WO2025015338A1

WO2025015338A1 - Rna-editing oligonucleotides and uses thereof

Info

Publication number: WO2025015338A1
Application number: PCT/US2024/038062
Authority: WO
Inventors: Derek Mark ERION; Christopher Brown; Venkat Krishnamurthy; Mateusz MACIEJEWSKI; Mohammad SHADID; Mallikarjuna Reddy Putta
Original assignee: Korro Bio Inc
Current assignee: Korro Bio Inc
Priority date: 2023-07-13
Filing date: 2024-07-15
Publication date: 2025-01-16
Anticipated expiration: 2026-01-13

Abstract

The present disclosure features useful compositions and methods to treat disorders (e.g., alpha-1-antitrypsin deficiency) for which deamination of an adenosine in an mRNA produces a therapeutic result in a subject in need thereof.

Description

RNA-EDITING OLIGONUCLEOTIDES AND USES THEREOF

INCORPORATION BY REFERENCE OF MATERIALS SUBMITTED ELECTRONICALLY

[0001] This application contains, as a separate part of the disclosure, a Sequence Listing in computer readable form (Filename: 50002_SeqListing.txt; Size: 1,185,167 bytes; Created: July 12, 2024), which is incorporated by reference in its entirety.

Background

[0002] Adenosine deaminases acting on RNA (ADAR) are enzymes which bind to double-stranded RNA (dsRNA) and convert adenosine to inosine through deamination. In RNA, inosine functions similarly to guanosine for translation and replication. Thus, conversion of adenosine to inosine in an mRNA can result in a codon change that may lead to changes to the encoded protein and its functions. There are three known ADAR proteins expressed in humans, ADAR1, ADAR2, and ADAR3. ADAR1 and ADAR2 are expressed throughout the body whereas ADAR3 is expressed only in the brain. ADAR1 and ADAR2 are catalytically active, while ADAR3 is thought to be inactive.

[0003] Synthetic single-stranded oligonucleotides have been shown capable of utilizing the ADAR proteins to edit target RNAs by deaminating particular adenosines in the target RNA. The oligonucleotides are complementary to the target RNA with the exception of at least one mismatch opposite the adenosine to be deaminated. However, the previously disclosed methods have not been shown to have the required selectivity and/or stability to allow for their use as therapies. Accordingly, new oligonucleotides capable of utilizing the ADAR proteins to selectively edit target RNAs in a therapeutically effective manner are needed.

Summary

[0004] The present disclosure features oligonucleotides, compositions and methods to deaminate adenosine in target RNAs, e.g., an adenosine which may be deaminated to produce a therapeutic result, e.g., in a subject in need thereof.

[0005] Adenosine deaminases that act on RNA (ADARs) are editing enzymes that recognize certain structural motifs of double-stranded RNA (dsRNA) and edit adenosine to inosine, resulting in recoding of amino acid codons that may lead to changes to the encoded protein and its function. The nucleobases surrounding the editing site, especially the one immediately 5' of the editing site and one immediately 3' to the editing site, which together with the editing site are termed the triplet, play an important role in the deamination of adenosine. A preference for U at the 5' position and G at the 3' position relative to the editing site, was revealed from the analysis of yeast RNAs efficiently edited by overexpressed human ADAR2 and ADAR1 . See Wang et al., (2018) Biochemistry, 57: 1640-1651, Eifler et al., (2013) Biochemistry, 52: 7857-7869, and Eggington et al., (2011) Nat. Commun., 319: 1-9. Recruiting ADAR to specific sites of selected transcripts and deamination of adenosine regardless of neighboring bases holds great promise for the treatment of disease. Based on structural and modeling studies of the editing site of dsRNA/ADAR complexes, several structural features that could be incorporated into guide oligonucleotides have been identified, whose properties could increase the recruitment of ADAR and increase the efficiency of editing of target RNA. Novel oligonucleotides with chemical modifications such as o-homo-DNA capable of recruiting ADAR proteins and deaminating adenosine with different surrounding base compositions in target RNA are shown. In addition, structure- activity relationship (SAR) studies revealed that a 2'-O-methyl (2’-0Me) modification of the ribose of some, but not all, nucleosides in the guide oligonucleotide, in addition to triplet modifications, are compatible with efficient ADAR engagement and editing.

[0006] In one aspect, described herein is an oligonucleotide comprising the structure:

[A_m]-X1-X2-X3-[B_n] wherein each A and B comprise (I) a nucleobase, (ii) a sugar ("an A/B sugar”), and (ill) an internucleotide linkage; each A/B sugar is independently a 2'-methoxyribose, a 2'-methoxyethylribose, a 2'-fluoroarabinose, a 2'-deoxy- 2'fluororibose, or a 2'-deoxyribose; m is 20 to 40; n is 4 to 15;

X¹ comprises (I) a nucleobase selected from uracil and thymine, (ii) a 2'deoxyribose sugar, and (ill) an internucleotide linkage;

X² comprises a structure

internucleotide linkage, wherein N is a nucleobase;

X³ comprises (I) a nucleobase selected from guanosine, hypoxanthine, and 7-deazaguinine, (ii) a sugar selected from 2'-deoxyribose and 2'-deoxy-2'-fluoroarabinose, and (ill) an internucleotide linkage; and the internucleotide linkages of the oligonucleotide comprise at least 30% phosphoramidate and/or phosphorothioate linkages.

[0007] 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 to which this invention belongs. Methods and materials are described herein for use in the present disclosure; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Definitions

[0008] For convenience, the meaning of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless stated otherwise, or implicit from context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments, and are not intended to limit the claimed technology, because the scope of the technology is limited only by the claims. 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 to which this technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided within the specification shall prevail.

[0009] In this application, unless otherwise clear from context, (I) the term "a” may be understood to mean "at least one”; (ii) the term "or” may be understood to mean "and/or”; and (ill) the terms "including” and "comprising” may be understood to encompass itemized components or steps whether presented by themselves or together with one or more additional components or steps.

[0010] As used herein, the terms "about” and "approximately” refer to a value that is within 10% above or below the value being described. For example, the term "about 5 nM” indicates a range of from 4.5 to 5.5 nM.

[0011] The term "at least” prior to a number or series of numbers is understood to include the number adjacent to the term "at least", and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21 -nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that "at least" can modify each of the numbers in the series or range.

[0012] As used herein, "no more than” or "less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, an oligonucleotide with "no more than 5 unmodified nucleotides” has 5, 4, 3, 2, 1, or 0 unmodified nucleotides. When "no more than” is present before a series of numbers or a range, it is understood that "no more than” can modify each of the numbers in the series or range.

[0013] As used herein, the term "administration” refers to the administration of a composition (e.g., a compound or a preparation that includes a compound as described herein) to a subject or system. Administration to an animal subject (e.g., to a human) may be by any appropriate route, such as the one described herein.

[0014] The term "oligonucleotide” as used herein, is a molecule including two or more nucleotides. The term "nucleotide” refers to a nucleobase, a sugar moiety, and an internucleotide linkage. Oligonucleotides are commonly made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to a sequence of the oligonucleotide, reference is made to the sequence or order of nucleobase moieties, or modifications thereof, of the covalently linked nucleotides or nucleosides. The oligonucleotide described herein may be man-made, and is chemically synthesized, and is typically purified or isolated. Oligonucleotide is also intended to include (I) compounds that have one or more furanose moieties that are replaced by furanose derivatives or by any structure, cyclic or acyclic, that may be used as a point of covalent attachment for the base moiety, (II) compounds that have one or more phosphodiester linkages that are either modified, as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by a suitable linking moiety as in the case of formacetal or riboacetal linkages, and/or (ill) compounds that have one or more linked furanose-phosphodiester linkage moieties replaced by any structure, cyclic or acyclic, that may be used as a point of covalent attachment for the nucleobase moiety. The oligonucleotide described herein may include one or more alternative nucleotides (e.g., including those described herein). It is also understood that oligonucleotide includes compositions lacking a sugar moiety or nucleobase but is still capable of forming a pairing with or hybridizing to a target sequence. Oligonucleotides as used herein comprise 100 or fewer nucleotides.

[0015] The terms "nucleobase” and "base” include the purine (e.g., adenine and guanine) and pyrimidine (e.g. uracil, thymine, and cytosine) moiety present in nucleosides and nucleotides that form hydrogen bonds in nucleic acid hybridization. The term nucleobase also encompasses alternative nucleobases that may differ from naturally- occurring nucleobases but are functional during nucleic acid hybridization. In this context "nucleobase” refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are for example described in Hirao et al (2012) Accounts of Chemical Research vol 45, page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl. 37 1.4.1.

[0016] “G,” “C,” "A,” “T,” and “U” each generally stand for a naturally-occurring nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a nucleobase, respectively. However, G, C, A, T and U can also refer to the guanine, cytosine, adenine, thymidine, and uracil nucleobase with a sugar moiety other than ribose (or deoxyribose). Such alternate sugar moieties are discussed herein.

[0017] In a some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine into a modified purine or pyrimidine, such as substituted purine or substituted pyrimidine, such as an "alternative nucleobase” selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl- cytosine, 5-propynyl-uracil, 5-bromouracil, 5-thiazolo-uracil, 2-thio-uracil, pseudouracil, 1 -methylpseudouracil, 5- methoxyuracil, 2'-thio-thymine, hypoxanthine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0018] The nucleobase moieties may be indicated by the letter code for each corresponding nucleobase, e.g. A, T, G, C, or U, wherein each letter may optionally include alternative nucleobases of equivalent function.

[0019] A "sugar” or "sugar moiety,” includes sugars having a furanose ring (e.g., ribose, deoxyribose, arabinose). A sugar also includes an "alternative sugar,” defined as a structure that is capable of replacing the furanose ring of a nucleoside. In certain embodiments, alternative sugars are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include a six-membered ring (e.g., a pyranose ring), or non-ring moieties such as those used in peptide nucleic acids. Alternative sugars may also include a morpholino, a pyranyl, or hexitol ring system. Sugar moieties useful in the preparation of oligonucleotides having motifs include, without limitation, p- D-ribose, p-D-2'-deoxyribose, methoxy-substituted sugars (e.g., p-D-2'methoxyribose), MOE-substituted sugars (e.g., p-D-2'methoxyethylribose), fluoro substituted sugars (e.g., 2'-deoxy-2-fluororibose and p-D-2'-deoxy-2'- fluoroarabinofurose, also referred to herein as 2'-fluoroarabinose), substituted sugars (such as 2', 5' and bis substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose), bicyclic alternative sugars (such as locked nucleic acid (LNA) having a 2'-O— CH2-4' or 2'-O— (CH2)2-4' bridged ribose derived bicyclic sugar) and sugar surrogates (such as when the ribose ring has been replaced with a morpholino, a pyran, or a hexitol ring system, such as a p-D-homoDNA). A p-D-homoDNA sugar moiety is a pyran ring substituted as shown in this structure:

[0020] The internucleotide linkage of the nucleotide can be a phosphate linkage. Other internucleotide linkages are known in the art, including, but not limited to, phosphorothioate or boronophosphate. Other internucleotide linkages include phosphotriester, phosphorothionate, phosphoramidate, and other variants of the phosphate backbone . [0021] The term "nucleoside” refers to a monomeric unit of an oligonucleotide or a polynucleotide having a nucleobase and a sugar moiety.

[0022] The oligonucleotide may be of any length that permits base modification of a target nucleobase (e.g., deamination of a target adenosine) on a desired target RNA through an ADAR-mediated pathway, and may range from about 27-50 base pairs in length, e.g., about 30-45 base pairs in length or about 35-45 base pairs in length, for example, about 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 base pairs in length. Ranges intermediate to the above recited ranges are also contemplated to be part of the oligonucleotides described herein.

[0023] As used herein, and unless otherwise indicated, the term "complementary," when used to describe a first nucleotide or nucleoside sequence in relation to a second nucleotide or nucleoside sequence, refers to the ability of an oligonucleotide or polynucleotide including the first nucleotide or nucleoside sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide including the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCI, 40 mM PIPES pH 6.4, 1 mM EDTA, 50 °C, or 70 °C, for 12-16 hours followed by washing (see, e.g., "Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides or nucleosides.

[0024] "Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson- Crick base pairs and/or base pairs formed from non-natural and alternative nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing. Complementary sequences between an oligonucleotide and a target sequence as described herein, include base-pairing of the oligonucleotide or polynucleotide including a first nucleotide sequence to an oligonucleotide or polynucleotide including a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as "fully complementary" with respect to each other herein. However, where a first sequence is referred to as "substantially complementary" with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally no more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., deamination of an adenosine. "Substantially complementary” can also refer to an oligonucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA having a target adenosine). For example, an oligonucleotide is complementary to at least a part of the mRNA of interest if the sequence is substantially complementary to a non-interrupted portion of the mRNA of interest.

[0025] As used herein, the term "region of complementarity" refers to the region on the oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, a sequence (e.g., a target sequence; e.g., a target sequence having a target nucleobase, e.g., adenosine), or processed mRNA, so as to interfere with expression of the endogenous gene. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5'- and/or 3'-terminus of the oligonucleotide.

[0026] The phrase "contacting a cell with an oligonucleotide," such as an oligonucleotide as described herein, includes contacting a cell by any possible means. Contacting a cell with an oligonucleotide includes contacting a cell in vitro with the oligonucleotide or contacting a cell in vivo with the oligonucleotide. The contacting may be done directly or indirectly. Thus, for example, the oligonucleotide may be put into physical contact with the cell by the individual performing the method, or alternatively, the oligonucleotide agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

[0027] Contacting a cell in vitro may be done, for example, by incubating the cell with the oligonucleotide. Contacting a cell in vivo may be done, for example, by injecting the oligonucleotide into or near the tissue where the cell is located, or by injecting the oligonucleotide agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the oligonucleotide may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the oligonucleotide to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an oligonucleotide and subsequently transplanted into a subject.

[0028] In one embodiment, contacting a cell with an oligonucleotide includes "introducing" or "delivering the oligonucleotide into the cell" by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an oligonucleotide can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an oligonucleotide into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, oligonucleotide s can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

[0029] As used herein, "lipid nanoparticle" or "LNP" is a vesicle including a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an oligonucleotide. LNP refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic, ionizable lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are described in, for example, U.S. Pat. Nos. 6,858,225; 6,815,432; 8,158,601; and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

[0030] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the oligonucleotide composition, although in some examples, it may. Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes including one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. [0031] "Micelles" are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

[0032] As used herein, the terms "effective amount,” "therapeutically effective amount,” and "a "sufficient amount” of an agent that results in a therapeutic effect (e.g., in a cell or a subject) described herein refer to a quantity sufficient to, when administered to the subject, including a human, effect beneficial or desired results, including clinical results, and, as such, an "effective amount” or synonym thereto depends on the context in which it is being applied. For example, in the context of treating a disorder, it is an amount of the agent that is sufficient to achieve a treatment response as compared to the response obtained without administration. The amount of a given agent will vary depending upon various factors, such as the given agent, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject (e.g., age, sex, and/or weight) or host being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. Also, as used herein, a "therapeutically effective amount” of an agent is an amount that results in a beneficial or desired result in a subject as compared to a control. As defined herein, a therapeutically effective amount of an agent may be readily determined by one of ordinary skill by routine methods known in the art. Dosage regimen may be adjusted to provide the optimum therapeutic response.

[0033] A "therapeutically-effective amount” includes an amount (either administered in a single or in multiple doses) of an oligonucleotide that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. Oligonucleotides employed in the methods as disclosed herein may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

[0034] By "determining the level of a protein” is meant the detection of a protein, or an mRNA encoding the protein, by methods known in the art either directly or indirectly. "Directly determining” means performing a process (e.g., performing an assay or test on a sample or "analyzing a sample” as that term is defined herein) to obtain the physical entity or value. "Indirectly determining” refers to receiving the physical entity or value from another party or source (e.g., a third-party laboratory that directly acquired the physical entity or value). Methods to measure protein level generally include, but are not limited to, western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescent polarization, phosphorescence, immunohistochemical analysis, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC)-mass spectrometry, microcytometry, microscopy, fluorescence activated cell sorting (FACS), and flow cytometry, as well as assays based on a property of a protein including, but not limited to, enzymatic activity or interaction with other protein partners. Methods to measure mRNA levels are known in the art.

[0035] "Percent (%) sequence identity” with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleotides or amino acids in a candidate sequence that are identical to the nucleotides or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For example, percent sequence identity values may be generated using the sequence comparison computer program BLAST. As an illustration, the percent sequence identity of a given sequence, A, to, with, or against a given sequence, B, (which can alternatively be phrased as a given sequence, A that has a certain percent sequence identity to, with, or against a given sequence, B) is calculated as follows:

100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program's alignment of A and B, and where Y is the total number of nucleotides or amino acids in B. It will be appreciated that where the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0036] By "level” is meant a level or activity of a protein, or mRNA encoding the protein, as compared to a reference. The reference can be any useful reference, as defined herein. By a "decreased level” or an "increased level” of a protein is meant a decrease or increase in protein level, as compared to a reference (e.g., a decrease or an increase by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500%, or more; a decrease or an increase of more than about 10%, about 15%, about 20%, about 50%, about 75%, about 100%, or about 200%, as compared to a reference; a decrease or an increase by less than about 0.01 -fold, about 0.02-fold, about 0.1 -fold, about 0.3-fold, about 0.5-fold, about 0.8-fold, or less; or an increase by more than about 1 .2-fold, about 1 .4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10- fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold, or more). A level of a protein may be expressed in mass/vol (e.g., g/dL, mg/mL, pg/mL, ng/mL) or percentage relative to total protein or mRNA in a sample.

[0037] The term "pharmaceutical composition,” as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient, and preferably manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); for intrathecal injection; for intracerebroventricular injections; for intraparenchymal injection; or in any other pharmaceutically acceptable formulation.

[0038] A "pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being substantially nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, sorbents, suspensing or dispersing agents, sweeteners, and waters of hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0039] As used herein, the term "pharmaceutically acceptable salt” means any pharmaceutically acceptable salt of an oligonucleotide as described herein. For example, pharmaceutically acceptable salts include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable organic acid.

[0040] Pharmaceutically acceptable salts may be acid addition salts involving inorganic or organic acids or the salts maybe prepared from inorganic or organic bases. Frequently, pharmaceutically acceptable salts are prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparation of the appropriate salts are well-known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, and valerate salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0041] By a "reference” is meant any useful reference used to compare protein or mRNA levels or activity. The reference can be any sample, standard, standard curve, or level that is used for comparison purposes. The reference can be a normal reference sample or a reference standard or level. A "reference sample” can be, for example, a control, e.g., a predetermined negative control value such as a "normal control” or a prior sample taken from the same subject; a sample from a normal healthy subject, such as a normal cell or normal tissue; a sample (e.g., a cell or tissue) from a subject not having a disease; a sample from a subject that is diagnosed with a disease, but not yet treated with a compound described herein; a sample from a subject that has been treated by a compound described herein; or a sample of a purified protein (e.g., any described herein) at a known normal concentration. By "reference standard or level” is meant a value or number derived from a reference sample. A "normal control value” is a pre-determined value indicative of non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y”), a high threshold ("no higher than X”), or a low threshold ("no lower than X”). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as "within normal limits” for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject not having a disease or disorder; a subject that has been treated with a compound described herein. In preferred embodiments, the reference sample, standard, or level is matched to the sample subject sample by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of levels of a purified protein, e.g., any described herein, within the normal reference range can also be used as a reference.

[0042] As used herein, the term "subject” refers to any organism to which oligonucleotides or compositions described herein may be administered, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may seek or be in need of treatment, require treatment, be receiving treatment, be receiving treatment in the future, or be a human or animal who is under care by a trained professional for a particular disease or condition. [0043] As used herein, the terms "treat," "treated," or "treating" mean both therapeutic treatment and prophylactic or preventative measures wherein the object is to prevent or slow down (lessen) an undesired physiological condition, disorder, or disease, or obtain beneficial or desired clinical results. Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms; diminishment of the extent of a condition, disorder, or disease; stabilized (i.e., not worsening) state of condition, disorder, or disease; delay in onset or slowing of condition, disorder, or disease progression; amelioration of the condition, disorder, or disease state or remission (whether partial or total), whether detectable or undetectable; an amelioration of at least one measurable physical parameter, not necessarily discernible by the patient; or enhancement or improvement of condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival as compared to expected survival if not receiving treatment.

[0044] As used herein, the terms "variant” and "derivative” are used interchangeably and refer to naturally- occurring, synthetic, and semi-synthetic analogues of a compound, peptide, protein, or other substance described herein. A variant or derivative of a compound, peptide, protein, or other substance described herein may retain or improve upon the biological activity of the original material.

[0045] The details of one or more embodiments described herein are set forth in the description below. Other features, objects, and advantages described herein will be apparent from the description and from the claims.

Brief Description of the Figures

[0046] Figure 1 : Editing percentage in M/Z primary hepatocytes 48 hours, 96 hours, 144 hours, and 192 hours after transfection. Cells were transfected with antisense oligonucleotides (ASOs) at indicated doses and incubated for indicated time point before mRNA extraction. Editing percentage was assessed using amplicon sequencing. Each condition has three technical replicates. Editing defined as A to I at the target site was observed at 10nM and 100nM. [0047] Figure 2: Editing percentage in zzHLC 48 hours after transfection. Cells were transfected with oligos disclosed herein at indicated doses and incubated for 48 hours in the presence of 1 U/piL recombinant human Interferon alpha before mRNA extraction. Editing percentage was assessed using amplicon sequencing. Experiment was performed 3 independent times while the figure included one representative experiment. Each condition has four technical replicates. At 10nM, 30-40% A to I editing at target site was achieved while at 1 nM, 10-20% A to I editing was achieved in zzHLC.

[0048] Figure 3A: Editing percentage in PiZ mice liver tissue 1, 4, 7 days after single dose administration. PiZ mice (n=3) were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASOs. Liver tissues were collected 1, 4, 7 days post dosing. Editing percentage at each timepoint was assessed by dPCR from mRNA harvested in pulverized liver tissue.

[0049] Figure 3B: WT-A1AT and Total-A1 AT measured in plasma of PiZ mice treated with oligos disclosed herein. PiZ mice (n=3) were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASOs. Plasma was isolated at appropriate time point, and WT-A1AT and Total-A1AT were measured using LC/MS-MS methods as described. [0050] Figure 4A: Editing percentage in PiZ mice liver tissue 7-days post either 1 , 2, 3, or 4 doses of Oligo #3 encapsulated in MC3 LNP PiZ mice (n=5) were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASOs for either 1,2,3, or 4 doses. Liver tissues were collected 7 days post dosing. Editing percentage at each timepoint was assessed by dPCR from mRNA harvested in pulverized liver tissue.

[0051] Figure 4B: WT-A1AT and Total-A1 AT measured in plasma of PiZ mice treated with oligos as disclosed herein. PiZ mice were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASO. Plasma was isolated at appropriate time point, and WT-A1AT and Total-A1AT were measured using LC/MS-MS methods as described. [0052] Figure 5A: Human metabolic assay using human liver homogenate was used to evaluate the metabolic stability of the different chemistries and modifications and can be used to select oligos with optimum drug-like properties for in vivo testing.

[0053] Figure 5B: Mouse metabolic assay using mouse liver homogenate was used to evaluate the metabolic stability of the different chemistries and modifications and can be used to select oligos with optimum drug-like properties for in vivo testing. (Same legend key as Figure 5A).

[0054] Figure 6 - Editing percentage in Def-HEP human hepatocyte-like cells (zzHLC). Cells were transfected with oligos disclosed herein at indicated doses and incubated for 48 hours in the presence of recombinant human Interferon alpha before mRNA extraction. Editing percentage was assessed using amplicon sequencing.

Detailed Description

[0055] Provided herein are oligonucleotides that can be used to modify a nucleobase on a target RNA.

Accordingly, the disclosure provides oligonucleotides, compositions containing the same, and methods to modify a target nucleobase (e.g., deaminate a target adenosine) on RNA, where the modification produces a therapeutic result, e.g., in a subject in need thereof. In some embodiments, the target RNA is mRNA. I. Disorders

[0056] The disclosure also provides oligonucleotides for use in a method for editing a target adenosine in a target RNA sequence (e.g., SERPINA1) in a mammalian cell, preferably human cell, as described herein. Similarly, the disclosure provides the use of these oligonucleotides in the manufacture of a medicament for editing a target adenosine in a target RNA in a mammalian cell, preferably human cell, as described herein.

[0057] The disclosure also relates to a method for the deamination of at least one specific target adenosine present in a target RNA sequence (e.g., SERPINA1) in a cell, said method including the steps of: allowing uptake by the cell of an oligonucleotide as disclosed herein; allowing annealing of the oligonucleotide to the target RNA sequence; allowing a mammalian ADAR enzyme including a natural dsRNA binding domain as found in the wild type enzyme to deaminate said target adenosine in the target RNA sequence to an inosine; and optionally identifying the presence of the inosine in the RNA sequence.

[0058] Hence, provided herein are oligonucleotides, compositions, and methods wherein two adenosines that are next to each other on a target RNA are co-deaminated by an RNA editing enzyme such as ADAR. In this particular case, the UAA stop codon is converted into a Ull Trp-encoding codon. Other examples of modifications resulting from deamination of target adenosines within a target codon are provided in Tables 1 and 2 below.

Table 1

Table 2. Triplet Base Composition and Resulting Edited Triplet

[0059] Because the deamination of the adenosine to an inosine may result in a protein that is no longer suffering from the mutated A at the target position, the identification of the deamination into inosine may be a functional readout, for instance an assessment on whether a functional protein is present, or even the assessment that a disease that is caused by the presence of the adenosine is (partly) reversed. The functional assessment for each of the diseases mentioned herein will generally be according to methods known to the skilled person. When the presence of a target adenosine causes aberrant splicing, the read-out may be the assessment of whether the aberrant splicing is still taking place, or not, or taking place less often. On the other hand, when the deamination of a target adenosine is intended to introduce a splice site, then similar approaches can be used to check whether the required type of splicing is indeed taking place. A suitable manner to identify the presence of an inosine after deamination of the target adenosine is RT-PCR and sequencing, using methods that are well-known to the person skilled in the art.

[0060] In general, mutations in any target RNA that can be reversed using oligonucleotides as disclosed herein are G-to-A mutations, and oligonucleotides can be designed accordingly. Mutations that may be targeted using oligonucleotides disclosed herein also include C to A, U to A (T to A on the DNA level) in the case of recruiting adenosine deaminases. Although RNA editing in the latter circumstances may not necessarily revert the mutation to wild-type, the edited nucleotide may give rise to an improvement over the original mutation. For example, a mutation that causes an in frame stop codon - giving rise to a truncated protein, upon translation - may be changed into a codon coding for an amino acid that may not be the original amino acid in that position, but that gives rise to a (full length) protein with at least some functionality, at least more functionality than the truncated protein.

[0061] The oligonucleotides described herein are particularly suitable for treating genetic diseases, such as alpha- 1 -antitrypsin (A1AT) deficiency. Oligonucleotides described herein may deaminate the adenosine mutation resulting in an increase in protein activity.

[0062] Alpha-1 -antitrypsin (A1AT) deficiency is a genetic disease caused by defects in the SERPINA1 gene (also known as PI; A1A; AAT; PH; A1AT; PRO2275; and alphal AT, set forth in Genbank Accession No. KJ897327.1) [0063] A1 AT deficiency is one of the most common genetic diseases in subjects of Northern European descent. Severe A1 AT deficiency causes emphysema, with subjects developing emphysema in their third or fourth decade. A1 AT deficiency can also cause liver failure and hepatocellular carcinoma, with up to 30% of subjects with severe A1 AT deficiency developing significant liver disease, including cirrhosis, fulminant liver failure, and hepatocellular carcinoma.

[0064] There are two predominant mutations in the SERPINA1 gene that cause A1 AT deficiency. These missense mutations affect protein conformation and secretion leading to reduced circulating levels of A1AT. The more common and more severe mutation is a glutamate to lysine substitution at amino acid position 342 (E342K, "Z mutation”) of the mature A1AT protein, which can arise from, e.g., c.1024G>A. Alleles carrying the Z mutation are identified as PiZ alleles. Subjects homozygous for the PiZ allele are termed PiZZ carriers, and express 10-15% of normal levels of serum A1 AT. Approximately 95% of subjects who are symptomatic for A1 AT deficiency have the PiZZ genotype. Subjects heterozygous for the Z mutation are termed Pi MZ mutants, and express 60% of normal levels of serum A1 AT. The other predominant mutation is a glutamate to valine substitution at position 264 (E264V, "S mutation”) of the mature A1 AT protein, which can arise from, e.g., c.791 A>T. Alleles with the S mutation are termed PIS. Subjects homozygous for the PIS allele are termed PISS carriers, and express 60% of normal levels of serum A1 AT. Subjects heterozygous for this mutation are termed Pi MS and express 80% of normal levels of serum A1 AT. Compound heterozygotes are represented as PISZ. PISZ subjects express 40% of normal levels of serum A1 AT. Normal SERPINA1 subjects are represented as PIMM.

[0065] Between 30,000 and 50,000 individuals in the United States have the PiZZ genotype. The prevalence of any one of the five genotypic classes of A1 AT mutations (PiZZ, Pi MZ, PISS, PIMS, and PISZ) is approximately 1 in 5,000- 7,000 in the United States. The prevalence is higher in Northern Europe, and may be as high as 1 in 1,500-3,000 in the Scandinavian population.

[0066] The pathophysiology of A1 AT deficiency varies by the organ affected. Liver disease is due to a gain-of- function mechanism. Abnormally folded A1AT, especially Z-type A1AT (Z-AT), aggregates and polymerizes within hepatocytes. A1 AT inclusions are found in PiZZ subjects and are thought to cause cirrhosis and, in some cases, hepatocellular carcinoma. Evidence for the gain-of-function mechanism in liver disease is supported by null homozygotes. These subjects produce no A1 AT and do not develop hepatocyte inclusions or liver disease. Lung disease has been classically thought to be due to a loss-of-function mechanism: lower A1 AT levels lead to unchecked activity of neutrophil elastase and subsequent alveolar destruction (Gadek et al. in The Metabolic Basis of Inherited Disease 1450-1457 (1982)). There is recent evidence that lung disease is also due in part to a gain-of- function mechanism. Z-type A1 AT has been identified within the lung parenchyma and has been shown to be a neutrophil chemoattractant (Mulgrew 2004). Z-AAT may contribute through a toxic gain-of-function mechanism to inflammation and destruction of the lung parenchyma in A1AT deficiency (Alam 2014; Mornex 1986).

[0067] Less common manifestations of A1 AT deficiency include membranoproliferative glomerular nephritis, rheumatoid arthritis, vascular disease including bleeding disorders, panniculitis, uveitis, and vasculitis.

[0068] A1 AT deficiency leads to lung disease due to reduced inhibition of neutrophil elastase in the lung. A1 AT enters the lung interstitium and alveolar lining fluid via passive diffusion from the plasma. Unchecked elastase activity in alveolae leads to destruction of the lung parenchyma. The primary manifestation of disease is emphysema with severe airflow obstruction. Subjects may also develop chronic bronchitis, bronchiectasis and asthma. Between 85 and 100% of subjects with the PiZZ genotype develop lung disease in their fourth or fifth decade. Other genotypes have a lower likelihood of developing lung disease and generally develop disease in their fifth decade or later. Smoking dramatically alters the phenotype in all A1 AT deficient subjects. Smokers are more likely to develop disease and to experience earlier onset (generally 20 years earlier) and more severe disease. Lung disease in A1 AT deficiency may additionally be due to monocyte and macrophage derived pathogenic Z-protein. Misfolded A1 AT in PiZZ or PISZ mutants is expressed by macrophages and has been demonstrated to be pro-inflammatory (Mulgrew 2004). The more severe lung phenotype of PiZZ and PISZ genotypes may be due to the secretion of an altered protein in lung tissue by macrophages and monocytes (Alam 2014; Mornex 1986).

[0069] Lung disease associated with A1 AT deficiency is currently treated with intravenous administration of human- derived replacement A1AT protein (Prolastin, Zemaira, or Aralast). The target A1 AT blood level in plasma is greater than or equal to 570 micrograms per milliliter, which generally corresponds to a dose of 60 mg/kg weekly. A1AT replacement therapy can be used for the prevention of lung disease prior to definitive clinical demonstration of efficacy in delaying the onset and/or progression of disease, e.g., to reduce loss in lung density. Other treatment methods currently in use include, but are not limited to, bronchodilators, antibiotics to treat respiratory infections, and vaccination against pneumococcus and influenza. Inhaled corticosteroids and long-acting bronchodilators are also used in subjects with asthmatic symptoms or airflow obstruction. A treatment that prevents the progression of lung disease in A1 AT deficiency without the need for monthly injections would be superior to the current standard of care. The inability of A1 AT replacement therapy to fully prevent the development of lung disease may be due to the fact that replacement therapy does not eliminate the mutant Z-protein from being expressed by circulating monocytes and macrophages. Thus, a treatment that prevents damage to lung tissue that may occur due to the expression of the Z allele by circulating monocytes and macrophages would also be superior to the current standard of care.

[0070] A1 AT deficiency leads to liver disease in up to 50% of A1 AT subjects and leads to severe liver disease in up to 30% of subjects. Liver disease may manifest as: (a) cirrhosis during childhood that is self-limiting, (b) severe cirrhosis during childhood or adulthood that requires liver transplantation or leads to death and (c) hepatocellular carcinoma that is often deadly. The onset of liver disease is bi-modal, predominantly affecting children or adults. Childhood disease is self-limiting in many cases but may be lead to end-stage, deadly cirrhosis. Up to 18% of subjects with the PiZZ genotype may develop clinically significant liver abnormalities during childhood. Approximately 2% of PiZZ subjects develop severe liver cirrhosis leading to death during childhood (Sveger 1988; Volpert 2000). Adult-onset liver disease may affect subjects with all genotypes, but presents earlier in subjects with the PiZZ genotype. Approximately 2-10% of A1AT deficient subjects develop adult-onset liver disease.

[0071] Screening for hepatocellular carcinoma (HCC) includes, e.g., serial hepatic ultrasounds to monitor for the appearance liver nodules. Subjects who develop hepatocellular carcinoma can be treated with chemotherapy and surgery. Subjects who develop liver failure can be treated with a liver transplant. The development of liver disease in A1 AT deficiency may be fatal in a large proportion of subjects: in one study, 40% of adult-onset liver disease subjects survived less than 2 years. A treatment that prevents the development of cirrhosis, liver failure, and hepatocellular carcinoma in subjects with A1 AT deficiency would be vastly superior to the current standard of care. [0072] In certain embodiments, treatment is performed on a subject who has been diagnosed with a mutation in the SERPINA1 gene, but does not yet have disease symptoms. In other embodiments, treatment is performed on an individual who has at least one symptom.

[0073] In certain embodiments, the oligonucleotide increases (e.g., an increase by 100%, 150%, 200%, 300%, 400%, 500%, 600%. 700%, 800%, 900%, 1000% or more, or an increase by more than 1.2-fold, 1.4-fold, 1.5-fold, 1.8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000- fold, or more) protein activity in vitro and/or in vivo.

[0074] In some embodiments, the oligonucleotide increases (e.g., an increase by 100%, 150%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or an increase by more than 1.2-fold, 1.4-fold, 1.5-fold,

I .8-fold, 2.0-fold, 3.0-fold, 3.5-fold, 4.5-fold, 5.0-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 100-fold, 1000- fold, or more) protein activity in the brain.

II. Oligonucleotides

[0075] The oligonucleotides described herein are complementary to target RNA with the exception of at least one mismatch capable of recruiting ADAR enzymes used to edit a target nucleobase on the target RNA, e.g., to deaminate a target adenosine on the target RNA (e.g., SERPINA1). In some embodiments, only one nucleobase (e.g., one adenosine) is edited (e.g., deaminated). In some embodiments, 1, 2, or 3 nucleobases are edited. The oligonucleotide includes a mismatch opposite the target base, e.g., at X² (see structure below). The oligonucleotides described herein may further include modifications (e.g., alternative nucleotides) to increase stability and/or increase deamination efficiency. In some embodiments, the oligonucleotides described herein comprises 1, 2, 3, 4, or 5 mismatches or wobbles.

[0076] In some embodiments, one or more of the nucleotides of an oligonucleotide described herein is chemically modified to enhance stability or other beneficial characteristics. Without being bound by theory, it is believed that certain modification can increase nuclease resistance and/or serum stability or decrease immunogenicity. For example, oligonucleotides described herein may contain nucleotides found to occur naturally in DNA or RNA (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) or may contain nucleotides that have one or more chemical modifications to one or more components of the nucleotide (e.g., the nucleobase, sugar, or internucletside linkage).

[0077] The oligonucleotides described herein comprise the structure:

[A_m]-X¹-X²-X³-[B_n] wherein each of A and B comprise (i) a nucleobase, (ii) a sugar ("an A/B sugar”), and (iii) an internucleotide linkage; each A/B sugar is independently a 2'-methoxyribose, a 2'-deoxy-2'-fluororibose, 2'-methoxyethyl ribose, a 2'- fluoroarabinose, or a 2'-deoxyribose; m is an integer from 20 to 40; n is an integer from 4 to 15; X¹ comprises (i) a nucleobase selected from uracil and thymine, a (ii) 2'deoxyribose sugar, and (iii) an internucleotide linkage; X² comprises a structure

homoDNA sugar with an “N” nucleobase) and an internucleotide linkage, wherein N is a nucleobase; X³ comprises (I) a nucleobase selected from guanosine, hypoxanthine, and 7- deazaguinine, (ii) a sugar selected from 2'-deoxyribose and 2'-deoxy-2'-fluoroarabinose, and (ill) an internucleotide linkage; and the internucleotide linkages of the oligonucleotide comprise at least 30% phosphoramidate and/or phosphorothioate linkages.

[0078] In some embodiments, N is a pyrimidine. In some embodiments, N is a cytosine.

O

R-N=P-OH i °

[0079] Phosphoramidate linkages (e.g., ' , where R is a suitable substituent on the nitrogen, such as an alkyl, sulfoxide, or the like), include mesyl phosphoramidate

Mesyl phosphoramidate is a preferred phosphoramidate internucleotide linkage.

[0080] In some embodiments, the internucleotide linkages of the oligonucleotide comprise at least 30% (e.g., at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or at least 85%) phosphoramidate and/or phosphorothioate linkages.

[0081] In some embodiments, the internucleotide linkages of the oligonucleotide comprise 0% phosphoramidate linkages. In some embodiments, the internucleotide linkages of the oligonucleotide comprise 10-15%, or 35-70%, or 40-85% phosphoramidate linkages. In some embodiments, the internucleotide linkages comprise 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% phosphoramidate linkages.

[0082] In some embodiments, the internucleotide linkages comprise 0% phosphorothioate linkages. In some embodiments, the internucleotide linkages comprise 10-15%, or 35-70%, or 40-85% phosphorothioate linkages. In some embodiments, the internucleotide linkages comprise 10%, 15%, 20%, 25%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85% phosphorothioate linkages. In some embodiments, the internucleotide linkages comprise at least 50% (e.g., 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 85%) phosphorothioate linkages.

[0083] In some embodiments, the internucleotide linkage between X¹ and X² is a phosphorothioate. In some embodiments, the internucleotide linkage between X² and X³ is a phosphorothioate. In some embodiments, the internucleotide linkage between X¹ and X² is a phosphorothioate, and the internucleotide linkage between X² and X³ is a phosphorothioate. [0084] The A/B sugars of the oligonucleotides disclosed herein are each individually 2'-methoxyribose

[0085] In some embodiments, 45-75% (e. g . , 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%, or 75%) of the A/B sugars of the oligonucleotides described herein are 2-methoxyribose. In some embodiments, 20-60% (e.g., 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%, or 60%) of the A/B sugars of the oligonucleotides described herein are 2'-methoxyethylribose. In some embodiments, no A/B sugar is a 2'-deoxyribose or a 2'-methoxyethylribose.

[0086] In some embodiments, 11-20 A/B sugars in the oligonucleotide are 2'-deoxy-2'fluororibose. In some embodiments, the oligonucleotide comprises 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 2'-deoxy-2'fluororibose. [0087] In some embodiments, 18-29 A/B sugars in the oligonucleotide are 2'-methoxyethylribose. In some embodiments, the oligonucleotide comprises 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or 29 2'-methoxyethylribose. [0088] In some embodiments, m is an integer ranging from 21-35. In some embodiments, m is 21, 22, 23, 24, 25, 26, 27, 28, 29. 30, 31, 32, 33, 34 or 35. In some embodiments, m is 30. In some embodiments, [A_m] has a sequence of SEQ ID NO: 142 (AAC AUG GCC CCA GCA GCU UCA GUC CCU UUC) or SEQ ID NO: 143 (AAC AUG GOO CCA GCA GCU UCA GUU CCU UUC) or SEQ ID NO: 144 (AAC AUG GCU CCA GCA GUU UCA GUU CCU UUC). [0089] In some embodiments, n is an integer ranging from 8 to 10. In some embodiments, n is 8, 9, or 10. In some embodiments, n is 9. In some embodiments, [B_n] has a sequence of UCG AUG GUC).

[0090] In some embodiments, the oligonucleotides described herein comprise 4-7 (e.g., 4, 5, 6 or 7) phosphoroamidate linkages. In some embodiments, the oligonucleotides described herein comprise 14 to 30 (e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) phosphorothioate linkages. In some embodiments, the phosphorothioate linkages are Sp phosphorothioate linkages. In other embodiments, the phosphorothioate linkages are Rp phosphorothioate linkages. In some embodiments, the phosphorothioate linkages are stereorandom.

[0091] Exemplary oligonucleotides described herein are shown in Table 3 below (in Hierarchical Editing Language for Macromolecules (HELM) syntax) (Zhang et al., J. Chem. Inf. Model. 2012, 52, 10, 2796-2806), where each nucleotide is noted by terms between periods (.); the first term indicates the sugar moiety, the next is the nucleobase, and last term is the internucleotide linkage. For clarity, the terms can be separated by punctuation, e.g., brackets and parentheses. Thus, a nucleotide designation of “,f(A)P.” means a 2'-deoxy-2'-fluororibose sugar moiety and an adenosine nucleobase that is then linked via a phosphodiester linkage. Sugar moiety designations are "f "for 2'- deoxy-2'-fluororibose, “m” for 2'-methoxyribose, "fana” for 2'-fluroarabinose, “d” is deoxyribose, "hD” or "hd” is beta- homoDNA, and "moe” is 2'-MOEribose. For linkages, "msPA” indicates a mesyl phosphroamidate internucleotide linkage, "sP” indicates a phosphorothioate linkage; and “P” indicates a phosphate linkage.

[0092] .

Table 3. Exemplary Oligonucleotides

[0093] In some embodiments, the oligonucleotides disclosed herein does not include a stem-loop structure. Stem loop structures can act as a recruitment domain for the ADAR enzyme (e.g., an ADAR-recruiting domain), yet the oligonucleotides as disclosed herein can affect ADAR recruitment and activity against a target adenosine in a target RNA without such a stem loop structure.

[0094] In some embodiments, the oligonucleotide described herein may further include a 5' cap structure. In some embodiments, the 5' cap structure is a 2,2,7-trimethylguanosine cap.

[0095] An oligonucleotide described herein can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

[0096] The oligonucleotide can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide including unnatural or alternative nucleotides can be easily prepared. Single-stranded oligonucleotides described herein can be prepared using solution-phase or solidphase organic synthesis or both.

[0097] It is contemplated that for any sequence identified herein, further optimization could be achieved by systematically either adding or removing linked nucleosides to generate longer or shorter sequences. Such optimized sequences can be adjusted by, e.g., the introduction of alternative nucleosides, alternative sugar moieties, and/or alternative internucleotide linkages as described herein or as known in the art, including alternative nucleosides, alternative sugar moieties, and/or alternative internucleotide linkages as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, and/or increasing interaction with RNA editing enzymes (e.g., ADAR)).

[0098] The nucleotides of oligonucleotides described herein are synthesized and/or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Representative U.S. patents that teach the preparation of the oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

[0099] Some embodiments include oligonucleotides with phosphorothioate backbones, and/or oligonucleotides with heteroatom backbones, and in particular -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2-[known as a methylene (methylimino) or MMI backbone], -CH2-O-N(CH₃)-CH₂-, -CH2-N(CH3)-N(CH₃)-CH₂- and -N(CH₃)-CH₂-CH₂-[wherein the native phosphodiester backbone is represented as -O-P-O-CH2-] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the oligonucleotides featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506. In some embodiments, the oligonucleotides described herein include phosphorodiamidate morpholino oligomers (PMO), in which the deoxyribose moiety is replaced by a morpholine ring, and the charged phosphodiester inter-subunit linkage is replaced by an uncharged phophorodiamidate linkage, as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997, 7:63-70.

[0100] An oligonucleotide described herein can include nucleobase (often referred to in the art simply as "base") alternatives (e.g., modifications or substitutions). Unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Alternative nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine, 5-hydroxymethylcytosine, 5- formylcytosine, 5-carboxycytosine, pyrrolocytosine, dideoxycytosine, uracil, 5-methoxyuracil, 5-hydroxydeoxyuracil, dihydrouracil, 4-thiouracil, pseudouracil, 1 -methyl-pseudouracil, deoxyuracil, 5-hydroxybutynl-2'-deoxyuracil, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanine, 7-methylguanine, 7- deazaguanine, 6-aminomethyl-7-deazaguanine, 8-aminoguanine, 2,2,7-trimethylguanine, 8-methyladenine, 8- azidoadenine, 7-methyladenine, 7-deazaadenine, 3-deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8- aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 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-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 8-azaguanine and 8- azaadenine, and 3-deazaguanine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligonucleotides disclosed herein. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5- propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6- 1.2°C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O- methoxyethyl sugar modifications.

[0101] Representative U.S. patents that teach the preparation of certain of the above noted alternative nucleobases as well as other alternative nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

[0102] Oligonucleotide Conjugated to Ligands

[0103] Oligonucleotides described herein may be chemically linked to one or more ligands, moieties, or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553- 6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let, 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett, 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac- glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett, 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett, 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229- 237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Then, 277:923-937).

[0104] In one embodiment, a ligand alters the distribution, targeting, or lifetime of an oligonucleotide agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ, or region of the body, as, e.g., compared to a species absent such a ligand.

[0105] Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly (L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2- hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly (2-ethy lacry llic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[0106] Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0107] Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1 -pyrene butyric acid, dihydrotestosterone, 1 ,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1 ,3-propanediol, heptadecyl group, palmitic acid, myristic acid, 03- (oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0108] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a coligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl- gulucosamine multivalent mannose, or multivalent fucose.

[0109] The ligand can be a substance, e.g., a drug, which can increase the uptake of the oligonucleotide agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0110] In some embodiments, a ligand attached to olgonucleotides described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that include a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases, or 20 bases, including multiple of phosphorothioate linkages in the backbone are also amenable as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

[0111] Ligand-conjugated oligonucleotides described herein may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

[0112] The oligonucleotides used in the conjugates described herein may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

[0113] In the ligand-conjugated oligonucleotides described herein, such as the ligand-molecule bearing sequencespecific linked nucleosides described herein, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0114] When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides described herein are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

[0115] Lipid Conjugates

[0116] In some embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

[0117] A lipid-based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney. [0118] In some embodiments, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. Exemplary vitamins include vitamin A, E, and K.

[0119] Cell Permeation Agents

[0120] In some embodiments, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

[0121] The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to oligonucleotide agents can affect pharmacokinetic distribution of the oligonucleotide, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

[0122] A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 145). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 146) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a "delivery" peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ; SEQ ID NO: 147) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK; SEQ ID NO: 148) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one- compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to an oligonucleotide agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0123] An RGD peptide for use in the compositions and methods described herein may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidomimetics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Some conjugates of this ligand target PECAM-1 or VEGF.

[0124] A cell permeation peptide is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an a- helical linear peptide (e.g., LL-37 or Ceropin P1 ), a disulfide bond-containing peptide (e.g., o-defensin, p-defensin, or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeon! et al., Nucl. Acids Res. 31 :2717-2724, 2003).

[0125] Carbohydrate Conjugates

[0126] In some embodiments, oligonucleotides described herein further includes a carbohydrate. The carbohydrate conjugated oligonucleotide is advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, "carbohydrate" refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include 05 and above (e.g., C5, 06, 07, or 08) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., 05, 06, 07, or 08).

[0127] In some embodiments, a carbohydrate conjugate is a monosaccharide.

[0128] In some embodiments, the carbohydrate conjugate further includes one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

[0129] Additional carbohydrate conjugates (and linkers) include those described in PCT Publication Nos. WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference. [0130] Linkers

[0131] In some embodiments, the conjugate or ligand described herein can be attached to an oligonucleotide with various linkers that can be cleavable or non-cleavable.

[0132] Linkers typically include a direct bond or an atom such as oxygen or sulfur, a unit such as NRs, 0(0), C(O)NH, SO, SO2, SO2NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroaryl alkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocycly I alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by 0, S, S(O), SO2, N(Rs), 0(0), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where Rs is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3- 24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0133] A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times, or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

[0134] Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential, or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selective for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

[0135] A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

[0136] A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a livertargeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

[0137] Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

[0138] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other nontarget tissues. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In some embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0139] Redox Cleavable Linking Groups

[0140] In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (-S-S-). To determine if a candidate cleavable linking group is a suitable "reductively cleavable linking group," or for example is suitable for use with a particular oligonucleotide moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one embodiment, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

[0141] Phosphate-Based Cleavable Linking Groups

[0142] In another embodiment, a cleavable linker includes a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are -O-P(O)(ORk)-O-, O P(S)(ORk) O-, -O-P(S)(SRk)-O-, -S-P(O)(ORk)-O-, -O- P(O)(ORk)-S-, -S-P(O)(ORk)-S-, O P(S)(ORk) S , -S-P(S)(ORk)-O-, -O-P(O)(Rk)-O-, -O-P(S)(Rk)-O-, -S-P(O)(Rk)-O- , -S-P(S)(Rk)-O-, S P(O)(Rk)-S-, -O-P(S)(Rk)-S-. These candidates can be evaluated using methods analogous to those described above.

[0143] Acid Cleavable Linking Groups

[0144] In another embodiment, a cleavable linker includes an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or --OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

[0145] Ester-Based Linking Groups

[0146] In another embodiment, a cleavable linker includes an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula -C(O)O-, or --OC(O)--. These candidates can be evaluated using methods analogous to those described above.

[0147] Peptide-Based Cleaving Groups

[0148] In yet another embodiment, a cleavable linker includes a peptide-based cleavable linking group. A peptide- based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (-C(O)NH-). The amide group can be formed between any alkylene, alkenylene, or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide-based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula -NHCHRAC(O)NHCHRBC(O)--, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

[0149] In some embodiments, oligonucleotides described herein are conjugated to a carbohydrate through a linker. Linkers include bivalent and trivalent branched linker groups. Exemplary oligonucleotide carbohydrate conjugates with linkers include, but are not limited to, those described in formulas 24-35 of PCT Publication No. WO 2018/195165.

[0150] Representative U.S. patents that teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046;

4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;

5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;

5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;

5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

[0151] In certain instances, the oligonucleotide described herein can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to oligonucleotides in order to enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61 ; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let, 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett, 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1 ,2-di-O- hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino- carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Then, 1996, 277:923). Representative United States patents that teach the preparation of such oligonucleotide conjugates have been listed above. Typical conjugation protocols involve the synthesis of an oligonucleotide bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the oligonucleotide still bound to the solid support or following cleavage of the oligonucleotide, in solution phase. Purification of the oligonucleotide conjugate by HPLC typically affords the pure conjugate.

III. Pharmaceutical Uses

[0152] The oligonucleotides described herein may be used to treat alpha-1 -antitrypsin deficiency in a subject in need thereof. In some embodiments, the oligonucleotides described herein, when administered to the subject, can result in correction of a guanosine to adenosine mutation. In some embodiments, the oligonucleotides described herein can result in turning off of a premature stop codon so that a desired protein (e.g., SERPINA1) is expressed. In some embodiments, the oligonucleotides described herein can result in inhibition of expression of an undesired protein (e.g., SERPINA1).

[0153] Deamination of an adenosine using the oligonucleotides disclosed herein includes any level of adenosine deamination, e.g., at least 1 deaminated adenosine within a target sequence (e.g., at least, 1, 2, 3, or more deaminated adenosines in a target sequence).

[0154] Adenosine deamination may be assessed by a decrease in an absolute or relative level of adenosines within a target sequence compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0155] Because the enzymatic activity of ADAR converts adenosines to inosines, adenosine deamination can alternatively be assessed by an increase in an absolute or relative level of inosines within a target sequence compared with a control level. Similarly, the control level may be any type of control level that is utilized in the art, e.g., pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

[0156] The levels of adenosines and/or inosines within a target sequence can be assessed using any of the methods known in the art for determining the nucleotide composition of a polynucleotide sequence. For example, the relative or absolute levels of adenosines or inosines within a target sequence can be assessed using nucleic acid sequencing technologies including but not limited to Sanger sequencing methods, Next Generation Sequencing (NGS; e.g., pyrosequencing, sequencing by reversible terminator chemistry, sequencing by ligation, and real-time sequencing) such as those offered on commercially available platforms (e.g., Illumina, Qiagen, Pacific Biosciences, Thermo Fisher, Roche, and Oxford Nanopore Technologies). Clonal amplification of target sequences for NGS may be performed using real-time polymerase chain reaction (also known as qPCR) on commercially available platforms from Applied Biosystems, Roche, Stratagene, Cepheid, Eppendorf, or Bio-Rad Laboratories. Additionally or alternatively, emulsion PCR methods can be used for amplification of target sequences using commercially available platforms such as Droplet Digital PCR by Bio-Rad Laboratories.

[0157] In certain embodiments, surrogate markers can be used to detect adenosine deamination within a target sequence. For example, effective treatment of a subject having a genetic disorder involving G-to-A mutations with an oligonucleotide of the present disclosure, as demonstrated by an acceptable diagnostic and monitoring criteria can be understood to demonstrate a clinically relevant adenosine deamination. In certain embodiments, the methods include a clinically relevant adenosine deamination, e.g., as demonstrated by a clinically relevant outcome after treatment of a subject with an oligonucleotide of the present disclosure.

[0158] Adenosine deamination in a gene of interest (e.g., SERPINA1) may be manifested by an increase or decrease in the levels of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a gene of interest is transcribed and which has or have been treated (e.g., by contacting the cell or cells with an oligonucleotide of the present disclosure, or by administering an oligonucleotide described herein to a subject in which the cells are or were present) such that the expression of the gene of interest is increased or decreased, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell (s) not treated with an oligonucleotide or not treated with an oligonucleotide targeted to the gene of interest). The degree of increase or decrease in the levels of mRNA of a gene of interest (e.g., SERPINA1) may be expressed in terms of:

(mRNA in control cells) — (mRNA in treated cells)

(mRNA in control cells)

[0159] In other embodiments, change in the levels of a gene may be assessed in terms of a reduction of a parameter that is functionally linked to the expression of a gene of interest, e.g., protein expression of the gene of interest or signaling downstream of the protein. A change in the levels of the gene of interest may be determined in any cell expressing the gene of interest, either endogenous or heterologous from an expression construct, and by any assay known in the art.

[0160] A change in the level of expression of a gene of interest may be manifested by an increase or decrease in the level of the protein produced by the gene of interest (e.g., SERPINA1) that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of mRNA suppression, the change in the level of protein expression in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

[0161] A control cell or group of cells that may be used to assess the change in the expression of a 3 gene of interest includes a cell or group of cells that has not yet been contacted with an oligonucleotide of the present disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an oligonucleotide.

[0162] The level of mRNA of a gene of interest (e.g., SERPINA1) that is expressed by a cell or group of cells may be determined using any method known in the art for assessing mRNA expression. In one embodiment, the level of expression of a gene of interest in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the gene of interest (e.g., SERPINA1). RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNEASY™ RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating mRNA of the gene of interest may be detected using methods the described in PCT Publication WC2012/177906, the entire contents of which are hereby incorporated herein by reference. In some embodiments, the level of expression of the gene of interest is determined using a nucleic acid probe. The term "probe," as used herein, refers to any molecule that is capable of selectively binding to a specific sequence, e.g. to an mRNA or polypeptide. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0163] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses, and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to the mRNA of a gene of interest. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an AFFYMETRIX gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of mRNA of a gene of interest.

[0164] An alternative method for determining the level of expression of a gene of interest in a sample involves the process of nucleic acid amplification and/or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizard! et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizard! et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects, the level of expression of a gene of interest is determined by quantitative fluorogenic RT-PCR (i.e., the TAQMAN™ System) or the DUAL-GLO® Luciferase assay.

[0165] The expression levels of mRNA of a gene of interest may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support including bound nucleic acids). See U.S. Pat. Nos. 5,770,722; 5,874,219; 5,744,305; 5,677,195; and 5,445,934, which are incorporated herein by reference. The determination of gene expression level may also include using nucleic acid probes in solution. [0166] In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of nucleic acids of the gene of interest.

[0167] The level of protein produced by the expression of a gene of interest may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of proteins produced by the gene of interest. Additionally, the above assays may be used to report a change in the mRNA sequence of interest that results in the recovery or change in protein function thereby providing a therapeutic effect and benefit to the subject, treating a disorder in a subject, and/or reducing of symptoms of a disorder in the subject.

[0168] In some embodiments, the oligonucleotides described herein are administered to a subject such that the oligonucleotide is delivered to a specific site within the subject. The change in the expression of the gene of interest may be assessed using measurements of the level or change in the level of mRNA or protein produced by the gene of interest in a sample derived from a specific site within the subject.

[0169] In other embodiments, the oligonucleotides described herein are administered in an amount and for a time effective to result in one of (or more, e.g., two or more, three or more, four or more of): (a) decrease the number of adenosines within a target sequence of the gene of interest, (b) delayed onset of the disorder, (c) increased survival of subject, (d) increased progression free survival of a subject, (e) recovery or change in protein function, and (f) reduction in symptoms of the disorder.

[0170] Treating disorders associated with G-to-A mutations can also result in a decrease in the mortality rate of a population of treated subjects in comparison to an untreated population. For example, the mortality rate is decreased by more than 2% (e.g., more than 5%, 10%, or 25%). A decrease in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating for a population the average number of disease-related deaths per unit time following initiation of treatment with a compound or pharmaceutically acceptable salt of a compound described herein. A decrease in the mortality rate of a population may also be measured, for example, by calculating for a population the average number of disease-related deaths per unit time following completion of a first round of treatment with a compound or pharmaceutically acceptable salt of a compound described herein.

A. Delivery of Oligonucleotides

[0171] The delivery of an oligonucleotide described herein to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an oligonucleotide described herein either in vitro or in vivo, and can be performed ex vivo. In vivo delivery may also be performed directly by administering a composition including an oligonucleotide to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the oligonucleotide. Combinations of in vitro and in vivo methods of contacting a cell are also possible. Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, e.g., a GalNAca ligand, or any other ligand that directs the oligonucleotide to a site of interest. Cells can include those of the central nervous system, or muscle cells. These alternatives are discussed further below.

[0172] Contacting of a cell with an oligonucleotide may be done in vitro or in vivo or ex vivo. For in vivo delivery, factors to consider in order to deliver an oligonucleotide molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an oligonucleotide can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the oligonucleotide molecule to be administered.

[0173] For administering an oligonucleotide systemically for the treatment of a disease, the oligonucleotide can include alternative nucleobases, alternative sugar moieties, and/or alternative internucleotide linkages, or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the oligonucleotide by endo- and exo-nucleases in vivo. Modification of the oligonucleotide or the pharmaceutical carrier can also permit targeting of the oligonucleotide composition to the target tissue and avoid undesirable off-target effects. Oligonucleotide molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. In an alternative embodiment, the oligonucleotide can be delivered using drug delivery systems such as a nanoparticle, a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an oligonucleotide molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an oligonucleotide by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an oligonucleotide, or induced to form a vesicle or micelle that encases an oligonucleotide. The formation of vesicles or micelles further prevents degradation of the oligonucleotide when administered systemically. In general, any methods of delivery of nucleic acids known in the art may be adaptable to the delivery of the oligonucleotides described herein. Methods for making and administering cationic oligonucleotide complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of oligonucleotides include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, "solid nucleic acid lipid particles" (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. Aug 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an oligonucleotide forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of oligonucleotides and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety. In some embodiments the oligonucleotides described herein are delivered by polyplex or lipoplex nanoparticles. Methods for administration and pharmaceutical compositions of oligonucleotides and polyplex nanoparticles and lipoplex nanoparticles can be found in U.S. Patent Application Nos. 2017/0121454; 2016/0369269; 2016/0279256; 2016/0251478; 2016/0230189; 2015/0335764; 2015/0307554; 2015/0174549; 2014/0342003; 2014/0135376; and 2013/0317086, which are herein incorporated by reference in their entirety. i. Membranous Molecular Assembly Delivery Methods

[0174] Oligonucleotides described herein can also be delivered using a variety of membranous molecular assembly delivery methods including polymeric, biodegradable microparticle, or microcapsule delivery devices known in the art. For example, a colloidal dispersion system may be used for targeted delivery an oligonucleotide agent described herein. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as delivery vehicles in vitro and in vivo. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the oligonucleotide are delivered into the cell where the oligonucleotide can specifically bind to a target RNA and can mediate RNase Fl- mediated gene silencing. In some cases, the liposomes are also specifically targeted, e.g., to direct the oligonucleotide to particular cell types. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids may also be used. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations.

[0175] A liposome containing an oligonucleotide can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the oligonucleotide and condense around the oligonucleotide to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of oligonucleotide.

[0176] If necessary, a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0177] Methods for producing stable oligonucleotide delivery vehicles, incorporating a oligonucleotide/cationic lipid complex as a structural component of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging oligonucleotide preparations into liposomes.

[0178] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0179] Liposomes entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0180] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0181] Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11 :417.

[0182] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems including non-ionic surfactant and cholesterol. Non-ionic liposomal formulations including NOVASOME™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466). [0183] Liposomes may also be sterically stabilized liposomes, including one or more specialized lipids that result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) includes one or more glycolipids, such as monosialoganglioside GMI, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0184] Various liposomes including one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglio side G^M1, galactocerebroside sulfate, and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes including (1) sphingomyelin and (2) the ganglioside GMI or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes including sphingomyelin. Liposomes including 1,2-sn- dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

[0185] In some embodiments, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver oligonucleotides to macrophages.

[0186] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated oligonucleotides in their internal compartments from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms," Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0187] A positively charged synthetic cationic lipid, N-[1 -(2,3-dioleyloxy)propyl]-N,N, N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of oligonucleotides (see, e.g., Feigner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

[0188] A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. LIPOFECTI N™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that include positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1 ,2-bis(oleoyloxy)-3,3- (trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

[0189] Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0190] Another cationic lipid conjugate includes derivatization of the lipid with cholesterol ("DC-Chol") which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L, (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

[0191] Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer oligonucleotides into the skin. In some implementations, liposomes are used for delivering oligonucleotides to epidermal cells and also to enhance the penetration of oligonucleotides into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2,405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176;

Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101 :512-527; Wang, C. Y. and Huang, L, (1987) Proc. Natl. Acad. Sci. USA 84:7851 -7855).

[0192] Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems including non-ionic surfactant and cholesterol. Non-ionic liposomal formulations including Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with oligonucleotide are useful for treating a dermatological disorder.

[0193] The targeting of liposomes is also possible based on, for example, organ-specificity, cell-specificity, and organelle-specificity and is known in the art. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting ligand in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting ligand. Additional methods are known in the art and are described, for example in U.S. Patent Application Publication No. 20060058255, the linking groups of which are herein incorporated by reference. [0194] Liposomes that include oligonucleotides can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include oligonucleotides can be delivered, for example, subcutaneously by infection in order to deliver oligonucleotides to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transfersomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often selfloading. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0195] Other formulations amenable to the disclosed oligonucleotides and methods are described in WO 2009/086558, and WO 2009/088891 . WO 2008/042973 also describes formulations that are amenable to the present oligonucleotides and methods.

[0196] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0197] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0198] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0199] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class. [0200] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N- alkylbetaines, and phosphatides.

[0201] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

[0202] The oligonucleotide for use in the methods described herein can also be provided as micellar formulations. Micelles are a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic. ii. Lipid Nanoparticle-Based Delivery Methods

[0203] Oligonucleotides described herein may be fully encapsulated in a lipid formulation, e.g., a lipid nanoparticle (LNP), or other nucleic acid-lipid particle. LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include "pSPLP," which include an encapsulated condensing agent- nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567;

5,981 ,501 ; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

[0204] In some embodiments, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to oligonucleotide ratio) will be in the range of from about 1 : 1 to about 50:1 , from about 1 :1 to about 25:1 , from about 3: 1 to about 15:1 , from about 4:1 to about 10:1 , from about 5:1 to about 9: 1 , or about 6: 1 to about 9: 1. Ranges intermediate to the above recited ranges are also contemplated to be part described herein.

[0205] Non-limiting examples of cationic lipid include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N- distearyl-N,N-dimethylammonium bromide (DDAB), N- (l-(2,3-dioleoyloxy)propyl)-N, N,N-trimethylammonium chloride (DOTAP), N— (l-(2,3-dioleyloxy)propyl)-N, N, N-trimethylammonium chloride (DOTMA), N, N-dimethyl-2,3- dioleyloxy)propylamine (DODMA), 1 ,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1 ,2-Dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), 1 ,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1 ,2- Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1 ,2- Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1 ,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1- Li nol eoyl-2-l i noleyloxy-3-d i methyl amin opropane (DLin-2-DMAP), 1 , 2-D i lin oleyloxy-3-tri methyl ami nopropane chloride salt (DLin-TMA.CI), 1 ,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1,2-Dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1 ,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)- 1 ,2-propanedio (DOAP), 1 ,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1 ,2- Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1 ,3]-dioxolane (DLin-K- DMA) or analogs thereof, (3aR,5s,6aS)-N, N-dimethyl-2,2-di((9Z, 12Z)-octadeca-9, 12-dienyetetrahydro— 3aH- cyclopenta[d][1 ,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-

(dimethylamino)bu- tanoate (MC3), 1 ,T-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)ami- no)ethyl)piperazin-1 -yeethylazanediyedidodecan-2-ol (Tech G1), or a mixture thereof. The cationic lipid can include, for example, from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

[0206] The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 -carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16- O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be, for example, from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

[0207] The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)- lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxy propyl (Ci2), a PEG-dimyristyloxypropyl (Ci4), a PEG-dipalmity loxy propyl (Cie), or a PEG-distearyloxy propyl (C]s). The conjugated lipid that prevents aggregation of particles can be, for example, from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

[0208] In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 50 mol % of the total lipid present in the particle.

IV. Pharmaceutical Compositions

[0209] The oligonucleotides described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0210] The oligonucleotides described herein may be administered, for example, by oral, parenteral, intrathecal, intracerebroventricular, intraparenchymal, buccal, sublingual, nasal, rectal, patch, pump, intratumoral, or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, intracerebroventricular, intraparenchymal, rectal, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0211] An oligonucleotide described herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard- or soft-shell gelatin capsules, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, an oligonucleotide described herein may be incorporated with an excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. An oligonucleotide described herein may also be administered parenterally. Solutions of an oligonucleotide described herein can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and in The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that may be easily administered via syringe. Compositions for nasal administration may conveniently be formulated as aerosols, drops, gels, and powders. Aerosol formulations typically include a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomizing device. Alternatively, the sealed container may be a unitary dispensing device, such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form includes an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air or an organic propellant, such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer. Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier, such as sugar, acacia, tragacanth, gelatin, and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base, such as cocoa butter. An oligonucleotide described herein may be administered intratumorally, for example, as an intratumoral injection. Intratumoral injection is injection directly into the tumor vasculature and is specifically contemplated for discrete, solid, accessible tumors. Local, regional, or systemic administration also may be appropriate.

[0212] The oligonucleotides described herein may be administered to an animal, e.g., a human, alone or in combination with pharmaceutically acceptable carriers, as noted herein, the proportion of which is determined by the solubility and chemical nature of the oligonucleotide, chosen route of administration, and standard pharmaceutical practice.

V. Dosages

[0213] The dosage of the compositions (e.g., a composition including an oligonucleotide) described herein, can vary depending on many factors, such as the pharmacodynamic properties of the compound; the mode of administration; the age, health, and weight of the recipient; the nature and extent of the symptoms; the frequency of the treatment, and the type of concurrent treatment, if any; and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compositions described herein may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. In some embodiments, the dosage of a composition (e.g., a composition including an oligonucleotide) is a prophylactically or a therapeutically effective amount.

VI. Kit

[0214] Provided herein are kits including (a) a pharmaceutical composition including an oligonucleotide that results in deamination of an adenosine in an mRNA in a cell or subject described herein, and (b) a package insert with instructions to perform any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition including an oligonucleotide that results in deamination of an adenosine in an mRNA in a cell or subject described herein, (b) an additional therapeutic agent, and (c) a package insert with instructions to perform any of the methods described herein.

[0215] Embodiments:

1 . An oligonucleotide comprising the structure:

[A_m]-X1-X2-X3-[Bn] wherein each A and B comprises a nucleobase, a sugar ("an A/B sugar”), and an internucleotide linkage; each A/B sugar is independently a 2'-methoxyribose, , a 2'-methoxyethylribose, a 2'-deoxy-2'fluororibose, a 2'- fluoroarabinose, or a 2'-deoxyribose; m is 20 to 40; n is 4 to 15;

X² comprises a structure

internucleotide linkage, wherein N is a nucleobase;

2. The oligonucleotide of embodiment 1 , wherein N is a pyrimidine.

3. The oligonucleotide of embodiment 1 or embodiment 2, wherein N is cytosine.

4. The oligonucleotide of any one of embodiment 1 to 3, wherein the internucleotide linkages comprise 0% phosphoramidate linkages.

5. The oligonucleotide of any one of embodiments 1 to 3, wherein the internucleotide linkages comprise 10-15% phosphoramidate linkages.

6. The oligonucleotide of any one of embodiments 1 to 5, wherein the internucleotide linkages comprise 35-70% phosphorothioate linkages.

7. The oligonucleotide of any one of embodiments 1 to 6, wherein the internucleotide linkages comprise at least 50% phosphorothioate linkages.

8. The oligonucleotide of any one of embodiments 1 to 7, wherein the internucleotide linkage between X¹ and X² is a phosphorothioate. 9. The oligonucleotide of any one of embodiments 1 to 8, wherein the internucleotide linkage between

X² and X³ is a phosphorothioate.

10. The oligonucleotide of any one of embodiments 1 to 9, wherein the internucleotide linkage between X¹ and X² is a phosphorothioate, and the internucleotide linkage between X² and X³ is a phosphorothioate.

11 . The oligonucleotide of any one of embodiments 1 to 10, wherein 45-75% of the A/B sugars are 2'- methoxy ribose.

12. The oligonucleotide of any one of embodiments 1 to 11 , wherein 20-60% of the A/B sugars are 2'- deoxy-2'-fluororibose.

13. The oligonucleotide of any one of embodiments 1 to 12, wherein no A/B sugar is a 2'-deoxyribose.

14. The oligonucleotide of any one of embodiments 1 to 13, wherein no A/B sugar is a 2'- methoxyethylribose.

15. The oligonucleotide of any one of embodiments 1 to 14, wherein m is 23 to 35.

16. The oligonucleotide of embodiment 15, wherein m is 30.

17. The oligonucleotide of embodiment 16, wherein [A_m] has a sequence of SEQ ID NO: 142 (AAC

AUG GCC CCA GCA GCU UCA GUC CCU UUC) or SEQ ID NO: 143 (AAC AUG GCC CCA GCA GCU UCA GUU

CCU UUC) or SEQ ID NO: 144 (AAC AUG GCU CCA GCA GUU UCA GUU CCU UUC).

18. The oligonucleotide of any one of embodiments 1 to 17, wherein n is 8 to 10.

19. The oligonucleotide of embodiment 18, wherein n is 9.

20. The oligonucleotide of embodiment19, wherein [B_n] has a sequence UCG AUG GUC.

21. The oligonucleotide of embodiment 16, 17, 19 or 20 having 4-7 phosphoramidate linkages.

22. The oligonucleotide of embodiment 16, 17, 19, 20, or 21, having 14-30 phosphorothioate linkages.

23. The oligonucleotide of embodiment 16, 17, 19, 20, 21 , or 22, wherein 11 -20 A/B sugars are 2'- deoxy-2'-fluororibose.

24. The oligonucleotide of embodiment 16, 17, 19, 20, 21, 22, or 23, wherein 18-29 A/B sugars are 2'- methoxy ribose.

25. The oligonucleotide of any one of embodiments 1 to 24, sufficiently complementary to part of a target RNA having a target adenosine and capable of forming a complex with the target RNA.

26. The oligonucleotide of embodiment 24, wherein, upon formation of the complex with the target RNA, the nucleotide of the oligonucleotide opposite the target adenosine is X². 27. The oligonucleotide of embodiment 25 or 26, capable of binding and recruiting an ADAR enzyme to perform editing on the target adenosine of the target RNA.

28. The oligonucleotide of any one of embodiment 1 to 27, wherein the oligonucleotide does not comprise a portion that is capable of forming an intramolecular stem-loop structure.

29. A complex comprising the oligonucleotide of any one of embodiment 1 to 28 and a target RNA, the complex formed by hybridization between the oligonucleotide and the target RNA.

30. The complex of embodiment 29, comprising 1 , 2, 3, 4, or 5 mismatches or wobbles.

31 . A method of editing a target adenosine in a target RNA in a cell comprising contacting the cell with the oligonucleotide of any one of embodiment 1 to 28 to (i) form a complex between the oligonucleotide and the target RNA such that X² of the oligonucleotide is opposite the target adenosine; and (ii) recruit an ADAR in the cell to the complex such that the ADAR edits the target adenosine.

32. A method of treating alpha-1 -antitrypsin deficiency in a subject in need thereof, comprising administering to the subject an effective amount of the oligonucleotide of any one of embodiments 1 to 28 to treat the alpha-1 -antitrypsin deficiency.

EXAMPLES

General Methods

[0216] All guide oligonucleotides were chemically synthesized on an automated RNA/DNA synthesizer using standard p-cyanoethylphosphoramidite chemistry and a universal solid support such as controlled pore glass (CPG). Phosphoramidites of N-protected p-homo-DNA was synthesized utilizing reported procedures. See Matheus Froeyen et al., (2001) Chem. Eur. J., 7: 5183-5794, Herdewijn, (2010) Chem. Biodivers., 7: 1-59, Jabgunde et al., (2019) Tetrahedron, 75: 1107-1114. Other 5'-O-DMT-3'-phosphoramidite RNA, 2’-O-methyl-RNA and DNA monomers, i.e., A, C, G, U, and T, were purchased from commercial sources. All oligonucleotides were synthesized by BioSpring GmbH (Frankfurt, Germany) at a 200 nmol scale. After synthesis, oligonucleotides were cleaved from the solid support, deprotected, and purified by a HPLC system using standard protocols. Oligonucleotides were desalted, dialyzed, and lyophilized. The purity of each lyophilized oligo was >95% as determined by analytical reversed-phase HPLC. The sequence integrity of the oligonucleotides was determined by ESI-MS. (The sequences of the various oligonucleotides are provided herein in Table 3).

[0217] Human ADAR2 sequence (NM_001112.4) was cloned into pcDNA3.1 plasmid under the control of the CMV promoter using BamHI and Xbal restriction sites (Quintara Bio, Berkeley, CA) and the correct insert was sequence verified. This plasmid henceforth will be denoted as ADAR2/pcDNA3.1 . For editing experiments, 2 pg of ADAR2/pcDNA3.1 plasmid were transfected into 5x10⁶ HEK293T cells (ATCC) using 25 pL of Lipofectamine 3000 and 24 pL of P3000 (Life Technologies) per 10 cm dish. After 4 hours, the culture media was replenished with fresh warmed media (DMEM High Glucose; Life Technologies). 12-16 hours after transfection, the transfected HEK293T cells were transfected with guide oligonucleotides such that the final concentration in the each well was 100 nM. All transfections were carried out with Lipofectamine 3000 (0.4 pL/per well) in a 96-well format, according to manufacturer's instructions. 12-16 hours after the second transfection, the cells were washed once with ice cold PBS and total mRNA isolation was performed using Dyna Beads mRNA Direct Kit (Life Technologies) adapted for KingFisher Flex Purification (Life Technologies), according to manufacturer's instructions. The samples were treated with TURBO DNase (Life Technologies) prior to elution. The resultant isolated mRNA was used for cDNA synthesis using SuperScript IV Vilo according to the manufacturer's instructions (Life Technologies). One l of the cDNA was used as template for PCR (Platinum II Hot-Start PCR Master Mix; Life Technologies) using gene specific primers to generate an amplicon for Sanger sequencing. Sanger sequencing was performed by Quintara Biosciences (Berkeley, CA). Adenosine to guanosine editing yields were quantified by measuring the peak height of adenosine and guanosine and dividing the guanosine peak height by the total peak height measurements of adenosine and guanosine combined.

Example 1 - In vitro Editing of the SERPINA1 gene

[0218] MZ Primary human hepatocyte (PHH) cells were washed in 37°C UCRM (Discovery Life Sciences: 81015) and spun down (100g, 10 min) before being resuspended in 4°C UPCM-A (Discovery Life Sciences: 81070) for plating. The cells were plated to 100% confluency (56,000 cells/well) in a collagen I coated 96-well and left to recover in 37°C incubator maintained at 95% balanced air and 5% CO2 for 4 hours prior to a media change to 90 ml of HIM media (Discovery Life Sciences, cat: 81018). The cells were transfected with compounds at the specified concentrations diluted in transfection reagent consisting of a 1 :35 ratio of OPTI-MEM (Gibco: 31985-0662) and lipofectamine RNAIMAX (Invitrogen: 13778-150). After a 15-minute room temperature incubation, 10 ml of the transfection reagents was added on top of the HIM media. The following morning, cells that were being cultured for greater than 2 days received a media change with HIM containing 0.3 mg/ml of Geltrex (Thermo Fisher A1413202). Collection of RNA was conducted on days 2, 4, and 7. The plate that went to day 7 had an additional media change with HIM (without Geltrex) on day 4.

[0219] mRNA Isolation: After culturing, the RNA was isolated using Oligo d(T) 25 Magnetic Dynabeads (New England BioLabs: S1419S). The beads were washed 3 times in lysis/binding buffer (Invitrogen: A33562), 10 ul/well. The washed beads were then resuspended in 200ul of lysis/binding buffer and added to the plate containing the cells for lysis and RNA binding. Post addition of the beads/buffer combination, the plate was mixed 10 times by pipetting and placed on a magnetic plate to hold the beads in place. After 5 minutes, the lysis buffer was aspirated and 150 ml of wash buffer A (Invitrogen: A33565) was added for a one-minute soak. This step was repeated one additional time. After the second wash, buffer A removed and 150 ml of wash buffer B (Invitrogen: 11900D) was added to soak for one minute. This step was repeated a second time. To elute, buffer B was removed and 50 ul of 10 mM Tris HCI, pH 7.5 (Boston BioProducts: C-9787K) was added. The plate was then placed onto a heat block at 80°C for 2 minutes. [0220] cDNA preparation: cDNA was generated using the SuperScript™ IV VILO™ Master Mix with ezDNase™ Enzyme kit (Thermo: 11766500). Genomic DNA was first removed by combining 16 ml of the isolated RNA with 2 ml of 10x EZ DNase buffer, 1 ml of EZ DNase enzyme, and 1 ml of dH2O for a total volume of 20 ml. The plate was then sealed and incubated in a thermocycler at 37°C for 2 minutes. Post incubation, a mix containing 2.5 ml of SuperScript IV Vilo MasterMix and 2.5 ml of dH2O was added to the reaction plate. Once resealed the following SuperScript incubation protocol was used in a thermocycler; 25°C for 10 minutes, 50°C for 10min, 85°C, for 5 minutes, and hold at 4°C forever.

[0221] NGS and editing analysis: The percent WT Serpin A1 transcript was measured via NGS amplicon sequencing. The amplicons were generated from the cDNA with forward primer PRI_KB-113F (5’- ACCTATGATCTGAAGAGCGTCCT-3', SEQ ID NO: 145) and reverse primer PRI_KB-355R (5’- TTCAATCATTAAGAAGACAAAGGGT-3', SEQ ID NO: 146). Percent editing was calculated by dividing the number of reads containing the WT by the total number of reads (WT+mutant) [WT reads I (WT + mutant) * 100 = percent editing]

[0222] As shown in Figures 1A and 1B, all oligonucleotides had significant editing efficiency of the Z allele conversion to the M allele on the mRNA in the primary M/Z human hepatocytes at both 10 and 100nM concentrations. More editing was observed at the 100nM concentration.

[0223] Next, Def-HEP human hepatocyte-like cells (zzHLC) (Definigen) were seeded into collagen-coated 384-well plates at 15000cells/well and underwent differentiation in hypoxia chamber following vendor's instruction. Two weeks post seeding, ASOs were transfected in cells at a final concentration of 10nM, and 1nM using Lipofectamine RNAIMAX (Life Technologies, #13778500) at a ratio of 1:25 (RNAIMax to OptiMEM) in the presence of HJ/piL recombinant human Interferon alpha (Millipore Sigma, #l F007). Cells were incubated for 48hrs at 37°C in hypoxia chamber. After 48hr incubation, Cell images were taken on EVOS M700 and mRNA was extracted from the transfected cells using the Dynabeads® Oligo (dT)25 (Life Technologies, 61005) and associated buffers adapted for purification on an EL406 plate washer (BioTek). When the isolation protocol was completed, each plate was heated at 80°C for 2 minutes to allow RNA to elute from beads, and 8uL of the elute was transferred into PCR plates for processing. 2uL of EZ DNase Master Mix (Life Technologies, #11766051) was added to each well on the plate. The plate was sealed and centrifuged at lOOOrpm for 1 minute and allowed to incubate in Thermocycler at 37°C for 10 minutes. 10uL of Vilo Master Mix (Life Technologies, # 11756050) was added to each well on the plate. The plate was sealed and centrifuged at lOOOrpm for 1 minute and allowed to incubate in Thermocycler at 25°C for 10 minutes, 50°C for 10 minutes, and 85°C for 5 minutes. After processing, 10uL of cDNA was stamped in a new 384-well PCR Plate and sent for Next Generation Sequencing (NGS), Amplicon Sequencing by Quintara Biosciences. The amplicons were generated from the cDNA with forward primer PRI_KB-113F (5'-ACCTATGATCTGAAGAGCGTCCT- 3’, SEQ ID NO: 145) and reverse primer PRI_KB-355R (5'-TTCAATCATTAAGAAGACAAAGGGT-3', SEQ ID NO: 146). Editing yields were quantified by counting the number of sequencing reads with A and I base calls at the target site, and dividing the number of reads containing a I by the total number of reads containing A and I. An empirical p- value for editing in each sample was calculated using kernel density estimation over the frequency distribution of errors across the amplicon. As shown in Figure 6, oligonucleotides #128-#197 also had significant editing. More editing was observed at the 100 nm concentration.

[0224] Cells were transfected with oligonucleotide #5, #7, #3, #6 and #4 at indicated doses in Figure A and incubated for 48hrs in the presence of HJ/piL recombinant human Interferon alpha before mRNA extraction. Editing percentage was assessed using amplicon sequencing. Experiment was performed 3 independent times while the figure included one representative experiment. Each condition has four technical replicates. At 10nM, 30-40% A to I editing at target site was achieved while at 1 nM, 10-20% A to I editing was achieved in zzHLC. [0225] A select number of oligonucleotides (e.g., oligonucleotide #5, #7, #3, #6 and #4) demonstrated at least 20% editing efficiency in hepatocyte-like cells (Figure 2) and at least 50% editing efficiency in M/Z primary hepatocytes (data not shown), in vitro. As shown in Figure 2, three oligos (oligo #3, oligo #5, and oligo #6) demonstrated similar in vitro editing 48h after transfection in zzHLC cells. At 10nM, oligo #3, oligo #6, and oligo #7 achieved 29%, 29% and 35% editing respectively, and at 1 nM, they achieved 13%, 15% and 16% editing respectively.

Example 2 - In vivo Editing of the SERPINA1 gene

[0226] Delivery In Vivo -All procedures for animal experimentation were approved by the Institutional Animal Care and Use Committee and conducted in accordance with their guidelines. PiZ female mice (age 6-8 weeks) were weighed and randomly distributed into groups of three mice per group. LNP formulations in MC3 were dosed via the lateral tail vein in a volume of 0.2 mL per animal at a 2mg/kg dose. Animals were euthanized by exsanguination and blood collected via cardiac puncture using a 1 mL syringe with a 25G needle. A minimum of 500uL of whole blood is collected and transferred to K2EDTA micro blood collection tubes. Liver tissue is also extracted from the mouse. [0227] Mouse liver tissue samples were pulverized with 2010 Geno/Grinder®following vendor manual. 5-10 mg of pulverized tissue was transferred to Fisherbrand™ Bead Mill 24 Homogenizer (Fisher Scientific, #15-340-163) tubes and homogenized with Trizol. Chroloform was added to each sample. After centrifuge, aqueous layer was transferred to a silica-membrane RNeasy 96 plate from RNeasy 96 Kit (Qiagen, #74181). RNA extraction was performed in Qiacube HT following standard miRNeasy protocol from vendor. The isolated mRNA was measured in nanodrop and cDNA was generated using up to 1 ug of RNA from each sample. 4 uL Superscipt VILO IV Master Mix (Life Technologies, # 11756050), 14 uL RNase-free water, 2 uL of diluted RNA (up to 1 ug of RNA) were added to each reaction in a 96 well plate. The plate was sealed and centrifuged at lOOOrpm for 1 minute and allowed to incubate in Thermocycler at 25°C for 10 minutes, 50°C for 10 minutes, and 85°C for 5 minutes. dPCR was performed on QIAcuity eight dPCR system (QIAGEN, #911056), on QIAcuity Nanoplate 8.5k 96-well (QIAGEN, #250021), using QIAcuity Probe PCR Kit (QIAGEN, #250103) following vendor protocol. Primer and probe for WT and mutant Serpin A1 are listed in the table below. Raw data was analyzed in Qiagen plate configurator and Editing percentage was calculated by the concentrations of each target.

[0228] As shown in Figure 3, several oligonucleotides successfully edited the SERPINA1 gene after 2 mg/kg dosing. Mouse liver tissue samples dosed with oligo #3 showed a relatively stable profile of around 20% on day 1 and 4, with a slight dip in editing to 16.7% and increased variability among mice on day 7. Oligo #6 performed 1.5 times as well as oligo #3 on day 1 and 4 around 30%, but showed a similar level of editing at 13% on day 7. Finally, oligo #5 showed a similar initial level of editing to oligo #6 (with increased variance on day 4) around 30%, and although it did show a decrease in editing on day 7, the observed level of 24% editing was still higher than the editing levels recorded on day 1 in oligo #3.

[0229] A1AT protein expression was assessed, mice (n=3) were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASOs. Plasma was isolated at appropriate time point, and WT-A1AT and Total-A1AT were measured using LC/MS-MS methods as described. Figure 3B provide the level and percentage, respectively, of wildtype A1 AT expression in plasma. As shown in Figure 3B, each of oligonucleotide #4, #5, #6, #7 and #3 increased the total A1 AT protein level in plasma of treated mice.

[0230] Next, editing percentage in PiZ mice liver tissue 7-days post either 1 , 2, 3, or 4 doses of oligo 3 encapsulated in MC3 LNP was assessed. PiZ mice (n=5) were dosed intravenously with vehicle (DPBS) or 2mg/kg indicated ASOs for either 1,2,3, or 4 doses. Liver tissues were collected 7 days post dosing. Editing percentage at each timepoint was assessed by dPCR from mRNA harvested in pulverized liver tissue. Multi-dosing with oligonucleotide #3 using MC3 in vivo resulted in -50% editing after 3 doses (Figure 4A), and provided high levels of M-A1AT concentration in serum (Figure 4B).

Example 3 - Oligonucleotide Stability Assay in Liver Homogenate

[0231] The stability of different oligos in human liver homogenate was assessed.

[0232] Preparation human liver homogenate: 8 mL of 20 mg/mL Human liver Homogenate was transferred to a clean vial and mixed with 8 mL 50 mM Tris-HCI, 150 mM KCI, pH 7.2. For each mL of homogenate, 10piL of 100mM MgCh and 20 piL 100x antibiotics was added)

[0233] 14 piL of oligo (100|JM) was mixed with 686 piL of 10 mg/mL protein human liver homogenate and stored at either -20°C or -80°C and stability was assessed at hour 0, at hour 24, hour 96, and hour 120, using Protein K digestion followed by liquid-liquid extraction, and LC-MS analysis. The data was analyzed and processed using Thermo Xcalibur software v4.0. All samples were run in Full Scan mode to identify their respective m/z fingerprints. The highest intensity ion (Q1) was selected, and the top three m/z ratios were used for quantitation. Based on the selected top three m/z ratios, the peak area ratio was calculated and determined.

[0234] The experiment was repeated using mouse liver homogenate in place of the human liver homogenate.

[0235] Results showed that significant stability was observed for oligo #3, while moderate stability was observed for oligo #5 and oligo #6. Figures 5A and 5B.

Other Embodiments

[0236] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.

[0237] While the invention has been described in connection with specific embodiments thereof, it will be understood that invention is capable of further modifications and this application is intended to cover any variations, uses, or adaptations described herein following, in general, the principles described herein and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth, and follows in the scope of the claims.

Claims

1 . An oligonucleotide comprising the structure:

[A_m]-X1-X2-X3-[B_n] wherein each A and B comprises a nucleobase, a sugar ("an A/B sugar”), and an internucleotide linkage; each A/B sugar is independently a 2'-methoxyribose, a 2'-methoxyethylribose, a 2'-deoxy-2'fluororibose, a 2'- fluoroarabinose, or a 2'-deoxyribose; m is 20 to 40; n is 4 to 15;

X¹ comprises (i) a nucleobase selected from uracil and thymine, (ii) a 2'deoxyribose sugar, and (iii) an internucleotide linkage;

X² comprises a structure

internucleotide linkage, wherein N is a nucleobase;

X³ comprises (i) a nucleobase selected from guanosine, hypoxanthine, and 7-deazaguinine, (ii) a sugar selected from 2'-deoxyribose and 2'-deoxy-2'-fluoroarabinose, and (iii) an internucleotide linkage; and the internucleotide linkages of the oligonucleotide comprise at least 30% phosphoramidate and/or phosphorothioate linkages.

2. The oligonucleotide of claim 1 , wherein N is a pyrimidine.

3. The oligonucleotide of claim 1 or claim 2, wherein N is cytosine.

4. The oligonucleotide of any one of claims 1 to 3, wherein the internucleotide linkages comprise 0% phosphoramidate linkages.

5. The oligonucleotide of any one of claims 1 to 3, wherein the internucleotide linkages comprise 10- 15% phosphoramidate linkages.

6. The oligonucleotide of any one of claims 1 to 5, wherein the internucleotide linkages comprise 35- 70% phosphorothioate linkages.

7. The oligonucleotide of any one of claims 1 to 6, wherein the internucleotide linkages comprise at least 50% phosphorothioate linkages.

8. The oligonucleotide of any one of claims 1 to 7, wherein the internucleotide linkage between X¹ and X² is a phosphorothioate.

9. The oligonucleotide of any one of claims 1 to 8, wherein the internucleotide linkage between X² and X³ is a phosphorothioate.

10. The oligonucleotide of any one of claims 1 to 9, wherein the internucleotide linkage between X¹ and X² is a phosphorothioate, and the internucleotide linkage between X² and X³ is a phosphorothioate.

11 . The oligonucleotide of any one of claims 1 to 10, wherein 45-75% of the A/B sugars are 2'- methoxy ribose.

12. The oligonucleotide of any one of claims 1 to 11, wherein 20-60% of the A/B sugars are 2'-deoxy- 2'-fluororibose.

13. The oligonucleotide of any one of claims 1 to 12, wherein no A/B sugar is a 2' -deoxyribose.

14. The oligonucleotide of any one of claims 1 to 13, wherein no A/B sugar is a 2'-methoxyethylribose.

15. The oligonucleotide of any one of claims 1 to 14, wherein m is 23 to 35.

16. The oligonucleotide of claim 15, wherein m is 30.

17. The oligonucleotide of claim 16, wherein [A_m] has a sequence of SEQ ID NO: 142 (AAC AUG GCC CCA GCA GCU UCA GUC CCU UUC) or SEQ ID NO: 143 (AAC AUG GCC CCA GCA GCU UCA GUU CCU UUC) or SEQ ID NO: 144 (AAC AUG GCU CCA GCA GUU UCA GUU CCU UUC).

18. The oligonucleotide of any one of claims 1 to 17, wherein n is 8 to 10.

19. The oligonucleotide of claim 18, wherein n is 9.

20. The oligonucleotide of claim 19, wherein [B_n] has a sequence UCG AUG GUC.

21. The oligonucleotide of claim 16, 17, 19 or 20 having 4-7 phosphoramidate linkages.

22. The oligonucleotide of claim 16, 17, 19, 20, or 21, having 14-30 phosphorothioate linkages.

23. The oligonucleotide of claim 16, 17, 19, 20, 21, or 22, wherein 11-20 A/B sugars are 2’ -deoxy-2’- fluororibose.

24. The oligonucleotide of claim 16, 17, 19, 20, 21, 22, or 23, wherein 18-29 A/B sugars are 2'- methoxy ribose.

25. The oligonucleotide of any one of claims 1 to 24, sufficiently complementary to part of a target RNA having a target adenosine and capable of forming a complex with the target RNA.

26. The oligonucleotide of claim 24, wherein, upon formation of the complex with the target RNA, the nucleotide of the oligonucleotide opposite the target adenosine is X².

27. The oligonucleotide of claim 25 or 26, capable of binding and recruiting an ADAR enzyme to perform editing on the target adenosine of the target RNA.

28. The oligonucleotide of any one of claims 1 to 27, wherein the oligonucleotide does not comprise a portion that is capable of forming an intramolecular stem-loop structure.

29. The oligonucleotide of any one of claims 1 and 25-28, comprising a nucleotide sequence set forth in any one of SEQ ID NOs: 1-141 and 151-222.

30. The oligonucleotide of any one of claims 1-29 capable of forming a complex with SERPINA1 as a target RNA.

31 . A complex comprising the oligonucleotide of any one of claims 1 to 30 and a target RNA, the complex formed by hybridization between the oligonucleotide and the target RNA.

32. The complex of claim 31 , comprising 1 , 2, 3, 4, or 5 mismatches or wobbles.

33. A method of editing a target adenosine in a target RNA in a cell comprising contacting the cell with the oligonucleotide of any one of claims 1 to 30 to (i) form a complex between the oligonucleotide and the target RNA such that X² of the oligonucleotide is opposite the target adenosine; and (ii) recruit an ADAR in the cell to the complex such that the ADAR edits the target adenosine.

34. A method of treating alpha-1 -antitrypsin deficiency in a subject in need thereof, comprising administering to the subject an effective amount of the oligonucleotide of any one of claims 1 to 30 to treat the alpha- 1 -antitrypsin deficiency.