an immune cell and another receptor on a virus-infected diseased cell. In specific embodiments, the antibody is a human or humanized antibody.

In another embodiment, the agonist of the co-stimulatory receptor is a ligand of the co-stimulatory receptor or a functional derivative thereof. In certain embodiments, the ligand is a fragment of a natural ligand. Specific examples of natural ligands include ICOSL, B7RP1, CD137L, OX, L, CD, herpes virus invasion mediator (HVEM), CD80 and CD86. Nucleotide sequences encoding natural ligands and amino acid sequences of natural ligands are known in the art.

In particular embodiments, the antagonist is an antagonist of an inhibitory molecule (e.g., an inhibitory receptor) found on immune cells such as T lymphocytes (e.g., cd4+ or cd8+ T lymphocytes), NK cells, and/or antigen presenting cells (e.g., dendritic cells or macrophages). Specific examples of inhibitory molecules include cytotoxic T lymphocyte-associated antigen 4 (CTLA-4 or CD 52), programmed cell death protein 1 (PD 1 or CD 279), B and T lymphocyte attenuation agents (BTLA), killer cell immunoglobulin-like receptor (KIR), lymphocyte activating gene 3 (LAG 3), T cell membrane protein 3 (TIM 3), CD 160, adenosine A2a receptor (A2 aR), T cell immune receptor (TIGIT) with immunoglobulin and ITIM domains, leukocyte-associated immunoglobulin-like receptor 1 (LAIR 1) and CD 160.

In another embodiment, the antagonist of the inhibitory receptor is an antibody (or antigen-binding fragment) that specifically binds to the natural ligand of the inhibitory receptor and prevents the natural ligand from binding to the inhibitory receptor and transducing an inhibitory signal. In certain embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is sc-Fv. In a specific embodiment, the antibody is a bispecific antibody that binds to two receptors on immune cells. In other embodiments, the bispecific antibody binds to a receptor on an immune cell and another receptor on a virus-infected diseased cell. In specific embodiments, the antibody is a human or humanized antibody.

In another embodiment, the antagonist of the inhibitory receptor is a soluble receptor or a functional derivative thereof that specifically binds to the natural ligand of the inhibitory receptor and prevents the natural ligand from binding to the inhibitory receptor and transducing an inhibitory signal. Specific examples of natural ligands for inhibitory receptors include PDL-1, PDL-2, B7-H3, B7-H4, HVEM, gal9 and adenosine. Specific examples of inhibitory receptors that bind to natural ligands include CTLA-4, PD-1, BTLA, KIR, LAG, TIM3 and A2aR.

In another embodiment, an antagonist of an inhibitory receptor is an antibody (or antigen binding fragment) or ligand that binds to the inhibitory receptor but does not transduce an inhibitory signal. Specific examples of inhibitory receptors include CTLA-4, PD1, BTLA, KIR, LAG, TIM3 and A2aR. In certain embodiments, the antibody is a monoclonal antibody. In other embodiments, the antibody is an scFv. In particular embodiments, the antibody is a human or humanized antibody. A specific example of an antibody to an inhibitory receptor is an anti-CTLA-4 antibody (Leach DR, et al Science 1996; 271:1734-1736). Another example of an antibody to an inhibitory receptor is an anti-PD-1 antibody (Topalian SL, NEJM 2012; 28:3167-75).

5.6.3 Patient population

In some embodiments, a nucleic acid described herein (e.g., a nucleic acid described in, e.g., section 5.4 or 6) is administered to a subject in need thereof. In some embodiments, the nucleic acid is non-naturally occurring. In some embodiments, the nucleic acid is mRNA. In some embodiments, the nucleic acid is a non-naturally occurring mRNA. In some embodiments, a vector described herein (e.g., a vector described in section 5.3.10) is administered to a subject in need thereof. In some embodiments, a lipid-containing composition comprising a nucleic acid described herein is administered to a subject in need thereof. In some embodiments, a nanoparticle composition described herein (e.g., a nanoparticle composition described in section 5.5 or 6) is administered to a subject in need thereof. In some embodiments, a protein described herein (e.g., a protein described in, for example, section 5.3 or 6) is administered to a subject in need thereof. In some embodiments, a fragment described herein (e.g., a fragment described in, for example, section 5.3 or 6) is administered to a subject in need thereof. In some embodiments, a mutant described herein (e.g., a mutant described in, for example, section 5.3 or 6) is administered to a subject in need thereof. In some embodiments, a fusion protein described herein (e.g., a fusion protein described in, for example, section 5.3 or 6) is administered to a subject in need thereof. In some embodiments, a pharmaceutical composition described herein (e.g., a pharmaceutical composition described in section 5.5.7) is administered to a subject in need thereof.

In some embodiments, the subject is a human. In some embodiments, the subject is a human adult. In some embodiments, the subject is a human at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, or at least 60 years old. In some embodiments, the subject is a human adult. In some embodiments, the subject is a human about 40 years old, about 41 years old, about 42 years old, about 43 years old, about 44 years old, or about 45 years old. In some embodiments, the subject is a human about 45 years old, about 46 years old, about 47 years old, about 48 years old, about 49 years old, or about 50 years old. In some embodiments, the subject is a human about 51 years old, about 52 years old, about 53 years old, about 54 years old, or about 55 years old. In some embodiments, the subject is a human about 56 years old, about 57 years old, about 58 years old, about 59 years old, or about 60 years old. In some embodiments, the subject is an elderly person. In some embodiments, the subject is a human infant. In some embodiments, the subject is a human infant. In some embodiments, the non-human mammal. In some embodiments, the subject (e.g., a human, such as, for example, a human adult or an elderly human) has a disorder that affects the immune system of the subject. In some embodiments, the subject (e.g., a human, such as, for example, a human adult or an elderly human) is immunodeficient or immunosuppressive. In some embodiments, a subject (e.g., a human, such as, for example, a human adult or an elderly human) is at risk of having shingles (herpes zoster) (or shingles). In some embodiments, the subject (e.g., a human, such as, for example, a human adult or an elderly human) has a latent VZV infection. In some embodiments, the subject (e.g., a human, such as, for example, a human adult or an elderly human) is susceptible to reactivation of a latent VZV infection.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject in need thereof.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a human subject. In some embodiments, the subject to whom the fragments, nucleic acids, or therapeutic nucleic acids described herein are administered, a vaccine composition comprising the fragments, nucleic acids, or therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, or a combination therapy described herein is an elderly person. In some embodiments, the subject to whom the fragments, nucleic acids, or therapeutic nucleic acids described herein are administered, a vaccine composition comprising the fragments, nucleic acids, or therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, or a combination therapy described herein is a human adult. In some embodiments, the subject to whom the fragments, nucleic acids, or therapeutic nucleic acids described herein are administered, a vaccine composition comprising the fragments, nucleic acids, or therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, or a combination therapy described herein is a human child. In some embodiments, the subject to whom the fragments, nucleic acids, or therapeutic nucleic acids described herein are administered, a vaccine composition comprising the fragments, nucleic acids, or therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, or a combination therapy described herein is a human pediatric. In some embodiments, the subject to whom the fragments, nucleic acids, or therapeutic nucleic acids described herein are administered, a vaccine composition comprising the fragments, nucleic acids, or therapeutic nucleic acids described herein, a lipid-containing composition (e.g., lipid nanoparticles) comprising the therapeutic nucleic acids described herein, or a combination therapy described herein is a human infant.

In some embodiments, the subject administered a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is a non-human mammal.

In some embodiments, the subject administered a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is a subject exhibiting at least one symptom associated with a VZV infection. In some embodiments, a subject receiving administration of a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein exhibits one or more symptoms of varicella, shingles, post-herpetic neuralgia (PHN), meningoemitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis, and pancreatitis.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy as described herein is administered to a subject without symptoms of VZV infection.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject at risk of or susceptible to VZV infection. In some embodiments, the subject at risk of or susceptible to VZV infection is elderly. In some embodiments, the subject at risk of or susceptible to VZV infection is a human adult. In some embodiments, the subject at risk of or susceptible to VZV infection is a human child. In some embodiments, the subject at risk of or susceptible to VZV infection is a human infant. In some embodiments, the subject at risk of or susceptible to VZV infection is a human infant. In some embodiments, the subject at risk of or susceptible to VZV infection is a human subject having an existing healthy condition affecting the immune system of the subject. In some embodiments, the subject at risk for or susceptible to VZV infection is a human subject having an existing healthy condition affecting a major organ of the subject. In some embodiments, the subject at risk for or susceptible to VZV infection is a human subject having an existing healthy condition affecting the lung function of the subject. In some embodiments, the subject at risk for or susceptible to VZV infection is an elderly subject having an existing healthy condition affecting the subject's immune system or major organs (such as lung function). In various embodiments described in this paragraph, the subject at risk for or susceptible to a VZV infection may be a subject exhibiting symptoms of a VZV infection or free of symptoms of a VZV infection.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject diagnosed with VZV infection positive. In some embodiments, the subject diagnosed as positive for a VZV infection is asymptomatic for a VZV infection, and the diagnosis is based on detecting the presence of viral nucleic acid or protein from a sample from the subject. In some embodiments, the diagnosis is based on clinical symptoms exhibited by the patient. Exemplary symptoms that may serve as a basis for diagnosis include, but are not limited to, upper respiratory tract infection, lower respiratory tract infection, lung infection, kidney infection, liver infection, intestinal infection, liver infection, nervous system infection, respiratory syndrome, pneumonia, gastroenteritis, encephalomyelitis, encephalitis, sarcoidosis, diarrhea, hepatitis, and demyelinating diseases. In some embodiments, diagnosis is based on a history of clinical symptoms exhibited by the subject in combination with the subject's contact with a geographic location, population, and/or individual believed to have a high risk of carrying VZV (such as contact with another individual diagnosed as positive for VZV infection).

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject who has not previously received administration of the fragment, the nucleic acid, or the therapeutic nucleic acid, the vaccine composition, the lipid-containing composition (e.g., a lipid nanoparticle), or the combination therapy.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject that has previously received administration of the fragment, the nucleic acid, or the therapeutic nucleic acid, the vaccine composition, the lipid-containing composition (e.g., a lipid nanoparticle), or the combination therapy. In particular embodiments, the subject has previously been administered a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy as described herein, once, twice, three times, or more.

In some embodiments, a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., a lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein is administered to a subject that has received therapy prior to administration of the fragment, the nucleic acid, or the therapeutic nucleic acid, the vaccine composition, the lipid-containing composition (e.g., a lipid nanoparticle), or the combination therapy. In some embodiments, a subject administered a fragment, nucleic acid, or therapeutic nucleic acid described herein, a vaccine composition comprising a fragment, nucleic acid, or therapeutic nucleic acid described herein, a lipid-containing composition (e.g., lipid nanoparticle) comprising a therapeutic nucleic acid described herein, or a combination therapy described herein experiences adverse side effects of a prior therapy or terminates a prior therapy due to unacceptable levels of toxicity to the subject.

5.6.4 Administration doses and frequency

The amount of fragment, nucleic acid or therapeutic nucleic acid or a combination thereof effective in controlling, preventing and/or treating an infectious disease will depend on the nature of the disease being treated, the route of administration, the general health of the subject, etc., and should be decided according to the discretion of the physician. Standard clinical techniques, such as in vitro assays, may optionally be employed to help identify optimal dosage ranges. However, suitable dosage ranges for therapeutic proteins or nucleic acids for administration as described herein are typically about 0.001mg, 0.005mg, 0.01mg, 0.05mg, 0.1mg, 0.5mg, 1.0mg, 2.0mg, 3.0mg, 4.0mg, 5.0mg, 10.0mg, 0.001mg to 10.0mg, 0.01mg to 1.0mg, 0.1mg to 1mg, and 0.1mg to 5.0mg. The therapeutic protein or nucleic acid or composition thereof may be administered to the subject as frequently as one, two, three, four or more times at intervals as desired. In some embodiments, a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6), a protein, fusion protein, fragment, or mutant described herein (e.g., a protein, fusion protein, fragment, or mutant described in chapter 5.3 or 6), a vector described herein (e.g., a vector described in chapter 5.4.10), a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), or a pharmaceutical composition described herein (e.g., in chapter 5.5.7) is administered to a subject at least once. In some embodiments, a nucleic acid described herein (e.g., a nucleic acid described in chapter 5.4 or 6), a protein, fusion protein, fragment, or mutant described herein (e.g., a protein, fusion protein, fragment, or mutant described in chapter 5.3 or 6), a vector described herein (e.g., a vector described in chapter 5.4.10), a nanoparticle composition described herein (e.g., a nanoparticle composition described in chapter 5.5 or 6), or a pharmaceutical composition described herein (e.g., in chapter 5.5.7) is administered to a subject twice. In some embodiments, the second administration is administered 1 to 2 months, 2 to 3 months, 2 to 6 months, 3 to 6 months, 6 to 12 months after the first administration. The effective dose can be inferred from dose response curves derived from in vitro or animal model test systems.

In certain embodiments, the therapeutic protein or nucleic acid or composition thereof is administered to the subject in a single dose followed by a second dose after 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 1 to 2 weeks. According to these embodiments, the subject may be administered a booster vaccination at intervals of 6 to 12 months after the second vaccination.

In certain embodiments, the therapeutic protein or nucleic acid or composition thereof may be repeatedly administered, and the administration may be at least 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days, 14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart. In other embodiments, the therapeutic protein or nucleic acid or composition thereof may be repeatedly administered, and the administration may be 1 to 14 days, 1 to 7 days, 7 to 14 days, 1 to 30 days, 15 to 45 days, 15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months, or 6 to 12 months apart. In some embodiments, a first therapeutic protein or nucleic acid or composition thereof is administered to a subject followed by administration of a second therapeutic protein or nucleic acid or composition thereof. In certain embodiments, the first and second therapeutic proteins or nucleic acids or compositions thereof may be spaced at least 1 day, 2 days, 3 days, 5 days, 6 days, 7 days, 10 days, 14 days, 15 days, 21 days, 28 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months apart. In other embodiments, the first and second therapeutic proteins or nucleic acids or compositions thereof may be spaced 1 to 14 days, 1 to 7 days, 7 to 14 days, 1 to 30 days, 15 to 45 days, 15 to 75 days, 15 to 90 days, 1 to 3 months, 3 to 6 months, 3 to 12 months, or 6 to 12 months apart.

In certain embodiments, a therapeutic protein or nucleic acid or composition thereof is administered to a subject in combination with one or more additional therapies (such as the therapies described in section 5.6.2). The dosage of the other additional therapy or therapies will depend on a variety of factors including, for example, the therapy, the nature of the infectious disease, the route of administration, the general health of the subject, etc., and should be determined at the discretion of the physician. In particular embodiments, the dose of the other therapy is the dose and/or frequency of administration of the therapy recommended for use as a single dose of therapy according to the methods disclosed herein. In other embodiments, the dosage of the other therapy is a lower dosage and/or less frequent administration of the therapy than recommended for the therapy used as a single agent according to the methods disclosed herein. Recommended dosages for approved therapies can be found in Physician' S DESK REFERENCE.

In certain embodiments, the therapeutic protein or nucleic acid or composition thereof is administered to the subject concurrently with one or more additional therapies. In other embodiments, the therapeutic protein or nucleic acid or composition thereof is administered to the subject every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 2 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks, and one or more additional therapies are administered every 3 to 7 days, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, or 1 to 2 weeks (as described in section 5.6.2). In certain embodiments, the therapeutic protein or nucleic acid or composition thereof is administered to the subject every 1-2 weeks, and one or more additional therapies are administered every 2-4 weeks (as described in section 5.6.2). In some embodiments, the therapeutic protein or nucleic acid or composition thereof is administered to the subject weekly, and one or more additional therapies (as described in section 5.6.2) are administered every 2 weeks.

5.7 Illustrative embodiments

5.7.1 Set 1

1. A fragment of the gE protein of VZV, wherein the fragment comprises a truncation of at least one and up to 49, 48, 47, 46, 45, 44, 43 or 42 amino acid residues from the C-terminus compared to the mature gE protein.

2. The fragment of embodiment 1, wherein the fragment comprises the substitution Y569A, positions numbered according to the full-length gE protein.

3. The fragment of embodiment 1 or 2, wherein the fragment comprises a truncation of up to 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus as compared to the mature gE protein.

4. The fragment of embodiment 3, wherein the fragment comprises the substitution Y582G, position numbered according to the full length gE protein.

5. The fragment of any one of embodiments 1 to 4, wherein the fragment comprises a truncation of up to 30 or 29 amino acid residues from the C-terminus compared to the mature gE protein.

6. The fragment of embodiment 5, wherein the fragment comprises the substitution S593A, positions numbered according to the full length gE protein.

7. The fragment of any one of embodiments 1 to 6, wherein the fragment comprises a truncation of up to 28 amino acid residues from the C-terminus compared to the mature gE protein.

8. The fragment of embodiment 7, wherein the fragment comprises the substitution S595A, positions numbered according to the full length gE protein.

9. The fragment of any one of embodiments 1 to 8, wherein the fragment comprises a truncation of up to 27 or 26 amino acid residues from the C-terminus compared to the mature gE protein.

10. The fragment of embodiment 9, wherein the fragment comprises the substitution T596A, positions numbered according to the full length gE protein.

11. The fragment of any one of embodiments 1 to 10, wherein the fragment comprises a truncation of up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues or up to 1 amino acid residue from the C-terminus compared to the mature gE protein.

12. The fragment of embodiment 11, wherein the fragment comprises the substitution T598A, positions numbered according to the full length gE protein.

13. The fragment of any one of embodiments 1 to 12, wherein the fragment comprises a truncation of at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acid residues from the C-terminus compared to the mature gE protein.

14. The fragment of any one of embodiments 1 to 12, wherein the fragment comprises a truncation of amino acid residues from the C-terminus 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44 compared to the mature gE protein.

15. The fragment of any one of embodiments 1 to 12, wherein the fragment comprises a truncation of 14 or 37 amino acid residues from the C-terminus compared to the mature gE protein.

16. The fragment of any one of embodiments 1 to 15, wherein the mature gE protein comprises the amino acid sequence set forth in SEQ ID No. 1 and/or the full length gE protein comprises the amino acid sequence set forth in SEQ ID No. 55.

17. The fragment of any one of embodiments 1 to 16, wherein the fragment comprises the amino acid sequence set forth in SEQ ID No. 3, 6, 8, 10 or 12, or an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 3, 6, 8, 10 or 12.

18. The fragment of any one of embodiments 1 to 17, wherein the N-terminus of the fragment is fused to the C-terminus of the signal peptide.

19. The fragment of any one of embodiments 1 to 18, wherein the N-terminus of the fragment is fused to a native signal peptide of the gE protein of VZV.

20. The fragment of embodiment 19, wherein the native signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 18.

21. The fragment of any one of embodiments 1 to 18, wherein the N-terminus of the fragment is fused to the C-terminus of a human tPA signal peptide.

22. The fragment of embodiment 21, wherein the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27.

23. The fragment of any one of embodiments 1 to 18, wherein the N-terminus of the fragment is fused to the C-terminus of a human IgE signal peptide.

24. The fragment of embodiment 23, wherein the human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 23.

25. A nucleic acid encoding the fragment of any one of embodiments 1 to 24.

26. The nucleic acid of embodiment 25, wherein the fragment is encoded by the nucleotide sequence set forth in SEQ ID No. 4, 5, 7, 9, 11 or 13 or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 4, 5, 7, 9, 11 or 13.

27. The nucleic acid of claim 25 or 26, wherein the natural signal peptide of the gE protein of VZV is encoded by the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22.

28. The nucleic acid of embodiment 25 or 26, wherein the human tPA signal peptide is encoded by the nucleotide sequence shown in SEQ ID No. 28, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID No. 28.

29. The nucleic acid of embodiment 25 or 26, wherein the human IgE signal peptide is encoded by the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26.

30. A non-naturally occurring nucleic acid comprising a coding nucleotide sequence encoding the fragment of any one of embodiments 1 to 24.

31. The non-naturally occurring nucleic acid of embodiment 30, wherein the coding nucleotide sequence has been codon optimized for expression in a cell of a subject, optionally wherein the subject is a non-human mammal or human.

32. The non-naturally occurring nucleic acid of embodiment 30 or 31, wherein the fragment is encoded by the nucleotide sequence set forth in SEQ ID No. 4, 5, 7, 9, 11 or 13 or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 4, 5, 7, 9, 11 or 13.

33. The non-naturally occurring nucleic acid of any of embodiments 30 or 32, wherein the natural signal peptide of the gE protein of VZV is encoded by the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22 or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 19, 20, 21 or 22.

34. The non-naturally occurring nucleic acid of any of embodiments 30 to 32, wherein the human tPA signal peptide is encoded by the nucleotide sequence shown in SEQ ID No. 28, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence shown in SEQ ID No. 28.

35. The non-naturally occurring nucleic acid of any one of claims 30 to 32, wherein the human IgE signal peptide is encoded by the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26.

36. The non-naturally occurring nucleic acid of any of embodiments 30 to 35,

The non-naturally occurring nucleic acid further comprises a 5' untranslated region (5 ' -UTR), wherein the 5' -UTR comprises the sequence shown in any one of SEQ ID NOs 29-38;

And/or

The non-naturally occurring nucleic acid further comprises a 3' untranslated region (3 ' -UTR), wherein the 3' -UTR comprises the sequence shown in any one of SEQ ID NOS 39-46,

Optionally, wherein the 3' -UTR further comprises a poly-a tail or a polyadenylation signal.

37. The non-naturally occurring nucleic acid of any of embodiments 30 to 36, wherein the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID No. 49, 50, 51, 52, 53, 54, 60, 61, 62, 63, or 64, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID No. 49, 50, 51, 52, 53, 54, 60, 61, 62, 63, or 64.

38. The non-naturally occurring nucleic acid of any of embodiments 30-37, wherein said nucleic acid is DNA or mRNA.

39. The non-naturally occurring nucleic acid of any of embodiments 30-38, wherein said nucleic acid comprises one or more functional nucleotide analogs.

40. The non-naturally occurring nucleic acid of embodiment 39, wherein said nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

41. The non-naturally occurring nucleic acid of any of embodiments 30 to 40, wherein said nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, or comprises a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the nucleotide sequence set forth in SEQ ID NO. 63, wherein all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU.

42. A vector comprising the nucleic acid of any one of embodiments 25 to 29 or the non-naturally occurring nucleic acid of any one of claims 30 to 41, preferably the vector is an IVT (in vitro transcription) plasmid.

43. A cell comprising the nucleic acid of any one of embodiments 25-29, the non-naturally occurring nucleic acid of any one of claims 30-41, or the vector of claim 42.

44. A pharmaceutical composition comprising a fragment according to any one of embodiments 1 to 24.

45. A pharmaceutical composition comprising the nucleic acid of any one of embodiments 25 to 29.

46. A pharmaceutical composition comprising the non-naturally occurring nucleic acid of any of embodiments 30-41 and at least a first lipid, optionally wherein the first lipid is a compound according to formula 01-I or formula 01-II, or a compound listed in table 01-1, or a compound according to formula 02-I, or a compound listed in table 02-1, or a compound according to formula 03-I, or a compound listed in table 03-1, or a compound according to formula 04-I, or a compound listed in table 04-1.

47. The pharmaceutical composition of embodiment 46, further comprising a second lipid, optionally wherein the second lipid is a compound according to formula 05-I.

48. The pharmaceutical composition of embodiment 46 or 47 formulated as lipid nanoparticles encapsulating nucleic acids in a lipid shell.

49. The pharmaceutical composition of any one of embodiments 46 to 48, wherein the composition is a vaccine.

50. A method for controlling, preventing or treating a disease or disorder caused by VZV or by infection with VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of the fragment of any one of embodiments 1-24, the nucleic acid of any one of claims 25-29, the non-naturally occurring nucleic acid of any one of claims 30-41, or the pharmaceutical composition of any one of embodiments 44-49.

51. The method of embodiment 50, wherein an immune response against VZV is elicited in the subject.

52. The method of embodiment 51, wherein the immune response comprises cytokine production in lymphocytes.

53. The method of embodiment 52, wherein the immune response comprises an increase in the proportion of lymphocytes expressing the cytokine.

54. The method of embodiment 53, wherein the lymphocytes are CD4 ⁺ T cells and/or CD8 ⁺ T cells, and/or wherein the cytokine is one or more of IFN- γ, IL-2, and TNF- α.

55. The method of embodiment 54, wherein cytokine production is increased in said lymphocytes.

56. The method of embodiment 55, wherein the immune response comprises the generation of antibodies that specifically bind to viral gE protein.

57. The method of any one of embodiments 50 to 56, wherein the disease or disorder caused by VZV is

(A) Varicella and/or shingles;

(b) Postherpetic neuralgia (PHN), and/or

(C) Meningoepitis, myelitis, cranial nerve paralysis, vascular disease, keratitis, retinopathy, ulcers, hepatitis and pancreatitis.

5.7.2 Set 2

1. A protein comprising a mutant of mature glycoprotein E (gE) of Varicella Zoster Virus (VZV), wherein the mutant comprises:

(a) (i) a truncation of 37 amino acid residues from the C-terminus of the mature gE, and (ii) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full length VSV gE;

(b) Amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full length VSV gE;

(c) Amino acid residue substitutions Y569A, Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue positions 569, 582, 593, 595, 596 and 598 are amino acid residue position numbers of full length VSV gE;

(d) Amino acid residue substitutions Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue positions 582, 593, 595, 596 and 598 are the amino acid residue position numbers of full length VSV gE, or

(E) (i) a truncation of 50 amino acid residues from the C-terminal end of the mature gE protein, and (ii) an amino acid residue substitution Y569A, wherein amino acid residue position 569 is the amino acid residue position number of full length VSV gE.

2. The protein of embodiment 1, wherein the mutant comprises the amino acid sequence of SEQ ID NO. 6, 8, 10, 12 or 3.

3. The protein of embodiment 1, wherein the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO. 6, 8, 10, 12 or 3.

4. The protein of embodiment 1, wherein the mutant comprises the amino acid sequence of SEQ ID NO. 6.

5. The protein of embodiment 1, wherein the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO. 6.

6. The protein of embodiment 1, wherein the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% identical to SEQ ID No. 6,8, 10 or 12.

7. The protein of embodiment 1, wherein the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID No. 6, 8, 10, or 12.

8. The protein of embodiment 1, wherein the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% identical to SEQ ID No. 6.

9. The protein of embodiment 1, wherein the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID No. 6.

10. The protein of any one of embodiments 1-9, further comprising a VZV gE signal peptide.

11. The protein of embodiment 10, wherein the signal peptide of VZV gE comprises the amino acid sequence set forth in SEQ ID NO. 18.

12. The protein of embodiment 11, wherein the signal peptide of VZV gE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID No. 18.

13. The protein of any one of embodiments 1-9, further comprising a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide.

14. The protein of embodiment 13, wherein the heterologous signal peptide is a human IgE signal peptide.

15. The protein of embodiment 14, wherein the human IgE signal peptide comprises (i) the amino acid sequence set forth in SEQ ID NO. 23, or (ii) an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence set forth in SEQ ID NO. 23.

16. The protein of embodiment 14, wherein the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO. 23.

17. The protein of embodiment 14, wherein the amino acid sequence of the mutant consists of the amino acid sequence set forth in SEQ ID NO. 6 and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence set forth in SEQ ID NO. 23.

18. The protein of embodiment 14 comprising the amino acid sequence set forth in SEQ ID NO. 59.

19. The protein of embodiment 14, wherein the amino acid sequence of the protein consists of the amino acid sequence set forth in SEQ ID NO. 59.

20. The protein of embodiment 13, wherein the heterologous signal peptide is a human tPA signal peptide.

21. The protein of embodiment 20, wherein the human tPA signal peptide comprises (i) an amino acid sequence set forth in SEQ ID NO:27, or (ii) an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence set forth in SEQ ID NO: 27.

22. The protein of any one of embodiments 1 to 21, wherein the mature gE comprises the amino acid sequence set forth in SEQ ID No.1 and/or the full length VZV gE comprises the amino acid sequence set forth in SEQ ID No. 55.

23. A fragment of a mature glycoprotein E (gE) of Varicella Zoster Virus (VZV), wherein the fragment comprises a truncation of at least one and up to 50, 49, 48, 47, 46, 45, 44, 43 or 42 amino acid residues from the C-terminus of the mature gE, optionally the truncation is a truncation of at least 2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48 or 49 amino acid residues from the C-terminus of the mature gE, or optionally the truncation is a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 amino acid residues from the C-terminus of the mature gE, or optionally the truncation is a truncation of 14 or 37 amino acid residues from the C-terminus of the mature gE.

24. The fragment of embodiment 23, wherein the fragment further comprises an amino acid residue substitution Y569A, and wherein amino acid residue position 569 is numbered according to the amino acid residue position of full-length VZV gE.

25. The fragment of embodiment 23 or 24, wherein the fragment comprises a truncation of up to 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, or 31 amino acid residues from the C-terminus of the mature gE.

26. The fragment of embodiment 25, wherein the fragment further comprises amino acid residue substitution Y582G, and wherein amino acid residue position 582 is numbered according to the amino acid residue position of the full-length VZV gE protein.

27. The fragment of any one of embodiments 23 to 26, wherein the fragment comprises a truncation of up to 30 or 29 amino acid residues from the C-terminus of the mature gE.

28. The fragment of embodiment 27, wherein the fragment further comprises amino acid residue substitution S593A, and wherein amino acid residue position 593 is numbered according to the amino acid residue position of full-length VZV gE.

29. The fragment of any one of embodiments 23 to 28, wherein the fragment comprises a truncation of up to 28 amino acid residues from the C-terminus of the mature gE.

30. The fragment of embodiment 29, wherein the fragment comprises amino acid residue substitution S595A, and wherein amino acid residue position 595 is numbered according to the amino acid residue position of full length VSV gE.

31. The fragment of any one of embodiments 23 to 30, wherein the fragment comprises a truncation of up to 27 or 26 amino acid residues from the C-terminus of the mature gE.

32. The fragment of embodiment 31, wherein the fragment further comprises an amino acid residue substitution T596A, and wherein amino acid residue position 596 is numbered according to the amino acid residue position of full-length VZV gE.

33. The fragment of any one of embodiments 23 to 32, wherein the fragment comprises a truncation of up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, or 2 amino acid residues or up to 1 amino acid residue from the C-terminus of the mature gE.

34. The fragment of embodiment 33, wherein the fragment further comprises an amino acid residue substitution T598A, and wherein amino acid residue position 598 is numbered according to the amino acid residue position of full-length VZV gE.

35. The fragment of embodiment 23, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of the mature gE.

36. The fragment of embodiment 23, wherein the fragment comprises (1) a truncation of 37 amino acid residues from the C-terminus of the mature gE, and (2) amino acid residue substitutions Y569A and Y582G, and wherein amino acid residue positions 569 and 582 are numbered according to the amino acid residue position of the full-length VZV gE.

37. The fragment of any one of embodiments 23 to 36, wherein the mature gE comprises the amino acid sequence set forth in SEQ ID No.1 and/or the full length VZV gE comprises the amino acid sequence set forth in SEQ ID No. 55.

38. The fragment of any one of embodiments 23 to 37, wherein the fragment comprises an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acid sequence set forth in SEQ ID No. 6, 3, 8, 10 or 12.

39. A fusion protein comprising the fragment of any one of embodiments 23 to 38 and a heterologous signal peptide, wherein the N-terminus of the fragment is fused to the C-terminus of the heterologous signal peptide.

40. The fusion protein of embodiment 39, wherein the heterologous signal peptide is a human tPA signal peptide.

41. The fusion protein of embodiment 40, wherein the human tPA signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 27.

42. The fusion protein of embodiment 39, wherein the heterologous signal peptide is a human IgE signal peptide.

43. The fusion protein of embodiment 42, wherein said human IgE signal peptide comprises the amino acid sequence set forth in SEQ ID NO. 23.

44. A fusion protein comprising a fragment of a mature glycoprotein (gE) of Varicella Zoster Virus (VZV) and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of the mature gE and the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide.

45. A nucleic acid encoding the protein of any one of embodiments 1 to 22, the fragment of any one of embodiments 23 to 38, or the fusion protein of any one of embodiments 39 to 44.

46. The nucleic acid of embodiment 45 comprising the nucleotide sequence set forth in SEQ ID No. 7, 9, 11, 13, 4, or 5, or a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the nucleotide sequence set forth in SEQ ID No. 7, 9, 11, 13, 4, or 5.

47. A nucleic acid encoding the protein of any one of embodiments 14 to 19 or the fusion protein of any one of embodiments 42 to 44, wherein the nucleotide sequence encoding the IgE signal peptide comprises (a) the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID No. 24, 25 or 26.

48. A nucleic acid encoding a protein according to any one of embodiments 10 to 12, wherein the nucleotide sequence of the signal peptide comprises (a) the nucleotide sequence set forth in SEQ ID NO:19, 20, 21 or 22, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO:19, 20, 21 or 22.

49. A nucleic acid encoding the protein of embodiment 20 or 21 or the fusion protein of embodiment 40 or 41, wherein the nucleotide sequence encoding a human tPA signal peptide comprises (a) the nucleotide sequence set forth in SEQ ID NO. 28, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO. 28.

50. A non-naturally occurring nucleic acid comprising a coding nucleotide sequence encoding the protein of any one of embodiments 1 to 22, the fragment of any one of embodiments 23 to 38, or the fusion protein of any one of embodiments 39 to 44.

51. The non-naturally occurring nucleic acid of embodiment 50, wherein said coding nucleotide sequence has been codon optimized for expression in a cell of a subject, optionally wherein said subject is a non-human mammal or human.

52. The non-naturally occurring nucleic acid of embodiment 50 or 51, comprising (a) a nucleotide sequence set forth in SEQ ID No. 7, 9, 11, 13, 4, or 5, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence set forth in SEQ ID No. 7, 9, 11, 13, 4, or 5.

53. A non-naturally occurring nucleic acid encoding the protein of any of embodiments 14 to 19 or the fusion protein of any of embodiments 42 to 44, wherein the nucleotide sequence encoding the IgE signal peptide comprises (a) the nucleotide sequence set forth in SEQ ID NO:24, 25 or 26, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence set forth in SEQ ID NO:24, 25 or 26.

54. A non-naturally occurring nucleic acid encoding the protein of any one of embodiments 10 to 12, wherein the nucleotide sequence of the signal peptide of the full length VSV gE protein comprises (a) the nucleotide sequence set forth in SEQ ID NO:19, 20, 21, or 22, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:19, 20, 21, or 22.

55. A non-naturally occurring nucleic acid encoding the protein of embodiment 20 or 21 or the fusion protein of embodiment 40 or 41, wherein the nucleotide sequence encoding a human tPA signal peptide comprises (a) the nucleotide sequence shown in SEQ ID NO:28, or (b) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO: 28.

56. A non-naturally occurring nucleic acid comprising the nucleotide sequence of SEQ ID No. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5.

57. The non-naturally occurring nucleic acid of embodiment 56, consisting of the nucleotide sequence of SEQ ID No. 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5.

58. The non-naturally occurring nucleic acid of any of embodiments 50 to 55, wherein the nucleic acid consists of, consists essentially of, or comprises (1) a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5, or (2) a nucleotide sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to a nucleotide sequence set forth in SEQ ID NO 63, 7, 51, 60, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5.

59. The non-naturally occurring nucleic acid of any of embodiments 50 to 58,

The non-naturally occurring nucleic acid further comprises a 5' untranslated region (5 ' -UTR), wherein the 5' -UTR comprises the nucleotide sequence depicted in any one of SEQ ID NOs 29-38;

And/or the non-naturally occurring nucleic acid further comprises a 3' untranslated region (3 ' -UTR), wherein the 3' -UTR comprises the nucleotide sequence depicted in any one of SEQ ID NOS 39-46,

60. The non-naturally occurring nucleic acid of any one of embodiments 50-59, wherein said nucleic acid is DNA.

61. The non-naturally occurring nucleic acid of any one of embodiments 50-60, wherein said nucleic acid comprises one or more functional nucleotide analogs.

62. The non-naturally occurring nucleic acid of any one of claims 50 to 59, wherein the nucleic acid is mRNA, and wherein thymine is replaced by uracil or a functional analogue thereof in the nucleic acid.

63. The non-naturally occurring nucleic acid of embodiment 62, wherein said nucleic acid comprises one or more functional nucleotide analogs selected from the group consisting of pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

64. The non-naturally occurring nucleic acid of embodiment 62, wherein said nucleic acid consists of, consists essentially of, or comprises (1) the nucleotide sequence set forth in SEQ ID NO:63, or (2) a nucleotide sequence at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence set forth in SEQ ID NO:63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine, and/or the first nucleotide G is substituted with m ⁷ GpppAmpU.

65. The non-naturally occurring nucleic acid of embodiment 62, wherein said nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is substituted with uracil (U) or N1-methyl pseudouridine and/or the first nucleotide G is substituted with m ⁷ GpppAmpU.

66. The non-naturally occurring nucleic acid of embodiment 62, wherein said nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence set forth in SEQ ID NO. 63, except that all thymine (T) is replaced with N1-methyl pseudouridine and the first nucleotide G is replaced with m ⁷ GpppAmpU.

67. A vector comprising the nucleic acid of any one of embodiments 45 to 49 or the non-naturally occurring nucleic acid of any one of embodiments 50 to 66, preferably the vector is an IVT (in vitro transcription) plasmid.

68. A host cell comprising the nucleic acid of any one of embodiments 45 to 49, the non-naturally occurring nucleic acid of any one of embodiments 50 to 66, or the vector of embodiment 67.

69. The host cell of embodiment 68, wherein said host cell is in vitro, ex vivo, or isolated.

70. A pharmaceutical composition comprising the protein of any one of embodiments 1 to 22, the fragment of any one of embodiments 23 to 38, or the fusion protein of any one of embodiments 39 to 44.

71. A pharmaceutical combination comprising the nucleic acid of any one of embodiments 45 to 49, the non-naturally occurring nucleic acid of any one of embodiments 50 to 66, or the vector of embodiment 67.

72. A pharmaceutical composition comprising the non-naturally occurring nucleic acid of any of embodiments 50-66 and at least a first lipid, optionally wherein the first lipid is a compound according to formula 01-I or formula 01-II, or a compound listed in table 01-1, or a compound according to formula 02-I, or a compound listed in table 02-1, or a compound according to formula 03-I, or a compound listed in table 03-1, or a compound according to formula 04-I, or a compound listed in table 04-1.

73. The pharmaceutical composition of embodiment 72, further comprising a second lipid, optionally wherein the second lipid is a compound according to formula 05-I.

74. The pharmaceutical composition of embodiment 72 or 73 formulated as a lipid nanoparticle encapsulating a nucleic acid in a lipid shell.

75. The pharmaceutical composition of any one of embodiments 70-74, wherein the composition is a vaccine.

76. A method for controlling, preventing or treating a disease or disorder caused by VZV or by infection of VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of the protein of any one of embodiments 1-22, the fragment of any one of embodiments 23-38, the fusion protein of any one of embodiments 39-44, the nucleic acid of any one of embodiments 45-49, the non-naturally occurring nucleic acid of any one of embodiments 50-66, the vector of embodiment 67, or the pharmaceutical composition of any one of embodiments 70-75.

77. The method of embodiment 76, wherein the method is for preventing a disease or disorder in a subject caused by VZV or by infection with VZV.

78. The method of embodiment 76 or 77, wherein an immune response is elicited in the subject against VZV.

79. The method of embodiment 78, wherein the immune response comprises cytokine production in lymphocytes.

80. The method of embodiment 78 or 79, wherein the immune response comprises an increase in the proportion of lymphocytes expressing the cytokine.

81. The method of embodiment 79 or 80, wherein the lymphocyte is a CD4 ⁺ T cell and/or a CD8 ⁺ T cell, and/or wherein the cytokine is one or more of IFN- γ, IL-2, and TNF- α.

82. The method of any one of embodiments 79 to 81, wherein cytokine production in lymphocytes is increased.

83. The method of any one of embodiments 78 to 82, wherein the immune response comprises production of an antibody that specifically binds to VZV gE.

84. The method of any one of embodiments 76 to 83, wherein the disease or disorder caused by VZV is

(A) Varicella and/or shingles;

(b) Postherpetic neuralgia (PHN), and/or

85. The method of any one of embodiments 76 to 84, wherein the subject is a human.

86. The method of embodiment 85, wherein the human is a human adult.

87. The method of embodiment 86, wherein the adult is at least 40 years old.

88. The method of embodiment 85, wherein the human is an elderly human.

89. A protein as defined in any one of embodiments 1 to 22, a fragment as defined in any one of embodiments 23 to 38, a fusion protein as defined in any one of embodiments 39 to 44, a nucleic acid as defined in any one of embodiments 45 to 49, a non-naturally occurring nucleic acid as defined in any one of embodiments 50 to 66, a vector as defined in embodiment 67, or a pharmaceutical composition as defined in any one of embodiments 70 to 75 for use in a method of controlling, preventing or treating a disease or disorder caused by VZV or by infection of VZV in a subject.

90. The protein, fragment, fusion protein, nucleic acid, non-naturally occurring nucleic acid, vector, or pharmaceutical composition for use according to embodiment 89, wherein the subject is a human, optionally a human adult or an elderly human.

91. Use of a protein as defined in any one of embodiments 1 to 22, a fragment as defined in any one of embodiments 23 to 38, a fusion protein as defined in any one of embodiments 39 to 44, a nucleic acid as defined in any one of embodiments 45 to 49, a non-naturally occurring nucleic acid as defined in any one of embodiments 50 to 66, a vector as defined in embodiment 67, or a pharmaceutical composition as defined in any one of embodiments 70 to 75, for the manufacture of a medicament for controlling, preventing or treating a disease or disorder caused by VZV or by infection of VZV in a subject.

92. The use of embodiment 91, wherein the subject is a human, optionally an adult or an elderly human.

6. Examples

The embodiments in this section are provided by way of illustration and not limitation.

The following examples are provided to illustrate the preparation of cationic lipid series 01 to series 04.

HPLC purification is typically performed on a Waters2767 equipped with a Diode Array Detector (DAD), on a INERTSIL PRE-C8 OBD column, typically using water with 0.1% TFA as solvent A and acetonitrile as solvent B.

LCMS analysis was performed on a Shimadzu (LC-MS 2020) system. Chromatography is performed on SunFire C18, typically using water with 0.1% formic acid as solvent a and acetonitrile with 0.1% formic acid as solvent B.

Preparation example 01 preparation of a compound of series 01.

Please refer to international patent application publication No. WO 2021/204175, which is incorporated by reference in its entirety.

Preparation example 02-1 preparation of Compound 02-1 (i.e., compound 1 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.90(m,12H),1.27-1.63(m,53H),1.97-2.01(m,2H),2.28-2.64(m,14H),3.52-3.58(m,2H),4.00-4.10(m,8H).LCMS:Rt:1.080min;MS m/z(ESI):826.0[M+H]⁺.

The following compounds were prepared in a similar manner to compound 02-1 using the corresponding starting materials.

Preparation example 02-2 preparation of Compound 02-2 (i.e., compound 2 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.90(m,12H),1.28-1.67(m,54H),1.88-2.01(m,7H),2.28-2.56(m,18H),3.16-3.20(m,1H),3.52-3.54(m,2H),4.00-4.10(m,8H).LCMS:Rt:1.060min;MS m/z(ESI):923.0[M+H]⁺.

Preparation example 02-3 preparation of Compound 02-4 (i.e., compound 4 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.90(m,9H),1.26-1.32(m,34H),1.41-1.49(m,4H),1.61-1.66(m,15H),2.00-2.03(m,1H),2.21-2.38(m,8H),2.43-2.47(m,4H),2.56-2.60(m,2H),3.50-3.54(m,2H),4.03-4.14(m,8H).LCMS:Rt:1.030min;MS m/z(ESI):798.0[M+H]⁺.

Preparation example 02-4 preparation of Compound 02-9 (i.e., compound 9 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.90(m,12H),1.28-1.30(m,33H),1.58-2.01(m,18H),2.30-2.54(m,18H),3.10-3.19(m,1H),3.52-3.68(m,8H),4.09-4.20(m,8H).LCMS:Rt:1.677min;MS m/z(ESI):927.7[M+H]⁺.

The following compounds were prepared in a similar manner to compounds 02-9 using the corresponding starting materials.

Preparation example 02-5 preparation of Compounds 02-10 (i.e., compound 10 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.90(m,12H),1.26-1.41(m,48H),1.51-1.72(m,11H),1.94-2.03(m,1H),2.29-2.32(m,6H),2.41-2.91(m,5H),3.51-3.76(m,2H),3.96-4.10(m,6H).LCMS:Rt:1.327min;MS m/z(ESI):782.6[M+H]⁺.

The following compounds were prepared in a similar manner to compounds 02-10 using the corresponding starting materials.

Preparation example 02-6 preparation of Compounds 02-12 (i.e., compound 12 in the scheme below).

¹H NMR(400MHz,CDCl₃)δ:0.86-0.89(m,18H),1.25-1.35(m,53H),1.41-1.48(m,8H),1.56-1.61(m,20H),1.95-2.01(m,2H),2.28-2.35(m,6H),2.43-2.46(m,4H),2.56-2.58(m,2H),3.51-3.54(m,2H),4.00-4.10(m,8H).LCMS:Rt:0.080min;MS m/z(ESI):1050.8[M+H]⁺.

Preparation examples 02-7 preparation of Compounds 02-20 (i.e., compound 20 in the scheme below).

Compound 20

¹HNMR(400MHz,CDCl3)δ：0.86-0.90(m,9H),1.25-1.36(m,48H),1.41-1.48(m,5H),1.60-1.62(m,8H),1.97-2.00(m,1H),2.27-2.32(m,6H),2.43-2.46(m,4H),2.56-2.59(m,2H),3.52-3.54(m,2H),4.01-4.10(m,6H).LCMS：Rt:0.093min;MS m/z(ESI):782.6[M+H]⁺.

Preparation example 03 preparation of the compounds of series 03.

Please refer to international patent application publication No. WO 2022/152109, which is incorporated by reference in its entirety.

Preparation example 04 preparation of the compounds of series 04.

Please refer to international patent application publication No. WO 2022/247755, which is incorporated by reference in its entirety.

Example 05 preparation and characterization of lipid nanoparticles

Briefly, cationic lipids (e.g., compound 01-1), DSPC, cholesterol, and PEG-lipids were dissolved in ethanol at molar ratios as described herein, and mRNA was diluted in 10 to 50mM citrate buffer (ph=4). LNP was prepared at a total lipid to mRNA weight ratio of about 10:1 to 30:1 by mixing lipid ethanol solution with mRNA aqueous solution at a volume ratio of 1:3 using a microfluidic device with a total flow rate ranging from 9-30 mL/min. Ethanol was thereby removed using dialysis and replaced with DPBS. Finally, the lipid nanoparticles were filtered through a 0.2 μm sterile filter.

Lipid nanoparticle size was determined by dynamic light scattering using Malvern Zetasizer Nano ZS (Malvern UK) using a 173 ° back scattering detection mode. The encapsulation efficiency of lipid nanoparticles was determined using the Quant-itRibogreen RNA quantitative assay kit (Thermo FISHER SCIENTIFIC, UK) according to the manufacturer's instructions.

As reported in the literature, the apparent pKa of an LNP formulation correlates with the efficiency of LNP delivery to nucleic acids in vivo. The apparent pKa of each formulation was determined using a fluorescence-based assay for 2- (p-toluidinyl) -6-naphthalene sulfonic acid (TNS). LNP formulations comprising cationic lipid/DSPC/cholesterol/DMG-PEG (50/10/38.5/1.5 mol%) in PBS were prepared as described above. TNS was prepared as a 300uM stock solution in distilled water. The LNP formulation was diluted to 0.1mg/mL total lipid in 3mL of buffer solution containing 50mM sodium citrate, 50mM sodium phosphate, 50mM sodium borate, and 30mM sodium chloride, wherein the pH range was 3 to 9. An aliquot of TNS solution was added to give a final concentration of 0.1mg/ml and after vortexing the fluorescence intensity was measured in a Molecular Devices Spectramax iD spectrometer at room temperature using an excitation wavelength of 325nm and an emission wavelength of 435 nm. The sigmoid curve best fit analysis was applied to the fluorescence data and the pKa value was measured as the pH at which half maximum fluorescence intensity was produced.

Example B1 mRNA synthesis and purification.

DNA linearization. Target sequences (e.g., SEQ ID NO:4, 5, 7, 9, 11, or 13), 5'-UTR (e.g., any of SEQ ID NO: 29-38), 3' -UTR (e.g., any of SEQ ID NO: 39-46) and IVT plasmid pJ241 (internally constructed, containing kanamycin resistance gene, T7 promoter sequence, poly (A) tag and unique type IIS restriction sites downstream of poly (A) sequence) encoding VZV gE proteins were linearized using type IIS restriction enzyme digestion (complete sequences of constructs are shown in Table 5). Each 10. Mu.g of plasmid was mixed with 10U Esp I/BsmBI and incubated at 37℃for 4 hours to ensure complete linearization. The reaction was quenched by the addition of 1/10 volume of 3M sodium acetate (pH 5.5) and 2.5 volumes of ethanol, thoroughly mixed and cooled at-20℃for 1 hour. Linearized DNA was precipitated by centrifugation at 13800g for 15 min at 4 ℃ and washed twice with 70% ethanol and resuspended in nuclease free H ₂ O.

In vitro transcription of mRNA. The contents of a typical 20 μl reaction mixture are shown in the table below:

Nuclease-free H ₂ O	Make up to 20 mu L
		RNase inhibitor (40U/. Mu.L)	0.5μL
RNTP mixture (100 mM each)	8 ΜL (final 10mM each)
		10XIVT reaction buffer	2μL
1MMgCl₂	0.8μL
		0.1MDTT	2μL
100U/mL inorganic pyrophosphatase	0.8μL
		100mMNaCl	1μL
Linearizing DNA	1μg
		T7RNA polymerase (50U/. Mu.L)	2μL

* Replacement of UTP with pseudo-UTP and N1-methyl-pseudo-UTP to synthesize modified mRNA.

The reaction mixture was incubated at 37℃for 6 hours, then 1. Mu.l DNase I (no RNase, 1U/. Mu.L) was added to remove the DNA template and incubated at 37℃for 30 minutes. The synthesized RNA was purified by adding 0.5 volume of 7.5M LiCl, 50mM EDTA and incubating at-20℃for 45 minutes, followed by centrifugation at 13800g at 4℃for 15 minutes to precipitate mRNA. The supernatant was then removed and the pellet was washed twice with 500 μl ice cold 70% ethanol, the mRNA was resuspended in nuclease free H ₂ O, adjusted to a concentration of 1mg/mL, and stored at-20 ℃.

MRNA capping. 10 μg of each uncapped mRNA was heated at 65℃for 10 min, placed on ice for 5min, and mixed with 10U vaccinia capping enzyme, 50U mRNA Cap 2' -O-methyltransferase, 0.2mM SAM, 0.5mM GTP, and 1U RNase inhibitor, and incubated at 37℃for 60 min to produce Cap1 modified structure. Modified mRNA was precipitated by LiCl as described previously and RNA was resuspended in nuclease free H ₂ O and stored at-20 ℃.

And (5) HPLC purification. RNA was purified by High Performance Liquid Chromatography (HPLC) using a C4 column (5 μm) (10 mm. Times.250 mm column). Buffer a contained 0.1M triethylammonium acetate (TEAA) (ph=7.0) and buffer B contained 0.1M TEAA (ph=7.0) and 25% acetonitrile.

As expected, mRNA molecules were successfully produced by the in vitro transcription and maturation processes described above, and purified from the reaction system using HPLC.

Example B2 in vitro transfection and antigen expression analysis.

The different mRNA molecules encoding mutants of VZV gE protein produced in example B1 were transfected into expression cell lines such as HEK293T culture cells to assess the in vitro expression efficiency of the mRNA molecules.

Expression analysis by FACS.

For assembly of the mRNA-lipid complex, the mRNA-lipid complex is assembled in two separate phasesMu.L of each was added to a 1.5mL microcentrifuge tube (Corning, #MCT-150-C-S)2000 (Invitrogen, # 11668019) and 141 μ LOpti-MEM ^TM I reduced serum medium (Gibco, # 11058021), as well as 9 μ gmRNA (or equal volumes of Opti-MEM ^TM I reduced serum medium for "Lipo only" blank) and Opti-MEM ^TM I reduced serum medium (added to 150 μL total). The contents of the two tubes were mixed together and incubated for 5 minutes at room temperature. 100 microliters of the resulting complex was added toEach well of a 6-well transparent TC-treated plate (Corning, # 3516) containing HEK293T cells grown to 80% confluency and 3mL fresh Opti-MEM ^TM I minus serum medium. Plates were incubated at 37 ℃ for 24 hours in a humidified 5% co ₂ incubator.

The cultures were transferred to 15mL centrifuge tubes (Thermo, # 339650) and centrifuged at 200 Xg for 5 min at room temperature. Cells were resuspended in PBS/1% BSA at a density of 1X 10 ⁶ cells/mL. 100 microliters of cell suspension was added toEach well of a 96-well transparent round bottom microwell plate (Corning, # 3788). Microplates were centrifuged at 300 Xg for 5 minutes at 4℃and the supernatant discarded. 100 microliters of a first antibody against the VZV gE protein (abcam, # Ab 272686) diluted to 10 μg/mL or 1 μg/mL in PBS/1% BSA was added. Microplates were incubated in the dark at 4 ℃ for 1 hour. Then, the microplate was centrifuged at 300×g at 4 ℃ for 5 minutes, and the supernatant was discarded. Cells were washed twice with 180. Mu. LPBS/1% BSA each. 100 microliters of a 1:100 dilution of secondary antibody goat anti-mouse IgG antibody (FITC) in PBS/1% BSA was added (Jackson, # 115-095-003). Microwell plates were incubated in the dark at 4 ℃ for 30 minutes. The microwell plates were then centrifuged at 300 Xg for 5 minutes at 4℃and the supernatant discarded. Cells were washed twice with 180. Mu. LPBS/1% BSA each. Cells were then resuspended with 100 μl PBS/1% bsa and prepared for FACS (BD, fortess).

As shown in FIG. 1, in vitro transcribed mRNA molecules encoding mutants of the VZV gE protein were efficiently transfected into HEK293T cells. Transfected HEK293T cells express the encoded viral antigen on the cell membrane at a higher level than the wild-type gE protein.

EXAMPLE B3 preparation of mRNA-LNP vaccine

An mRNA-LNP-containing vaccine was prepared according to the procedure provided in example 05 above, wherein lipids were prepared according to the procedure provided in examples 01 to 04 above, and mRNA was prepared according to the procedure provided in example B1 above.

EXAMPLE B4 antigen immunogenicity analysis

The purpose of the following experiments was to evaluate the immunogenicity of mutants of the VZV gE protein expressed by the gE-mRNA-LNP of the invention.

Female C57BL/6 mice (LINGCHANG SYSTEMS, shanghai, china) of 6-8 weeks of age and body weight ranging from 17.5-21.0g were randomly assigned to each group on day 0 with minimal average body weight change between groups (10 mice per group). Mice were vaccinated on days 0, 21 and 42 by intramuscular injection of 50 μl of LNP formulation containing 0.03 μg/μl mRNA encoding the VZV gE protein mutant. Group 1 (shintrix (GSK) positive control) was vaccinated on day 0 and day 21 and PBS was used as placebo. Blood samples were collected on days 20, 35, 65 (as before vaccination where applicable) and spleens were collected from half of each group on day 35 for example B5.

Serum samples were collected from mice on days 20, 35 and 65 for use in determining gE-specific IgG titers by ELISA. In this experiment, 100 μl of coating solution (0.1 μg/ml VZV gE protein in PBS) (Bionintron, #b 483001) was added to each well of a 96-well microplate (Costar, # 42592), sealed and incubated at4 ℃ for 16 hours. After three washes with 0.5% pbst, 200 μl of blocking solution (5% bsa) was added to each well, sealed and incubated at 37 ℃ for 1 hour. After three washes, 100 μl of diluted serum sample (4-fold serial dilution in 1% bsa) or control was added to each well, gently shaken to mix well, sealed and incubated at 37 ℃ for 1 hour. After three washes, 100 μl of secondary antibody solution (peroxidase conjugated affinipure donkey anti-mouse IgG, jackson, # 715-035-151) was added to each well, gently shaken to mix well, sealed and incubated at 37 ℃ for 1 hour. After washing three times, 100 μl of a developer solution (TMB substrate, solarbio, #pr 1200) was added to each well, gently shaken to mix well, and developed for 3 minutes at room temperature in the dark. After three washes, 100 μl of stop solution (Solarbio, #c1058) was added to each well and gently shaken to mix well. The results were measured within 10 minutes. The wavelength of the microplate reader (SpectraMax ID5, molecular Devices) was set at 450nm. The maximum dilution factor that detected as positive was selected, and the titer result was the OD value of the maximum positive dilution factor/0.1 x the corresponding dilution factor.

As shown in FIG. 2, all candidates stimulated strong humoral responses, and 115S-5 induced the highest antibody titers among the drug candidates and Sringrix (GSK) after each immunization.

Example B5. cytokine production in mice vaccinated with mRNA-LNP vaccine

Individual spleen cells were prepared from spleen and resuspended in complete medium (RMPI 1640 with 2% fbs, gibco, #22400-089, # 10099141C) at 3×10 ⁷ cells/mL. mu.L of spleen cell suspension was added to each well of a 96-well culture plate (Corning, # 3788). Then, 100. Mu.L of complete medium containing each peptide in the 113 15-mer gE peptide library at a final concentration of 2.5. Mu.g/mL, 2. Mu.g/mL of anti-mCD 28 antibody (bioleged, cat# 102102) and 2. Mu.g/mL of anti-mCD 49d antibody (BD, cat# 553153) was added for stimulation. Plates were incubated at 37 ℃ for 2 hours in a 5% co ₂ incubator, then brefeldin a (Biolegend, cat# 420601) was added to a final concentration of 10 μg/mL. Plates were incubated for an additional 4 hours. For positive control, 4 μ LPHARMINGEN ^TM leukocyte activation mix (BD Biosciences, # 550583) was added for stimulation and the plates were incubated in a 5% co ₂ incubator for 6 hours at 37 ℃.

Stimulated cells were washed with FACS buffer (PBS with 2% fbs, # 10099141C) and resuspended in 100 μl of antibody mixture per well.

For cell surface staining, 100. Mu.L of antibody mixtures (Zombie green fixable viability kit, biolegend, cat#423112; BV 510-labeled anti-mCD 45, biolegend, cat#103138;AlexaFluor 700-labeled anti-mCD 3, biolegend, cat#100216; perCP/Cyanine 5.5-labeled anti-mCD 4, biolegend, cat#100434; APC/Cyanine 7-labeled anti-mCD 8, biolegend, cat#100714; all 1:50 dilutions) were added to each well and the cells were incubated in the dark for 30min at 4 ℃. Cells were then washed twice with 200 μ LFACS buffer per well.

For intracellular staining, cells were resuspended in 200. Mu.L of 1 Xfixing/permeabilizing buffer (Thermo Fisher, # 88-8824-00) and incubated in the dark at room temperature for 30 min. The cells were washed 2 times with 2mL of each 1 Xpermeabilization buffer (Thermo Fisher, # 88-8824-00). mu.L of antibody mixture (PE-labeled anti-mIFN-. Gamma., biolegend, cat# 505508; APC-labeled anti-mIL-2, biolegend, cat#503810; PE/Cyanine 7-labeled anti-mIL-4, biolegend, cat#504118; BV 421-labeled anti-mTNF-. Alpha., biolegend, cat#506328; all 1:50 dilutions) was added and the cells were incubated for 30 minutes at room temperature in the dark. Then, the cells were washed twice with 200. Mu.L of FACS buffer each. Cells were resuspended in 200 μ LFACS buffer for data acquisition using BD Fortessa flow cytometer.

The ratio of CD4+ and CD8+ cells expressing IL-2, IL-4, TNF- α or IFN- γ in the data of the stained samples was analyzed using Flowjo software. Analysis showed that mRNA vaccines induced IL-2, TNF- α and IFN- γ expression above Shingrix, with gE-115S-1 and gE-115S-5 showing the highest CD4+ T cell responses. The frequency of gE-specific CD8 ⁺ T cells was generally lower than that of CD4 ⁺ T cells. However, some gE-mRNA-LNP vaccines (e.g., gE-115S-1-1, gE-115S-5) induced a higher CD8+ T cell response than other candidate vaccines and Shingrix. Few IL-4 secreting T cells were detected, indicating that mRNA vaccines induced a strong Th 1-based T cell response.

Example B6. screening of codon optimized mRNA sequences by in vitro expression

Different codon optimized mRNA molecules encoding VZV gE variant proteins were transfected into expression cell lines such as HEK293T culture cells to assess expression efficiency in vitro.

Expression analysis by FACS

For assembly of the mRNA-lipid complex, the mRNA-lipid complex is assembled in two separate phasesMu.L of each was added to a 1.5mL microcentrifuge tube (Corning, #MCT-150-C-S)2000 (Invitrogen, # 11668019) and 141 μ LOpti-MEM ^TM I reduced serum medium (Gibco, # 11058021), as well as 9 μ gmRNA (or equal volumes of Opti-MEM ^TM I reduced serum medium for "Lipo only" blank) and Opti-MEM ^TM I reduced serum medium (added to 150 μL total). The contents of the two tubes were mixed together and incubated for 5 minutes at room temperature. 100. Mu.L of the resulting complex was added toEach well of a 6-well transparent TC-treated plate (Corning, # 3516) containing HEK293T cells grown to 80% confluency and 3mL fresh Opti-MEM ^TM I minus serum medium. Plates were incubated at 37 ℃ for 24 hours in a humidified 5% co ₂ incubator.

The cultures were transferred to 1.5mL microcentrifuge tubes (Corning, #MCT-150-C-S) and centrifuged at 200Xg for 5 minutes at room temperature. Cells were resuspended in PBS/1% BSA at a density of 1X10 ⁶ cells/mL. 100. Mu.L of the cell suspension was added toEach well of a 96-well transparent round bottom microwell plate (Corning, # 3788). Microplates were centrifuged at 300xg for 5 minutes at 4 ℃ and the supernatant discarded. mu.L of a first antibody against the VZV gE protein (abcam, # Ab 272686) diluted to 10. Mu.g/mL or 1. Mu.g/mL in PBS/1% BSA was added. Microplates were incubated in the dark at 4 ℃ for 1 hour. The microwell plates were then centrifuged at 300Xg for 5 minutes at 4℃and the supernatant discarded. Cells were washed twice with 180. Mu.L each of PBS/1% BSA. mu.L of a goat anti-mouse IgG antibody (FITC) as a secondary antibody diluted 1:100 in PBS/1% BSA was added (Jackson, # 115-095-003). Microwell plates were incubated in the dark at 4 ℃ for 30 minutes. The microwell plates were then centrifuged at 300Xg for 5 minutes at 4℃and the supernatant discarded. Cells were washed twice with 180. Mu. LPBS/1% BSA each. Cells were then resuspended with 100 μ LPBS/1% BSA and prepared for FACS (BD, aria).

As shown in fig. 5 and 6, mRNA molecules encoding VZV gE proteins were efficiently transfected into HEK293T cells. All optimized mRNA sequences translated equivalent target proteins.

Example B7. selection of codon optimized nucleic acid sequences by immunogenicity

The purpose of the following experiments was to evaluate the immunogenicity of the different gE-mRNA sequences encapsulated by LNP.

On day 0, 6-8 week old female C57BL/6 mice were randomly assigned to each group with minimal average weight change between groups (5 mice per group). Mice were vaccinated on days 0 and 21 by intramuscular injection of 50 μl of LNP formulation containing 0.01 μg/μl mRNA encoding VZV gE protein. Shangrix (GSK) served as positive control at a dose of 1.7 μg and PBS served as placebo for vaccination. 115S molecule is a wild-type gE protein and 115S-5 has a similar nucleic acid sequence to 115S, but with individual base mutations. Blood samples were collected on days 20 and 29 and spleens were collected on day 35 to analyze T cell responses.

1. Candidate-induced gE-specific IgG titers

Serum samples were collected on days 20 and 29 to analyze gE-specific IgG titers by ELISA. 100. Mu.L of coating solution (0.1. Mu.g/ml VZV gE protein) (Bionintron #B 483001) was added to each well of a microplate (Costar, # 42592), sealed and incubated at 4℃for 16 hours. After three washes, 200 μl of blocking solution (5% bsa) was added to each well, sealed and incubated at 37 ℃ for 1 hour. After three washes, 100 μl of diluted sample (4-fold serial dilution in 1% bsa) or control was added to each well, gently shaken to mix well, sealed and incubated at 37 ℃ for 1 hour. After three washes, 100 μl of secondary antibody solution (peroxidase conjugated affinipure donkey anti-mouse IgG, jackson, # 715-035-151) was added to each well, gently shaken to mix well, sealed and incubated at 37 ℃ for 1 hour. After washing three times, 100 μl of TMB substrate (Solarbio, #pr1200) was added to each well, gently shaken to mix well, and incubated in the dark for 3 minutes at room temperature. After three washes, 100 μl of stop solution (Solarbio, #c1058) was added to each well and gently shaken to mix well. The results were measured within 10 minutes. The wavelength of the microplate reader (SpectraMax ID5, molecular Devices) was set at 450nm. The maximum dilution factor that detected as positive was selected, and the titer result was the OD value of the maximum positive dilution factor/0.1 x the corresponding dilution factor.

As shown in FIG. 7, all candidates were able to stimulate strong humoral responses, and the titer induced by 115S-5-5 was the highest of all candidates.

2. Specific T cell response induced by candidate

Spleen cells were prepared from spleen and resuspended in complete medium (RMPI 1640, gibco, # 22400-089) at 2x10 ⁷ cells/mL. mu.L of spleen cell suspension was added to each well of a 96-well culture plate (Corning, # 3788). Then, 100. Mu.L of complete medium containing 2.5. Mu.g/mL of a library of 113 15 mer peptides, 2. Mu.g/mL of anti-mCD 28 antibody (bioleged, cat# 102102) and 2. Mu.g/mL of anti-mCD 49d antibody (BD, cat# 553153) was added for stimulation, and the plates were incubated in a 5% CO ₂ incubator for 2 hours at 37 ℃. Brefeldin a (Biolegend, cat# 420601) was added to 10 μg/mL and the plates were incubated for an additional 4 hours. The positive control was Pharmingen ^TM leukocyte activation mix (BD Biosciences, # 550583). Stimulated cells were pelleted and resuspended in 3 mLPBS. One to two million living cells were transferred into the flowtube.

For cell surface staining, 100. Mu.L of antibody mixture (ef 780 labeled live/dead marker, eBiosciences, cat#65-0865-18; perCP/CY5.5 labeled anti-mCD 45 rat IgG2b, kappa, clone 17A2, bioleged, cat#103132; BUV395 labeled anti-mCD 3 rat IgG2b, kappa, clone GK1.5, BD, cat#740268; BV421 labeled anti-mCD 4 rat IgG2b, kappa, clone 53-6.7, bioleged, cat#100438; PE-eFluor610 labeled anti-mCD 8 rat IgG2a, kappa, clone 30-F11, eBiosciences, cat#61-0081-82; all 1:50 diluted) was added and the cells were incubated in the dark for 30min at 4 ℃. Then, the cells were washed twice with 2mLPBS each. Cells were resuspended in 250 μ LPBS, ready for FACS.

For intracellular staining, cells were resuspended in 200. Mu.L of 1 Xfixing/permeabilizing buffer (Thermo Fisher, # 88-8824-00) and incubated in the dark for 30min at room temperature. The cells were washed twice with 2mL of each 1 Xpermeabilization buffer (Thermo Fisher, # 88-8824-00). mu.L of the antibody mixture (PE-labeled anti-mIFN-. Gamma., rat IgG1,. Kappa., clone XMG1.2, eBiosciences, cat#12-7311-82; APC-labeled anti-mIL-2, rat IgG2b,. Kappa., clone JES6-5H4, eBiosciences, cat#17-7021-82; FITC-labeled anti-mIL-4, rat IgG1,. Kappa., clone BVD6-24G2, eBiosciences, cat#11-7042-82; BVR#421-labeled anti-mNF-. Alpha., rat IgG1,. Kappa., clone MP6-XT22, eBiosciences, cat#11-7042-82, all 1:50) was added and the cells were incubated in the dark for 30 minutes at room temperature. Then, the cells were washed twice with 2mLPBA each. Cells were resuspended in 250 μ LPBS, ready for FACS.

After staining, flow cytometry was performed on stimulated spleen cells. The proportion of CD4+ cells expressing IL-2, IL-4, TNF- α or IFN- γ in the data was analyzed using Flowjo as in FIG. 8. The highest IFN- γ+ response was observed in 115S-5-5, and other Th-1 indications that the percentage of cytokine positivity in 115S-5-5 was acceptable. All cytokine positive percentages stimulated by mRNA vaccines (except IL-4) showed higher than all cytokine positive percentages stimulated by GSK.

Example B8.293T distribution of variant gE antigens in cells

Cytoplasmic regions of the gE protein containing three specific motifs (motifs) signal the internalization of the protein by endocytosis and its Targeting Golgi Network (TGN), thereby promoting assembly of VZV viral particles. These three motifs are YAGL (amino acids 582 to 585, capable of mediating the internalization of the gE into the cell), AYRV (amino acids 568 to 571, which direct the gE to the TGN region) and SSTT (amino acids 588 to 601, which contain phosphorylation sites), respectively, which, after phosphorylation, initiate the relevant signaling pathway, which also contributes to endocytosis of the gE protein. Confocal microscopy was used to evaluate whether the 115S-5-5 mutation site involved in the above three motifs increased the expression level of gE protein on the cell membrane and further enhanced its ability to activate immunogenicity.

293T cells were transfected with constructs as described above. Transfected cells were stained with antibody against gE (Abcam, cat#ab 272686) and the golgi marker GM130 (Abcam, cat#ab 195303), and nuclei were stained with DAPI (Beyotime, cat#p0131). The dyeing method follows the manufacturer's protocol. Confocal microscopy was used to observe stained cells. FIG. 9 shows the results of higher levels of gE expressed on cell membranes in the 115S-5-5 group than in the wild type construct.

Example B9. in vitro bridging assay between uridine modified and unmodified molecules

1. In vitro inflammatory response inhibition experiments

MRNA vaccines require uridine chemical modification and/or modification of the production process to prevent activation of cellular innate immunosensors and concomitant reduction in, e.g., dsRNA. Cytoplasmic RNA sensor retinoic acid-induced gene I (RIG-I) binds to dsRNA and induces expression of pro-inflammatory cytokines such as IFN- β by activating the adaptor protein mitochondrial antiviral signaling protein (MAVS). The minimal stimulation of the immune endpoint was reported to be immunostimulatory using m1ψ prepared by the modified method, whereas mRNA containing canonical uridine was immunostimulatory regardless of the method. The RIG-I signaling cell lines and BJ cell lines are used herein to test RIG-I pathway activation and IFN- β release to evaluate inflammatory responses induced by modified and unmodified mRNA vaccines.

The innovative technique of abogen was used to replace 115S-5-5 molecular uridine with N1-methyl-pseudouridine to reduce inflammatory responses. In particular, for in vitro transcription of 115S-5-5 (m1ψ, cap analogue: m7 GpppAmpU), UTP was replaced with m1ψ in rNTP mixture, and capping was performed by adding m7 GpppAmpU according to HiScribe T high yield RNA synthesis kit (NEB, # E2040S) instructions to synthesize mRNA with Cap1 "Cap" structure.

BJ fibroblasts were purchased from ATCC and cultured in EMEM medium supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS, life Technologies, thermofisher). Prior to transfection, cells were seeded into 96-well flat bottom cell culture plates (Corning) at a density of 20,000 cells per well for 24 hours. mRNA was transfected into cells using Lipofectamine 2000 (Thermo FISHER SCIENTIFIC). After 48 hours transfection period, cell culture supernatants were collected and analyzed for IFN- β expression levels using ELISA kits (R & D Systems).

HEK-Lucia TM RIG-I cells were purchased from Invivogen and cultured in DMEM medium supplemented with L-glutamine and 10% heat-inactivated fetal bovine serum (Gibco-FBS) (Life Technologies, thermo Fisher). Prior to transfection, cells were seeded into 96-well flat bottom cell culture plates (Corning) at a density of 50,000 cells per well and incubated for 24 hours. mRNA was transfected into cells using Lipofectamine 2000 (Thermo FISHER SCIENTIFIC). About 24 hours after transfection, 20. Mu.l of supernatant was aspirated and 50. Mu.l QUANTILuc ^TM 4/Lucia/Gaussia luciferase assay reagent (Invivogen) was added. Chemiluminescent signals are then detected using a microplate reader (MD).

The results as shown in fig. 10 demonstrate that the innovative modification significantly reduced the level of RIG-I signaling pathway activation and reduced the release of the pro-inflammatory factor IFN- β after transfection with the corresponding mRNA.

2. In vitro bridging of expression levels between modified and unmodified molecules

To compare the modified and unmodified molecular expression levels, the two mRNA molecules were transfected into HEK293T cell lines separately and expressed using FACS analysis as described above.

The results as shown in figures 11 and 12 show that the modified and unmodified molecules expressed comparable antigens, indicating that the modified bases had no effect on expression in vitro.

3. Immune response to modified mRNA vaccine in vivo

The purpose of the following experiments was to compare the immunogenicity of other mRNA vaccines with 115S-5-5 (m 1. Phi., cap analogue: m7 GpppAmpU). Assays were performed in mice undergoing LAV and in a mouse model of the primary trial, respectively.

Animal model for initial test

On day 0, 6-8 week old female C57BL/6 mice were randomly assigned to each group with minimal average weight change between groups (5 mice per group). Mice were vaccinated on days 0 and 14 by intramuscular injection of 50 μl at a dose of 0.5 μg. Shangrix (GSK) was vaccinated with 1.7 μg dose as positive control and PBS as placebo. Moderna-128 and Moderna-178 represent other company mRNA constructs. Blood samples were collected on days 13 and 21 and spleens were collected on day 28 for analysis of T cell responses.

Animal model experiencing LAV

On day 0, 6-8 week old female C57BL/6 mice were randomly assigned to each group with minimal average weight change between groups (5 mice per group). Mice were immunized with 1/10 human dose per mouse (2000 pfu) on day 0 by subcutaneous injection with an attenuated live varicella (LAV) vaccine (Ganwei, BCHT) and on days 28 and 42 with an mRNA vaccine at a dose of 0.5 μg per mouse. Shangrix (GSK) was vaccinated with 1.7 μg dose as positive control and PBS as placebo. Blood samples were collected on days 27, 41 and 49 and spleens were collected on day 56 for analysis of T cell responses.

3.1 Serum titre analysis

Serum titre assay protocols were as described above.

As shown in FIGS. 13A and 13B, the IgG titers on day 13 (priming) and day 21 (boosting) similarly revealed that the GMT of mice receiving 115S-5-5 was significantly higher than the gE (1-573 aa, Y569A, codon optimization 1) and gE (1-573 aa, Y569A, codon optimization 2) of mice that were initially subjected to the experiment.

As shown in fig. 13C and 13D, igG titers at day 34 (priming) and day 49 (boosting) showed a similar trend as titers in the mice that were initially subjected to the experiment. The GMT in the 115S-5-5 group was higher than in gE (1-573 aa, Y569A, codon optimization 1) and gE (1-573 aa, Y569A, codon optimization 2), but there was no significant difference.

3.2T cell response analysis

T cell response analysis protocol was as described above.

The proportion of CD4+ cells expressing IL-2, IL-4, TNF- α or IFN- γ in the data was analyzed using Flowjo as in FIGS. 14A and 14B. The results revealed that in both animal models, the strong Th-1 bias indicated by IFN-gamma, IL-2, TNF-alpha positive and IL-4 negative in the group receiving the RNA vaccine, the response induced by Shingrix was small in the mice that were initially tested, but strong in the mice that underwent LAV. In both animal models, 115S-5-5 stimulated a significantly higher T cell response compared to gE (1-573 aa, Y569A, codon optimization 2).

Claims

1. A protein comprising a mutant of mature glycoprotein E (gE) of varicella-zoster virus (VZV), wherein the mutant comprises:

(a) (i) a truncation of 37 amino acid residues from the C-terminus of the mature gE, and (ii) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full-length VSV gE;

(b) amino acid residue substitutions Y569A and Y582G, wherein amino acid residue positions 569 and 582 are the amino acid residue position numbers of full-length VSV gE;

(c) amino acid residue substitutions Y569A, Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue positions 569, 582, 593, 595, 596 and 598 are the amino acid residue position numbers of full-length VSV gE;

(d) amino acid residue substitutions Y582G, S593A, S595A, T596A and T598A, wherein amino acid residue positions 582, 593, 595, 596 and 598 are the amino acid residue position numbers of full-length VSV gE; or

(e) (i) a truncation of 50 amino acid residues from the C-terminus of the mature gE protein, and (ii) an amino acid residue substitution of Y569A, wherein amino acid residue position 569 is the amino acid residue position number of full-length VSV gE.

2. The protein of claim 1, wherein the mutant comprises the amino acid sequence of SEQ ID NO: 6, 8, 10, 12 or 3.

3. The protein according to claim 1, wherein the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO: 6, 8, 10, 12 or 3.

4. The protein of claim 1, wherein the mutant comprises the amino acid sequence of SEQ ID NO:6.

5. The protein according to claim 1, wherein the amino acid sequence of the mutant consists of the amino acid sequence of SEQ ID NO: 6.

6. The protein of claim 1, wherein the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94% or at least 95% identical to SEQ ID NO: 6, 8, 10 or 12.

7. The protein of claim 1, wherein the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO: 6, 8, 10 or 12.

8. The protein of claim 1, wherein the mutant comprises an amino acid sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, or at least 95% identical to SEQ ID NO:6.

9. The protein of claim 1, wherein the mutant comprises an amino acid sequence that is at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO:6.

10. The protein of any one of claims 1 to 9, further comprising a VZV gE signal peptide.

11. The protein of claim 10, wherein the signal peptide of the VZV gE comprises the amino acid sequence shown in SEQ ID NO: 18.

12. The protein of claim 11, wherein the signal peptide of the VZVgE comprises an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:18.

13. The protein of any one of claims 1 to 9, further comprising a heterologous signal peptide, wherein the N-terminus of the mutant is fused to the C-terminus of the heterologous signal peptide.

14. The protein of claim 13, wherein the heterologous signal peptide is a human IgE signal peptide.

15. The protein of claim 14, wherein the human IgE signal peptide comprises: (i) the amino acid sequence shown in SEQ ID NO:23, or (ii) an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:23.

16. The protein of claim 14, wherein the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO: 23.

17. The protein of claim 14, wherein the amino acid sequence of the mutant consists of the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of the human IgE signal peptide consists of the amino acid sequence shown in SEQ ID NO:23.

18. The protein of claim 14, comprising the amino acid sequence shown in SEQ ID NO:59.

19. The protein of claim 14, wherein the amino acid sequence of the protein consists of the amino acid sequence shown in SEQ ID NO:59.

20. The protein of claim 13, wherein the heterologous signal peptide is a human tPA signal peptide.

21. The protein of claim 20, wherein the human tPA signal peptide comprises: (i) the amino acid sequence shown in SEQ ID NO:27, or (ii) an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence shown in SEQ ID NO:27.

22. The protein of any one of claims 1 to 21, wherein the mature gE comprises the amino acid sequence shown in SEQ ID NO: 1, and/or the full-length VZV gE comprises the amino acid sequence shown in SEQ ID NO: 55.

23. A fragment of the mature glycoprotein E (gE) of varicella-zoster virus (VZV), wherein the fragment comprises a truncation of at least one and at most 50, 49, 48, 47, 46, 45, 44, 43 or 42 amino acid residues from the C-terminus of the mature gE, optionally wherein the truncation is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 0, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 or 49 amino acid residues from the C-terminus of the mature gE, or, optionally, the truncation is a truncation of 11, 12, 13, 14, 15, 16, 17, 18, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or 44 amino acid residues from the C-terminus of the mature gE, or, optionally, the truncation is a truncation of 14 or 37 amino acid residues from the C-terminus of the mature gE.

24. The fragment of claim 23, wherein the fragment further comprises the amino acid residue substitution Y569A, and wherein amino acid residue position 569 is numbered according to the amino acid residue position of full-length VZV gE.

25. The fragment of claim 23 or 24, wherein the fragment comprises a truncation of up to 41, 40, 39, 38, 37, 36, 35, 34, 33, 32 or 31 amino acid residues from the C-terminus of the mature gE.

26. The fragment of claim 25, wherein the fragment further comprises the amino acid residue substitution Y582G, and wherein amino acid residue position 582 is numbered according to the amino acid residue position of the full-length VZV gE protein.

27. The fragment of any one of claims 23 to 26, wherein the fragment comprises a truncation of up to 30 or 29 amino acid residues from the C-terminus of the mature gE.

28. The fragment of claim 27, wherein the fragment further comprises the amino acid residue substitution S593A, and wherein amino acid residue position 593 is numbered according to the amino acid residue position of the full-length VZV gE.

29. The fragment of any one of claims 23 to 28, wherein the fragment comprises a truncation of up to 28 amino acid residues from the C-terminus of the mature gE.

30. The fragment of claim 29, wherein the fragment comprises the amino acid residue substitution S595A, and wherein amino acid residue position 595 is numbered according to the amino acid residue position of the full-length VSVgE.

31. The fragment of any one of claims 23 to 30, wherein the fragment comprises a truncation of up to 27 or 26 amino acid residues from the C-terminus of the mature gE.

32. The fragment of claim 31, wherein the fragment further comprises the amino acid residue substitution T596A, and wherein amino acid residue position 596 is numbered according to the amino acid residue position of the full-length VZV gE.

33. A fragment as described in any one of claims 23 to 32, wherein the fragment comprises a truncation of up to 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 or 2 amino acid residues or up to 1 amino acid residue from the C-terminus of the mature gE.

34. The fragment of claim 33, wherein the fragment further comprises the amino acid residue substitution T598A, and wherein amino acid residue position 598 is numbered according to the amino acid residue position of the full-length VZV gE.

35. The fragment of claim 23, wherein the fragment comprises a truncation of the C-terminal 37 amino acid residues from the mature gE.

36. The fragment of claim 23, wherein the fragment comprises: (1) a truncation of 37 amino acid residues from the C-terminus of the mature gE, and (2) the amino acid residue substitutions Y569A and Y582G, and wherein amino acid residue positions 569 and 582 are numbered according to the amino acid residue positions of the full-length VZV gE.

37. The fragment of any one of claims 23 to 36, wherein the mature gE comprises the amino acid sequence shown in SEQ ID NO: 1 and/or the full-length VZV gE comprises the amino acid sequence shown in SEQ ID NO: 55.

38. A fragment as described in any one of claims 23 to 37, wherein the fragment comprises an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequence shown in SEQ ID NO:6, 3, 8, 10 or 12.

39. A fusion protein comprising the fragment of any one of claims 23 to 38 and a heterologous signal peptide, wherein the N-terminus of the fragment is fused to the C-terminus of the heterologous signal peptide.

40. The fusion protein of claim 39, wherein the heterologous signal peptide is a human tPA signal peptide.

41. The fusion protein of claim 40, wherein the human tPA signal peptide comprises the amino acid sequence shown in SEQ ID NO:27.

42. The fusion protein of claim 39, wherein the heterologous signal peptide is a human IgE signal peptide.

43. The fusion protein of claim 42, wherein the human IgE signal peptide comprises the amino acid sequence shown in SEQ ID NO:23.

44. A fusion protein comprising a fragment of the mature glycoprotein (gE) of varicella-zoster virus (VZV) and a human IgE signal peptide, wherein the fragment comprises a truncation of 37 amino acid residues from the C-terminus of the mature gE, and the N-terminus of the fragment is fused to the C-terminus of the human IgE signal peptide.

45. A nucleic acid encoding the protein of any one of claims 1 to 22, the fragment of any one of claims 23 to 38, or the fusion protein of any one of claims 39 to 44.

46. A nucleic acid as described in claim 45, wherein the nucleic acid comprises the nucleotide sequence shown in SEQ ID NO:7, 9, 11, 13, 4 or 5, or a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:7, 9, 11, 13, 4 or 5.

47. A nucleic acid encoding the protein of any one of claims 14 to 19 or the fusion protein of any one of claims 42 to 44, wherein the nucleotide sequence encoding the IgE signal peptide comprises: (a) the nucleotide sequence shown in SEQ ID NO:24, 25 or 26, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:24, 25 or 26.

48. A nucleic acid encoding the protein of any one of claims 10 to 12, wherein the nucleotide sequence of the signal peptide comprises: (a) the nucleotide sequence shown in SEQ ID NO: 19, 20, 21 or 22, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 19, 20, 21 or 22.

49. A nucleic acid encoding the protein of claim 20 or 21 or the fusion protein of claim 40 or 41, wherein the nucleotide sequence encoding the human tPA signal peptide comprises: (a) the nucleotide sequence shown in SEQ ID NO:28, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:28.

50. A non-naturally occurring nucleic acid comprising a nucleotide sequence encoding the protein of any one of claims 1 to 22, the fragment of any one of claims 23 to 38, or the fusion protein of any one of claims 39 to 44.

51. The non-naturally occurring nucleic acid of claim 50, wherein the encoding nucleotide sequence has been codon-optimized for expression in a cell of a subject, optionally wherein the subject is a non-human mammal or a human.

52. The non-naturally occurring nucleic acid of claim 50 or 51, comprising: (a) the nucleotide sequence shown in SEQ ID NO:7, 9, 11, 13, 4 or 5, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:7, 9, 11, 13, 4 or 5.

53. A non-naturally occurring nucleic acid encoding the protein of any one of claims 14 to 19 or the fusion protein of any one of claims 42 to 44, wherein the nucleotide sequence encoding the IgE signal peptide comprises: (a) the nucleotide sequence shown in SEQ ID NO:24, 25 or 26, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:24, 25 or 26.

54. A non-naturally occurring nucleic acid encoding the protein of any one of claims 10 to 12, wherein the nucleotide sequence of the signal peptide of the full-length VSVgE protein comprises: (a) the nucleotide sequence shown in SEQ ID NO: 19, 20, 21 or 22, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO: 19, 20, 21 or 22.

55. A non-naturally occurring nucleic acid encoding the protein of claim 20 or 21 or the fusion protein of claim 40 or 41, wherein the nucleotide sequence encoding the human tPA signal peptide comprises: (a) the nucleotide sequence shown in SEQ ID NO:28, or (b) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence shown in SEQ ID NO:28.

56. A non-naturally occurring nucleic acid comprising the nucleotide sequence of SEQ ID NO: 63, 7, 51, 60, 62, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5.

57. The non-naturally occurring nucleic acid of claim 56, which consists of the nucleotide sequence of SEQ ID NO: 63, 7, 51, 60, 62, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4 or 5.

58. The non-naturally occurring nucleic acid of any one of claims 50 to 55, wherein the nucleic acid consists of, consists essentially of, or comprises: (1) the nucleotide sequence shown in SEQ ID NO:63, 7, 51, 60, 62, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5; or (2) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO:63, 7, 51, 60, 62, 62, 64, 9, 11, 13, 52, 53, 54, 58, 49, 50, 4, or 5.

59. The non-naturally occurring nucleic acid of any one of claims 50 to 58,

The non-naturally occurring nucleic acid further comprises a 5' untranslated region (5'-UTR), wherein the 5'-UTR comprises the nucleotide sequence shown in any one of SEQ ID NOs: 29-38;

and/or,

The non-naturally occurring nucleic acid further comprises a 3' untranslated region (3'-UTR), wherein the 3'-UTR comprises the nucleotide sequence shown in any one of SEQ ID NOs: 39-46,

Optionally, the 3'-UTR further comprises a poly-A tail or a polyadenylation signal.

60. The non-naturally occurring nucleic acid of any one of claims 50 to 59, wherein the nucleic acid is DNA.

61. The non-naturally occurring nucleic acid of any one of claims 50 to 60, wherein the nucleic acid comprises one or more functional nucleotide analogs.

62. The non-naturally occurring nucleic acid of any one of 50 to 59, wherein the nucleic acid is an mRNA, and wherein thymine is replaced by uracil or a functional analog thereof in the nucleic acid.

63. The non-naturally occurring nucleic acid of claim 62, wherein the nucleic acid comprises one or more functional nucleotide analogs selected from pseudouridine, 1-methyl-pseudouridine, and 5-methylcytosine.

64. The non-naturally occurring nucleic acid of claim 62, wherein the nucleic acid consists of, consists essentially of, or comprises: (1) the nucleotide sequence shown in SEQ ID NO:63, or (2) a nucleotide sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the nucleotide sequence shown in SEQ ID NO:63; except that all thymines (T) are replaced by uracils (U) or N1-methylpseudouridine and/or the first nucleotide G is replaced by ^m7GpppAmpU .

65. The non-naturally occurring nucleic acid of claim 62, wherein the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence shown in SEQ ID NO:63, except that all thymines (T) are replaced by uracils (U) or N1-methylpseudouridine and/or the first nucleotide G is replaced by ^m7GpppAmpU .

66. The non-naturally occurring nucleic acid of claim 62, wherein the nucleic acid consists of, consists essentially of, or comprises the nucleotide sequence shown in SEQ ID NO:63, except that all thymines (T) are replaced with N1-methylpseudouridine and the first nucleotide G is replaced with ^m7GpppAmpU .

67. A vector comprising the nucleic acid of any one of claims 45 to 49 or the non-naturally occurring nucleic acid of any one of claims 50 to 66; preferably, the vector is an IVT (in vitro transcription) plasmid.

68. A host cell comprising the nucleic acid of any one of claims 45 to 49, the non-naturally occurring nucleic acid of any one of claims 50 to 66, or the vector of claim 67.

69. The host cell of claim 68, wherein the host cell is in vitro, ex vivo or isolated.

70. A pharmaceutical composition comprising the protein of any one of claims 1 to 22, the fragment of any one of claims 23 to 38, or the fusion protein of any one of claims 39 to 44.

71. A pharmaceutical composition comprising the nucleic acid of any one of claims 45 to 49, the non-naturally occurring nucleic acid of any one of claims 50 to 66, or the vector of claim 67.

72. A pharmaceutical composition comprising a non-naturally occurring nucleic acid as described in any one of claims 50 to 66 and at least a first lipid, optionally wherein the first lipid is a compound according to Formula 01-I or Formula 01-II; or a compound listed in Table 01-1; or a compound according to Formula 02-I; or a compound listed in Table 02-1; or a compound according to Formula 03-I; or a compound listed in Table 03-1; or a compound according to Formula 04-I; or a compound listed in Table 04-1.

73. A pharmaceutical composition as described in claim 72, further comprising a second lipid, optionally, wherein the second lipid is a compound according to Formula 05-I.

74. The pharmaceutical composition of claim 72 or 73, formulated as lipid nanoparticles encapsulating the nucleic acid in a lipid shell.

75. A pharmaceutical composition as described in any one of claims 70 to 74, wherein the composition is a vaccine.

76. A method for controlling, preventing or treating a disease or condition caused by VZV or infection with VZV in a subject, the method comprising administering to the subject a therapeutically effective amount of a protein as described in any one of claims 1 to 22, a fragment as described in any one of claims 23 to 38, a fusion protein as described in any one of claims 39 to 44, a nucleic acid as described in any one of claims 45 to 49, a non-naturally occurring nucleic acid as described in any one of claims 50 to 66, a vector as described in claim 67, or a pharmaceutical composition as described in any one of claims 70 to 75.

77. The method of claim 76, wherein the method is used to prevent a disease or condition caused by VZV or by infection with VZV in the subject.

78. The method of claim 76 or 77, wherein an immune response against the VZV is elicited in the subject.

79. The method of claim 78, wherein the immune response comprises the production of cytokines in lymphocytes.

80. The method of claim 78 or 79, wherein the immune response comprises an increase in the proportion of lymphocytes expressing cytokines.

81. The method of claim 79 or 80, wherein the lymphocytes are CD4 ⁺ T cells and/or CD8 ⁺ T cells, and/or wherein the cytokines are one or more of IFN-γ, IL-2, and TNF-α.

82. The method of any one of claims 79 to 81, wherein the production of cytokines in lymphocytes is increased.

83. The method of any one of claims 78 to 82, wherein the immune response comprises the production of antibodies that specifically bind to VZV gE.

84. The method of any one of claims 76 to 83, wherein the disease or condition caused by VZV is

(a) Chickenpox and/or herpes zoster;

(b) postherpetic neuralgia (PHN); and/or

(c) one or more of meningoencephalitis, myelitis, cranial nerve palsy, vasculopathy, keratitis, retinopathy, ulcer, hepatitis and pancreatitis.

85. The method of any one of claims 76 to 84, wherein the subject is a human.

86. The method of claim 85, wherein the human is a human adult.

87. The method of claim 86, wherein the adult is at least 40 years old.

88. The method of claim 85, wherein the human is an elderly human.

89. A protein as defined in any one of claims 1 to 22, a fragment as defined in any one of claims 23 to 38, a fusion protein as defined in any one of claims 39 to 44, a nucleic acid as defined in any one of claims 45 to 49, a non-naturally occurring nucleic acid as defined in any one of claims 50 to 66, a vector as defined in claim 67 or a pharmaceutical composition as defined in any one of claims 70 to 75 for use in a method of controlling, preventing or treating a disease or condition caused by VZV or infection with VZV in a subject.

90. The protein, fragment, fusion protein, nucleic acid, non-naturally occurring nucleic acid, vector or pharmaceutical composition for use according to claim 89, wherein the subject is a human, optionally a human adult or elderly.

91. Use of a protein as defined in any one of claims 1 to 22, a fragment as defined in any one of claims 23 to 38, a fusion protein as defined in any one of claims 39 to 44, a nucleic acid as defined in any one of claims 45 to 49, a non-naturally occurring nucleic acid as defined in any one of claims 50 to 66, a vector as defined in claim 67 or a pharmaceutical composition as defined in any one of claims 70 to 75 for the manufacture of a medicament for controlling, preventing or treating a disease or condition caused by VZV or infection with VZV in a subject.

92. The use of claim 91, wherein the subject is a human, optionally an adult or elderly person.