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WO2018160690A1 - Formulations pharmaceutiques d'arn pour le traitement prophylactique et thérapeutique d'une infection par le virus zika - Google Patents

Formulations pharmaceutiques d'arn pour le traitement prophylactique et thérapeutique d'une infection par le virus zika Download PDF

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WO2018160690A1
WO2018160690A1 PCT/US2018/020220 US2018020220W WO2018160690A1 WO 2018160690 A1 WO2018160690 A1 WO 2018160690A1 US 2018020220 W US2018020220 W US 2018020220W WO 2018160690 A1 WO2018160690 A1 WO 2018160690A1
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composition
sirna
mammal
zikv
sirna molecule
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Yibin CAI
Shenggao TANG
Patrick Yang Lu
Jian Guan
Alan Yang LU
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Guangzhou Nanotides Pharmaceuticals Co., Ltd.
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Priority to CN201880013887.4A priority Critical patent/CN110520155A/zh
Publication of WO2018160690A1 publication Critical patent/WO2018160690A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Synthetic bilayered vehicles, e.g. liposomes or liposomes with cholesterol as the only non-phosphatidyl surfactant
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes or liposomes coated or grafted with polymers
    • A61K9/1273—Polymersomes; Liposomes with polymerisable or polymerised bilayer-forming substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
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    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/31—Combination therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24111—Flavivirus, e.g. yellow fever virus, dengue, JEV
    • C12N2770/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This invention presents novel prophylactic and therapeutic agents for prevention and treatment of Zika virus (ZIKV) infection and methods for their use.
  • ZIKV Zika virus
  • the Zika virus (a member of the flaviviridae family including West Nile Virus, Dengue and Chikungunya viruses) has recently garnered great interest because of a link between infection in pregnant women and miscarriage or birth defects in their infants. Transmission is via the bite of an infected Aedes mosquito, and a rapid increase in transmission observed in Brazil since late 2014 preceded an increase in the number of cases of microcephaly in infants reported to the Brazil Ministry of Health in 2015 [1, 2]. As of November 2016, a total of more than 4, 100 suspected microcephaly cases had been reported, many of which occurred in infants born to women who lived in or had visited areas where Zika virus transmission was observed [3].
  • ZIKA virus infections have now been identified and validated in the mainland US in Florida and gulf coast states, and the need for suitable prophylactic or therapeutic options is significant.
  • ZIKV is a single-strand positive RNA virus, belonging to family Flaviviridae; genus Flavivirus.
  • the genome of ZIKV consists of about 10K nucleotides, which code at least three structural proteins (capsid (C), membrane (prM; processed to M) and envelope (E)), and seven non-structural proteins (NSl, NS2A, NS2B, NS3, NS4A, NS4B and NS5).
  • C structural proteins
  • prM membrane
  • E envelope
  • NSl seven non-structural proteins
  • NS2A, NS2B, NS3, NS4A, NS4B and NS5 seven non-structural proteins
  • the single ORF is flanked by 5'- and 3'- untranslated regions (UTRs), respectively ( Figure 1).
  • RNA elements of the 3' UTR greatly enhance viral RNA synthesis.
  • ZIKV infection initiates from envelope (E) glycoproteins on the surface of the ZIKV virion binding to a host cell receptor, such as AXL and broadly neutralizing antibodies have been demonstrated against this E protein region [5].
  • E envelope
  • AXL broadly neutralizing antibodies have been demonstrated against this E protein region [5].
  • ZIKV enters the host cell by endocytosis, and then its genome escapes from the endosome into the cytoplasm after viral-host membrane fusion driven by the fusion loop.
  • the viral genome is first translated into a large polyprotein, and then cleaved by host signal peptidases and a viral serine protease (during and after translation) to generate the viral proteins. Also in the cytoplasm, the positive strand RNA genome produces a negative strand RNA template to generate new copies of the genome.
  • AXL a member of the receptor tyrosine kinase subfamily, has been identified as the major receptor for ZIKV binding.
  • the study showed that blocking or silencing AXL dramatically reduced ZIKV replication in fibroblasts and alveolarepithelial cells [6].
  • the function of AXL. includes transducing signals from the extracellular matrix into the cytoplasm by binding growth factors, such as vitamin K-dependent protein growth-arrest-specific gene 6 (GAS6), as well as being involved in the stimulation of cell proliferation.
  • GAS6 vitamin K-dependent protein growth-arrest-specific gene 6
  • Zika strains and similarities For a prophylactic or therapeutic to have viability it is important to ensure that it is able to function against a wide array (if not all) strains of a virus.
  • ZIKV has circulated in both Africa and Asia since at least the 1950s. Haddow et al determined the nucleotide sequences of the open reading frames of five isolates from
  • mRNA is a nontoxic molecule that allows transient protein expression of a desired protein product in nearly all transducable cell types. Compared with traditional plasmid and viral-based approaches, this approach allows design of patient personalized mRNAs that also benefit by not needing to pass through the nuclear membrane (as DNA does) and thus carries little to no risk of genomic integration. mRNA vaccines have also been demonstrated to promote immunogenicity in both very young and very old mice. If this translates to humans, then this would be beneficial since these demographics typically do not mount a robust immunological reaction to vaccines. Recently, self-amplifying mRNA vaccines have been demonstrated to be safe and efficacious against other viruses (e.g.
  • mRNA vaccines may benefit in that they have been demonstrated to induce balanced, long-lived and protective immunity to influenza A virus infections in even very young and very old mice.
  • Vaccines based on mRNA or RNA replicons have also been shown to be immunogenic in a variety of animal models, including nonhuman primates [10]. Target selection for mRNA vaccine against ZIKV
  • the ZIKV E protein contains three distinct domains (shown in Figure 1C): a central ⁇ -barrel domain (Domain I; aa. 1-51, 132-192 and 280-295), an extended dimerization domain (Domain II; aa. 52-131 and 193-279), and an immunoglobulin-like domain (Domain III; aa. 297-406) ( Figure 1C.) [5].
  • the fusion loop a hydrophobic sequence located in Domain II (aa.
  • the fusion loop of Envelope protein Domain II may stimulate Antibody Dependent Enhancement (ADE) of immune response (when non-neutralizing antiviral proteins facilitate virus entry into host cells and increase infectivity in these cells resulting in higher viremia and more severe symptoms).
  • ADE Antibody Dependent Enhancement
  • stains of different DENV serotypes totally 4 identified serotypes
  • the antibodies to the old strain interfered with the immune response to the new strain and resulted in more virus uptake [11].
  • siRNA a flexible molecular platform for prophylactic and therapeutic efficacy against ZIKA virus RNA interference (RNAi) is a naturally occurring, highly specific mode of gene regulation that has broad potential applications in both research and therapeutic settings. The mechanics of RNAi are both extraordinar and highly discriminating.
  • RNAi short (19-21bp) double stranded RNA sequences (referred to as short interfering RNAs, siRNAs) associate with the cytoplasmically localized RNA Interference Silencing Complex (RISC).
  • RISC cytoplasmically localized RNA Interference Silencing Complex
  • the resultant ribonucleoprotein complex searches the resident population of messenger RNAs (mRNAs) for complementary sequences, eventually degrading (and/or attenuating translation of) these transcripts and effectively down-regulating the expression of the targeted gene.
  • mRNAs messenger RNAs
  • RNAi represents a highly flexible platform by which researchers and clinicians can control diseases, including infectious diseases.
  • RNA interference has previously been employed to target a range of human pathogenic viruses, including HIV, hepatitis, respiratory syncytial virus, polio virus, SARS coronavirus, Marburg, dengue, foot-and-mouth disease and Influenza.
  • RNAi has the ability to (1) efficiently limit viral replication without reliance on host immune functions, and (2) target multiple genes and/or sequences simultaneously, making this an ideal therapeutic approach for viruses like Zika (where a large number of strains may need to be targeted and no other options currently exist) or against other viruses (like Influenza), which have rapidly evolving genomes.
  • siRNAs By performing recursive analyses of siRNAs against 28 sequenced Zika strains (including both African and Asian lineages), we identified siRNAs that were able to cover an extensive number of strains (Table 2). Furthermore, careful selection of multiple siRNAs can be seen to provide complete coverage against all strains used in our predictions (Table 3).
  • siRNA #62 and siRNA #62A can provide 100% coverage as seen by combining siRNA #62 and siRNA #62A (Table 3).
  • siRNAs targeting different regions of the viral genome can produce synergistic silencing and antiviral efficacy compared with multiple siRNAs targeting the same gene. Therefore, while we can see complete coverage with certain combinations, we may see higher potency against the selected strains using a combination (such as 13 & 62) targeting distinct regions of the virus.
  • a combination such as 13 & 62
  • siRNA #62 and siRNA #62a can provide 100% coverage as seen by combining siRNA #62 and siRNA #62a.
  • siRNAs targeting different regions of the viral genome can produce synergistic silencing and antiviral efficacy compared with multiple siRNAs targeting the same gene. Therefore, while we can see complete coverage with certain combinations, we may see higher potency against the selected strains using a combination (such as 13 & 62) targeting distinct regions of the virus.
  • a combination such as 13 & 62
  • ZIKV is a single-strand positive RNA virus, belonging to family Flaviviridae; genus Flavivirus.
  • the genome of ZIKV consists of about 10K nucleotides, which code at least three structural proteins (capsid (C), membrane (prM; processed to M) and envelope (E)), and seven non-structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5).
  • C structural proteins
  • prM membrane
  • E envelope
  • NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5 seven non-structural proteins
  • the single ORF is flanked by 5'- and 3'- untranslated regions (UTRs), respectively.
  • the ZIKV E protein contains three distinct domains (shown in Figure 1): a central ⁇ -barrel domain (Domain I; aa. 1-51, 132-192 and 280-295), an extended dimerization domain (Domain II; aa. 52-131 and 193-279), and an
  • HKP/siRNA can self-assemble into nanoparticles (average 150nm in diameter).
  • the nanoparticles can be dissolved in aqueous solution, can be lyophilized into dry powder, and can be redissovled and mixed with methylcellulose, or with RNAse free water.
  • C HKP/siRNA nanoparticle delivery to mouse respiratory track: upper airway, bronchi, alveoli.
  • FIG. 3 Comparison of target knockdown of lung endogenous gene among HKP, DOTAP and D5W after oral tracheal deliveries of siRNA with three different dosing regimens. HKP demonstrated the efficient siRNA-mediated knockdown of the target gene at the 20 ⁇ g dose.
  • FIG. 5 The structures of Spermine-Lipid Conjugates (SLiC) species.
  • the synthesis route for each of the five molecules are listed with the specific liposome chain, such as, Ri, R 2 , R 3 , R 4 and R 5 , conjugated at the location of RiH, R 2 H, R 3 H, R 4 H and R 5 H respectively.
  • the structures of the five SLiC species are illustrated with a spermine head and two lipid legs.
  • TM4-packaged siRNA specific to cyclophilin-B was selected for being tested in a Balb/c mouse model through a respiratory route of delivery.
  • a HKP package cyclophilin-B siRNA was used as a positive control.
  • Three different dosage: 25, 40 and 50 ⁇ g were tested. Both 40 and 50 ⁇ g siRNA dosages achieved significant target gene silencing (N 3, *P ⁇ 0.05).
  • FIG. 7 Evaluation of the cytokine response in the mouse lung after HKP-siRNA nanoparticles delivery. HKP-siRNA at different dosages were oraltracheally administrated in the mouse lungs. The total lung tissue was harvested for protein isolation and cytokine measurements by ELIS A assay.
  • FIG. 1 Standard curve to measure IFN-a concentration was prepared according to in-house SOP (Lowry Method); B. IFN-a concentration in each sample was determined and normalized to total protein.
  • the present invention provides RN A pharmaceutical formulations comprising either siRNA or mRNA sequences for the prevention and treatment of Zika virus infection.
  • One formulation comprises siRNA sequences targeting the viral genome RNA and mRNA for knocking down the viral activity, and a carrier, such as Histidine-Lysine Co-polymers (HKP) or Spermine-liposome Conjugates (SLiC).
  • Another formulation comprises mRNA sequences coding viral proteins critical for Zika viral infection and replication, and a carrier serving as an adjuvant for amplifying an immune response against the Zika virus within the host.
  • the invention also provides methods of use for the pharmaceutical formulations for the prevention and therapeutic treatment of Zika virus Infection.
  • siRNA molecule or an “siRNA duplex” is a duplex
  • oligonucleotide that is a short, double-stranded polynucleotide, that interferes with the expression of a gene in a cell, after the molecule is introduced into the cell, or interferes with the expression of a viral gene.
  • it targets and binds to a complementary nucleotide sequence in a single stranded (ss) target RNA molecule.
  • siRNA molecules are chemically synthesized or otherwise constructed by techniques known to those skilled in the art. Such techniques are described in U.S. Pat. Nos. 5, 898,031, 6,107,094, 6,506,559, 7,056,704 and in European Pat. Nos. 1214945 and 1230375, which are incorporated herein by reference in their entireties.
  • the sequence refers to the sense strand of the duplex molecule.
  • One or more of the ribonucleotides comprising the molecule can be chemically modified by techniques known in the art.
  • the backbone of the oligonucleotide can be modified.
  • siRNA molecules of the invention target a conserved region of the genome of a ZIKV.
  • target or “targets” means that the molecule binds to a
  • a "conserved region" of a ZIKV gene is a nucleotide sequence that is found in more than one strain of the virus, is identical among the strains, rarely mutates, and is critical for viral infection and/or replication and/or release from the infected cell.
  • the targeted conserved region of the genome comprises sequences coding for envelope protein or nonstructural proteins NS1, NS3, NS4B, or NS5.
  • the siRNA molecule can target envelope gene expression, NS1 gene expression, NS3 gene expression, NS4B gene expression, or NS5 gene expression.
  • the targeted conserved region of the genome is the 3' untranslated region (3'-UTR) of the virus.
  • the siRNA molecule can target the 3' untranslated region (3'-UTR) of the virus.
  • the siRNA molecule is a double-stranded oligonucleotide with a length of about 19 to about 29 base pairs. In one aspect of this embodiment, the molecule is a double-stranded oligonucleotide with a length of 19 to 21 base pairs. In another aspect of this embodiment, it is a double-stranded oligonucleotide with a length of 25 base pairs. In all of these aspects, the molecule may have blunt ends at both ends, or sticky ends with overhangs at both ends (unpaired bases extending beyond the main strand), or a blunt end at one end and a sticky end at the other. In one particular aspect, it has blunt ends at both ends. In another particular aspect, the molecule has a length of 25 base pairs (25 mer) and has blunt ends at both ends. In another particular aspect, the siRNA molecule is one of those identified in Table 4.
  • the siRNA molecules of the invention also include ones derived from those listed in Table 4 or otherwise disclosed herein.
  • the derived molecules can have less than the 25 base pairs shown for each molecule, down to 17 base pairs, so long as the "core" contiguous base pairs remain. That is, once given the specific sequences shown herein, a person skilled in the art can synthesize molecules that, in effect, "remove” one or more base pairs from either or both ends in any order, leaving the remaining contiguous base pairs, creating shorter molecules that are 24, 23, 22, 21, 20, 19, 18, or 17 base pairs in length, if starting with the 25 base pair molecule.
  • the derived molecules of the 25 mer molecules disclosed in Table 4 includes: a) 24 contiguous base pairs of any one or more of the molecules; b) 23 contiguous base pairs of any one or more of the molecules; c) 22 contiguous base pairs of any one or more of the molecules; b) 21 contiguous base pairs of any one or more of the molecules; d) 20 contiguous base pairs of any one or more of the molecules; e) 19 contiguous base pairs of any one or more of the molecules; f) 18 contiguous base pairs of any one or more of the molecules; and g) 17 contiguous base pairs of any one or more of the molecules.
  • the derived molecules can have more than the 25 base pairs shown for each molecule, so long as the initial 25 contiguous base pairs remain. That is, once given the specific sequences disclosed herein, a person skilled in the art can synthesize molecules that, in effect, "add" one or more base pairs to either or both ends in any order, creating molecules that are 26 or more base pairs in length and containing the original 25 contiguous base pairs.
  • compositions comprising One or More siRNA Molecules
  • the invention includes a pharmaceutical composition
  • a pharmaceutical composition comprising an siRNA molecule that targets a conserved region of the genome of a ZIKV and a pharmaceutically acceptable carrier.
  • the carrier condenses the molecules to form a nanoparticle.
  • the composition may be formulated into nanoparticles.
  • the compositions may be lyophilized into a dry powder.
  • the pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.
  • the composition comprises at least two different siRNA molecules that target one or more conserved regions of the genome of a ZIKV and a pharmaceutically acceptable carrier.
  • compositions are sometimes referred to herein as a "cocktail.”
  • the gene sequences in the conserved regions of the ZIKV are critical for the viral infection of a mammal.
  • the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).
  • the mammal is a human.
  • composition can include one or more additional siRNA molecules that target still other conserved regions of the ZIKV genome.
  • composition comprises siRNA molecules that target the
  • Envelope (E) gene of the ZIKV genome the molecules are selected from the group consisting of:
  • ZIKV14 CCUUGACAAGCAAUCAGACACUCAA
  • ZIKV17 CCGGAACUCCACACUGGAACAACAA .
  • the composition comprises siRNA molecules that target the NSl gene of the ZIKV genome.
  • the molecule comprises ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA.
  • the composition comprises siRNA molecules that target the NS3 gene of the ZIKV genome.
  • the molecules are selected from the group consisting of:
  • ZIKV62 GCCUAUCAGGUUGCAUCUGCCGGAA ,
  • ZIKV63 CCUAUCAGGUUGCAUCUGCCGGAAU ,
  • the composition comprises siRNA molecules that target the NS4B gene of the ZIKV genome.
  • the molecule comprises ZIKV74: CCACUUCAUACAACAACUACUCCUU.
  • the composition comprises siRNA molecules that target the NS5 gene of the ZIKV genome.
  • the molecule comprises ZIKV103 : GGUGCGCAGGAUCAUAGGUGAUGAA.
  • the composition comprises siRNA molecules that target the 3'-UTR region of the ZIKV genome.
  • the molecule comprises: ZIKV105: C C GAG A AC GC C AUGGC AC GG A AG A A .
  • the composition comprises a cocktail, MSTZIKV13, wherein a first siRNA molecule comprises ZIKV13 : GGUGAAGCCUACCUUGACAAGCAAU and a second siRNA molecule comprises ZIKV30: GCCAUGGCACAGUGAAGAGCUUGAA.
  • the composition comprises a cocktail, MSTZIKV62, wherein a first siRNA molecule comprises ZIKV62: GCCUAUCAGGUUGCAUCUGCCGGAA and a second siRNA molecule comprises ZIKV74: CCACUUCAUACAACAACUACUCCUU.
  • composition comprises a cocktail, MSTZIKV62B, wherein a first siRNA molecule comprises ZIKV62B:
  • a second siRNA molecule comprises ZIKV17: CCGGAACUCCACACUGGAACAACAA.
  • composition comprises a cocktail, MSTZIKV103, wherein a first siRNA molecule comprises ZIKV103 :
  • a second siRNA molecule comprises KIKV63 : CCUAUCAGGUUGCAUCUGCCGGAAU
  • a third siRNA molecule comprises ZIKVl 05 : CCGAGAACGCCAUGGC ACGGAAGAA.
  • a pharmaceutically acceptable carrier comprises a polymeric nanoparticle or a liposomal nanoparticle.
  • the siRNA molecules comprise 25 mer blunt-end siRNA molecules and the carrier comprises a Histidine-Lysine copolymer or Spermine-Lipid Conjugate (SLiC) and cholesterol.
  • Pharmaceutically Acceptable Carriers for the siRNA Molecules include saline, sugars, polypeptides, polymers, lipids, creams, gels, micelle materials, and metal nanoparticles.
  • the carrier comprises at least one of the following: a glucose solution, a polycationic binding agent, a cationic lipid, a cationic micelle, a cationic polypeptide, a hydrophilic polymer grafted polymer, a non-natural cationic polymer, a cationic polyacetal, a hydrophilic polymer grafted polyacetal, a ligand functionalized cationic polymer, a ligand
  • the polymers comprise a biodegradable histidine-lysine polymer, a biodegradable polyester, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), and poly(lactic-co-glycolic acid) (PLGA), a polyamidoamine (PAMAM) dendrimer, a cationic lipid, or a PEGylated PEL Cationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE.
  • PLA poly(lactic acid)
  • PGA poly(glycolic acid)
  • PLGA poly(lactic-co-glycolic acid)
  • PAMAM polyamidoamine dendrimer
  • a cationic lipid or a PEGylated PEL Cationic lipids
  • PEGylated PEL Cationic lipids include DOTAP, DOPE, DC-Chol/DOPE, DOTMA, and DOTMA/DOPE
  • the carrier is a polymer.
  • the polymer comprises a histidine-lysine copolymer (HKP).
  • HTP histidine-lysine copolymer
  • Such copolymers are described in U.S. Pat. Nos. 7,070,807 B2, 7,163,695 B2, and 7,772,201 B2, which are incorporated herein by reference in their entireties.
  • the HKP forms a nanoparticle with the siRNA molecule, wherein the diameter of the nanoparticle is about lOOnm to about 400 nm.
  • the HKP and the siRNA molecules self-assemble into nanoparticles or can be formulated into nanoparticles.
  • the carrier is a liposome.
  • the liposome comprises a cationic lipid conjugated with cholesterol.
  • the cationic lipid comprises a spermine head and one or two oleyl alcoholic tails.
  • the liposome comprises a Spermine-Liposome-Conjugate (SLiC) and cholesterol. Examples of such molecules are disclosed in Figure 5.
  • the liposome and the siRNA molecules self-assemble into nanoparticles or can be formulated into nanoparticles.
  • the invention also includes methods of using the siRNA molecules and
  • compositions containing them to prevent or treat ZIKV disease As used herein "treat” or “treatment” refers to reducing the severity of or curing ZIKV disease.
  • a therapeutically effective amount of the composition of the invention is administered to a mammal.
  • the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).
  • rodent e.g., rat, mouse, or guinea pig
  • ferret e.g., a monkey
  • non-human primate e.g., a monkey
  • the mammal is a laboratory animal, such as a rodent.
  • the mammal is a non-human primate, such as a monkey.
  • the mammal is a human.
  • a "therapeutically effective amount” is an amount that prevents, reduces the severity of, or cures ZIKV disease. Such amounts are determinable by persons skilled in the art, given the teachings contained herein.
  • a therapeutically effective amount of the pharmaceutical composition administered to a human comprises about 1 mg of the siRNA molecules per kilogram of body weight of the human to about 5 mg of the siRNA molecules per kilogram of body weight of the human.
  • mRNA Vaccines The invention provides a vaccine comprising an mRNA molecule that codes for an amino acid sequence encoded by a conserved region of the genome of a ZIKV and a pharmaceutically acceptable carrier comprising a polymer or a liposome.
  • a "conserved region" of a ZIKV gene is a nucleotide sequence that is found in more than one strain of the virus, is identical among the strains, rarely mutates, and is critical for viral infection and/or replication and/or release from the infected cell.
  • the gene sequence in the conserved region of the ZIKV genome is critical for the viral infection of a mammal.
  • the conserved region of the genome comprises gene sequences coding for the Envelope protein of ZIKV.
  • the gene sequences code for amino acid sequences within Domain III of the Envelope protein.
  • the polymer comprises a Histidine-Lysine co-polymer (HKP).
  • the HKP and the mRNA molecules self-assemble into nanoparticles.
  • the HKP and mRNA molecules are formulated into nanoparticles.
  • the liposome comprises a Spermine-Lipid Conjugate (SLiC) and cholesterol.
  • the SLiC and cholesterol and the siRNA self-assemble into nanoparticles.
  • the SLiC and cholesterol and the mRNA molecules are formulated into nanoparticles.
  • the SLiC and cholesterol also acts as an adjuvant for amplifying the immune response.
  • the mRNA vaccines of the invention are used to prevent a ZIKV infection or reduce its severity.
  • the invention includes method of preventing or reducing the severity of a ZIKV infection in a mammal comprising administering to the mammal a therapeutically effective amount of the vaccine prior to infection.
  • the vaccine is administered to the mammal through injection instillation or intradermal, intravenous, intraperitoneal, intravenous, intravaginal, or subcutaneous administration.
  • the mammal is a human, rodent (e.g., rat, mouse, or guinea pig), ferret, or non-human primate (e.g., a monkey).
  • the mammal is a laboratory animal, such as a rodent.
  • the mammal is a non-human primate, such as a monkey.
  • the mammal is a human.
  • Example 1 Design mRNA targeting the envelope protein of ZIKV
  • ZIKV E protein represents a major target for development of a neutralizing antibody.
  • the ZIKV E protein contains three distinct domains (shown in Figure 1).
  • the mRNA constructs containing Envelope Domain II of ZIKV will be transfected into human cells in vitro using a variety of commercially available transfection agent.
  • Cells to be used for these studies included HEK293T, VERO cells, A549 cells and others.
  • electroporation using MaxCyte technology
  • the various delivery processes are aimed at determining which will give good uptake into a variety of cells and to see subsequent expression of the construct. The aim is to determine protein production by each construct and also to determine whether the product is secreted from the cells. This process is not necessarily identified a clinically viable delivery process.
  • mRNA will be detected in live cells using SmartFlare probes (Millipore) or through use of QRTPCR. mRNA of GFP was taken as the positive control ( Figure 9).
  • Example 3 Detection of mRNA uptaking into cells using SmartFlare technology
  • These smart flares are beads that have a sequence attached that, when recognizing the RNA sequence in the cell, produce an increase in fluorescence. Smartflares will be designed against several regions along the constructs in case steric hindrance reduces signal from one region.
  • the protein expressed by the mRNA construct will be identified and quantitated by RPHPLC using an analytical C18 column (250mm x 2.1mm; Phenomenex). Protein detection will use a dual wavelength detector. A gradient of 0.1%TFA/Acetonitrile will be adjusted over time to allow analytical separation of protein peaks. In initial experiments, fractions will be collected and submitted for Mass Spectrometry to determine the presence of the expected sequence. The secreted product and the product manufactured within the cells will be compared using protein sequencing. To mitigate enzyme degradation of the sample, we will use enzyme inhibitors in the media and concentrated media from multiple wells in order to detect the product on HPLC.
  • Example 5 Examination of siRNAs against Zika in a CPE assay
  • siRNAs for testing will be provided to Immuquest (Frederick, MD) for analysis in their Zika CPE assay.
  • control and test siRNAs will be administered at select intervals ahead of the virus and the degree of effect monitored by examining the change in the CPE value.
  • Example 6 Determine best nanoparticle for delivery
  • branched polypeptides e.g. Histidine Lysine Ploymer or HKP
  • various derivatives that, like the HKP, have been modified with targeting ligands to allow tissue specific delivery or PEGylated varieties that should assist in uptake across the mucosa of the vagina.
  • HKP Histidine Lysine Ploymer
  • a spermine/spermidine co-polymer carrier and a liposomal delivery agent that, like the HKP, protect the siRNA and provide high efficiency delivery to tissues.
  • the various peptide/lipid formulations will be evaluated for their ability to form nanoparticles with single siRNAs or combinations of siRNAs against different targets. Binding with the nucleic acid will be evaluated by gel electrophoresis. Nanoparticle formation will be studied by particle size measurements using DLS (Dynamic Light Scattering) and the charge/size distribution measured using a nanoparticle size/charge instrument (Malvern instruments D9000).
  • TEM electron microscopy
  • Example 7 SLiC/mRNA/siR A nanoparticle SLiC Liposome Preparation Regular methods will be tried at first to prepare liposomes with newly synthesized SLiC molecules, such as thin film method, solvent injection and so on without much success.
  • Norbert Maurer et al reported a method of liposome preparation in which siRNA or oligonucleotide solution will be slowly added under vortexing to the 50% ethanol solution (v/v) of liposome and ethanol was later removed by dialysis.
  • the nanoparticles thus derived will be small in size and homogeneous.
  • mRNA and siRNA will be directly wrapped by cationic lipids during formation of liposome, while in most other methods mRNA or siRNA are loaded (or entrapped) into preformed liposome, such as Lipofectamine 2000. Lipids dissolved in ethanol are in so-called metastable state in which liposomes are not very stable and tend to aggregate. We will then prepare un-loaded or pre-formed liposomes using modified Norbert Maurer's method. (Maurer, N., A., Mori, L., Palmer, M. A., Monck, K. W. C, Mok, B., Mui, Q. F., Akhong, and P. R., Cullis. 1999.
  • Liposomes were prepared by addition of lipids (cationic SLiC /cholesterol, 50:50, mol %) dissolved in ethanol to sterile dd-LbO.
  • the ethanolic lipid solution need to be added slowly under rapid mixing. Slow addition of ethanol and rapid mixing were critical for the success in making
  • SLiC liposomes as the process allows formation of small and more homogeneous liposomes. Unlike conventional methods in which mRNA or siRNAs are loaded during the process of liposome formulation and ethanol or other solvent is removed at end of manufacturing, our SLiC liposomes were formulated with remaining ethanol still in the solution so that liposomes were thought to be still in metastable state. When mRNA or siRNA solution was mixed/loaded with liposome solution cationic groups lipids will interact with anionic siRNA and condense to form core. SLiC liposomes' metastable state helped or facilitated liposome structure transformation to entrap mRNA or siRNA more effectively. Because of the entrapment of mRNA/siRNA, SLiC liposomes become more compact and homogeneous.
  • SLiC liposome formation After the liposome formation, we have developed an array of assays to characterize the physicochemical properties of SLiC liposome, including particle size, surface potential, morphology study, mRNA or siRNA loading efficiency and biological activity, etc.
  • the particle size and zeta-potentials of SLiC liposomes were measured with Nano ZS Zeta Sizer (Malvern Instruments, UK). Each new SLiC liposome was tested for particle size and zeta-potential when ethanol contents changed from 50% to 25% and to 12.5%. Data were derived from formulations of different ethanol contents. All SLiC liposomes were prepared at lmg/ml in concentration and loaded with siRNA (2: 1, w/w).
  • SLiC Liposomes were composed of cationic SLiC and cholesterol dissolved in ethanol at 12.5%, e.g. TM2 (12.5).
  • TM2 cationic SLiC
  • the average particle sizes of three sequential measurements and the average zeta-potentials of three sequential measurements were illustrated in Table 5.
  • liposomes particle sizes became much smaller when they were loaded with mRNA or siRNA at 2: 1 (w/w) resulting in particle sizes in the range of 110 to 190nm in diameter and much lower PDI values.
  • Conventional consideration of liposomal structure dictates that mRNA or siRNA is loaded or interacted with cationic lipids through electrostatic forces and liposomes wraps mRNA or siRNA to form spherical particles in shape in order to reduce surface tension. As the result, the liposomes particle sizes became much smaller after loaded with mRNA or siRNA. Liposomes formulated with mRNA or siRNA also have lower surface charge, which could be explained by neutralizing effect from loaded mRNA or siRNA.
  • Example 8 Mouse model study for mRNA vaccine
  • mice will be five-week old at the beginning of this study.
  • 20 mice of prophylactic group will be intravenously injected with mRNA combination encapsidated with HKP-SLiC nanoparticle system from the tail vein.
  • the other 20 mice of control group will be injected with PBS. 14 days later, the serum of all mice will be collected, and the mice from both prophylactic group and control group will be intravenously injected with ZIKV.
  • mice All mice will be weighed and the survival of each group will be counted daily.
  • the serum of infected mice will be taken at 1, 3, 5, 7, 9 and 14-day post-infection.
  • the tumor necrosis factor alpha (TNF-a) will be detected using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the tissues including testes, spleen, liver, heart, brain and kidney will be collected at 24 and 72 h post-infection.
  • the total RNA from the tissues will be extracted, one-step quantitative real-time PCR and 5'-RACE assay will be performed as described in the above in vitro study part.
  • the viral titers in the sera and tissues will be detected in Vero cells with serially dilution.
  • the results from vaccination group will be compared with the control groups to evaluate the protective efficiency of the mRNA vaccine candidates.
  • siRNAs By performing recursive analyses of siRNAs against 28 sequenced Zika strains (including both African and Asian lineages) we identified siRNAs that were able to cover an extensive number of strains (Table 2). Furthermore, careful selection of multiple siRNAs can be seen to provide complete coverage against all strains used in our predictions (Table 3). Further sequence analysis showed that the 11 elected anti-ZIKV siRNA target the highly homologous regions of Envelope (E) protein, non-structure protein NS1, NS3, NS4B, NS5 and 3'-UTR in all ZIKV strains (Table 4 and Figure 1). If the mRNA translation and genome replication of ZIKV mimic DENV and take place in the cytoplasm, the siRNA candidates should work efficiently to degrade both positive and negative strand of the viral RNAs in ZIKV.
  • E Envelope
  • Example 10 Cell Culture Based Screening for Potent Anti-ZIKV siRNA Oligos siRNAs will be tested for their silencing activities using a reporter assay where the regions of the ZIKV genome to be silenced are incorporated into an expression vector. An appropriate ZIKV gene segment will be cloned into psiCheckTM-2 (Promega). The cells will be co-transfected with reporter plasmids and siRNAs and the expression of renilla luciferase will be normalized to firefly luciferase expression (transfection efficiency control).
  • RTPCR RT-PCR
  • Example 11 Identify potent combinations of siRNAs to improve strain coverage.
  • siRNA mixtures for evaluation in the CPE assay.
  • siRNAs will selecte the siRNAs to include in the mixtures based on their efficacy as well as the degree of overlap between Zika strains.
  • Example 12 Develop and characterize nanoparticles for in vivo delivery of siRNAs.
  • the most critical and challenging aspect of developing an siRNA-based therapeutic is the delivery of the siRNA into the cells where ZIKV infection can be transmitted. Since ZIKV has been shown to be transmitted by sexual intercourse we have elected to pursue intravaginal siRNA delivery as a means of validating this as a preliminary approach to therapy. Once more is understood about the etiology of the disease we may be able to adjust the delivery approach to treat therapeutically.
  • the vaginal tract is a suitable site for the administration of both local and systemic acting drugs.
  • vaginal products on the market such as those approved for contraception, treatment of yeast infection, hormonal replacement therapy, and feminine hygiene. However, there are many biological barriers and factors that protect the vagina from foreign particles, such as a thick and elastic mucus layer that may bind and inhibit access of these agents.
  • PLGA, PEG or PEG-PEI to this HKP nanoparticle or evaluation of a spermine/spermidine copolymer that exhibits suitable molecular characteristics for delivery including size, charge and hydrophobicity.
  • Previous studies have shown significant success with PLGA, PEG and other modifications to existing nanoparticle formulations and we have demonstrated functionalization of our nanoparticles with these moieties while retaining this optimal size, ability to carry and protect the siRNAs while penetrating the mucus layer. Finally, others have shown that spray dried powders can enhance intravaginal siRNA delivery [16]. We have also demonstrated that we can lyophilize our HKP nanoparticle complexed with siRNA without adversely affecting its characteristic size or delivery capacity.
  • Example 13 Mice model study for anti-ZIKV siRNA
  • AG129 mice will be used as the animal model. We will perform all mouse studies in Biosafety level-2 conditions. All AG129 mice will be five-week old at the beginning of this study. 20 mice of treatment group will be intravenously injected with siRNA combination encapsidated with HKP-SLiC nanoparticle system from the tail vein. The other 20 mice of control group will be injected with PBS. 24 h later, mice from treatment group and control group will be infected intravenously with ZIKV, respectively. siRNA or PBS will be intravenously injected into the treatment or control group at 0, 24, 48 and 72 h post-infection.
  • mice All mice will be weighed and the survival of each group was counted daily.
  • the serum of infected mice will be taken at 1, 3, 5, 7, 9 and 14-day post-infection.
  • the tumor necrosis factor alpha (TNF-a) will be detected using enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • the studies are expected to demonstrate that the cell cultures infected with ZIKV exhibit up-regulated expression of tumor necrosis factor-a (TNF-a), and interleukin- ⁇ (IL- ⁇ ).
  • the tissues including testes, spleen, liver, heart, brain and kidney will be collected at 24, 28 and 72 h post-infection.
  • the total RNA from the tissues were extracted, one-step quantitative real-time PCR and 5 '-RACE assay will be performed.
  • the viral titers in the sera and tissues will be detected in Vero cells with serially dilution.
  • the results from treatment groups will be compared with the control
  • Table 2 Table showing strains used for siRNA prediction and siRNA sequence overlap.
  • Table 4 The list of sequence and targets for anti-ZIKV siRNA candidates Table 5 Characterization indexes of five SLiC species and five SLiC-siRNA nanoparticles, including particle sizes, poly-dispersity index (PDI) and Zeta-potential.
  • PDI poly-dispersity index
  • Zika vs Dengue virus serotype 3 Zika vs Dengue virus serotype 4
  • Zanluca C Melo VC, Mosimann AL, Santos GI, Santos CN, Luz K. First report of autochthonous transmission of Zika virus in Brazil. Mem Inst Oswaldo Cruz.

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Abstract

La présente invention concerne des compositions pharmaceutiques prophylactiques et thérapeutiques comprenant soit des molécules d'ARNsi, soit des molécules d'ARNm, destinées à la prévention et au traitement d'une infection par le virus Zika, ainsi que des méthodes pour leur utilisation.
PCT/US2018/020220 2017-02-28 2018-02-28 Formulations pharmaceutiques d'arn pour le traitement prophylactique et thérapeutique d'une infection par le virus zika WO2018160690A1 (fr)

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