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WO2025166047A1 - Composition et méthode de prévention ou de traitement de la grippe - Google Patents

Composition et méthode de prévention ou de traitement de la grippe

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Publication number
WO2025166047A1
WO2025166047A1 PCT/US2025/013848 US2025013848W WO2025166047A1 WO 2025166047 A1 WO2025166047 A1 WO 2025166047A1 US 2025013848 W US2025013848 W US 2025013848W WO 2025166047 A1 WO2025166047 A1 WO 2025166047A1
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WO
WIPO (PCT)
Prior art keywords
pharmaceutical composition
rna
sipbl
sipb2
sirnas
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Pending
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PCT/US2025/013848
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English (en)
Inventor
Kevin V. Morris
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Gene Co Pty Ltd
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Gene Co Pty Ltd
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Publication of WO2025166047A1 publication Critical patent/WO2025166047A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • 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
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • 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
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • 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
    • 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]
    • 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
    • C12N2320/31—Combination therapy

Definitions

  • RNA molecules for repressing expression of influenza (Flu) virus genes Provided herein are RNA molecules for repressing expression of influenza (Flu) virus genes, pharmaceutical compositions comprising the RNA molecules, and methods of preventing or treating influenza in a subject in need thereof.
  • influenza viruses While the annual flu vaccine may mitigate some viral spread, zoonotic infections with novel influenza viruses of avian or swine origin continue to pose pandemic threats.
  • influenza viruses have frequent and small changes and the ease of generating new viral variants by reassortment or mixing of genetic material between different strains all contribute to the epidemiologic characteristic of influenza, so that they can easily escape from protective immunity induced by a previous exposure to a different variant of the virus. It is also the reason why there is still no effective therapy for influenza and the existing vaccines still cannot contain the epidemic properly.
  • the present disclosure relates to a pharmaceutical composition that represses or inhibits a gene expression of an influenza virus.
  • the present disclosure also provides a pharmaceutical composition comprising siRNAs and/or antisense RNAs targeted to ultra-conserved sites in the human, avian, and swine influenza PB 1 (polymerase basic protein 1) and PB2 (polymerase basic protein 2) genes, and is a potent means to selectively target a virus family, rather than specific viral variants.
  • the pharmaceutical composition of the present disclosure is capable of repressing various influenza virus variants and is refractory to the emergence of viral resistance.
  • the present disclosure also provides a pharmaceutical composition comprising an RNA combination therapeutic that is refractory to the emergence of viral mutation, resulting in endemic in influenza viruses, and also able to treat virtually any influenza virus in human, swine, or avian.
  • the pharmaceutical composition comprises a plurality of RNA molecules and a pharmaceutically acceptable carrier thereof, wherein the plurality of RNA molecules comprises at least two RNA molecules selected from the group consisting of RNA molecules having at least 80% sequence identity to SEQ ID NOs.: 1 to 38.
  • the RNA molecule is a double stranded RNA. In at least one embodiment of the present disclosure, the RNA molecule is a small interference RNA (siRNA) or a short hairpin RNA (shRNA). In at least one embodiment of the present disclosure, the RNA molecule is about 10 to 50 nucleotides long, about 16 to 30 nucleotides long, about 18 to 28 nucleotides long, or about 20 to 27 nucleotides long.
  • siRNA small interference RNA
  • shRNA short hairpin RNA
  • the RNA molecule is a single stranded RNA. In at least one embodiment of the present disclosure, the RNA molecule is an antisense RNA (asRNA). In at least one embodiment of the present disclosure, the RNA molecule is about 50 to 400 nucleotides long, about 75 to 300 nucleotides long, or about 100 to 250 nucleotides long.
  • asRNA antisense RNA
  • the pharmaceutical composition comprises a plurality of RNA molecules, and the plurality of RNA molecules is a combination of siRNAs. In at least one embodiment of the present disclosure, the plurality of RNA molecules is a combination of shRNAs. In at least one embodiment of the present disclosure, the combination of siRNAs or shRNAs further comprises at least one asRNA.
  • the pharmaceutically acceptable carrier of the pharmaceutical composition is an extracellular vesicle or a lipid nanoparticle. In at least one embodiment of the present disclosure, the pharmaceutically acceptable carrier of the pharmaceutical composition is an extracellular vesicle. In at least one embodiment of the present disclosure, the extracellular vesicle is an exosome.
  • Also provided in the present disclosure are methods of repressing an expression of an influenza gene in a cell, comprising contacting the cell with any of the above pharmaceutical compositions.
  • Also provided in the present disclosure are methods of preventing or treating influenza in a subject in need thereof, comprising administering to the subject an effective amount of any of the above pharmaceutical compositions.
  • the subject is a human, swine, or avian.
  • the present disclosure provides a use of any of the above pharmaceutical compositions for preventing or treating influenza in a subject in need thereof.
  • the present disclosure also provides a use of any of the above pharmaceutical compositions for manufacture of a medicament for preventing or treating influenza in a subject in need thereof.
  • FIG. 1 shows the vector map of the PB1 reporter plasmid used in assessing siRNA repression of influenza virus gene PB 1.
  • FIG. 2 shows the vector map of the PB2 reporter plasmid used in assessing siRNA repression of influenza virus gene PB2.
  • FIG. 3 shows the vector map of antisense RNA as 1 expressing plasmid.
  • FIG. 4 shows the vector map of antisense RNA as2 expressing plasmid.
  • FIG. 5 shows the vector map of antisense RNA as3 expressing plasmid.
  • FIG. 6 shows the vector map of antisense RNA as4 expressing plasmid.
  • FIG. 7 shows the vector map of shRNA expressing plasmid expressing shPBl-GC- 2 shRNA.
  • FIG. 8 shows the vector map of shRNA expressing plasmid expressing shPB 1-GC- 22 shRNA.
  • FIG. 9 shows the vector map of shRNA expressing plasmid expressing shPB 1-GC- 2 and shPBl-GC-22 shRNAs.
  • FIG. 10 shows the relative locations of the siRNAs (e.g., siPBl-GC-1 to siPBl- GC-5, siPBl-GC-8, siPBl-GC-16, siPB l-GC-17, siPBl-GC-21, and siPB l-GC-22) and the conserved and ultra-conserved sites in PB 1 gene of avian, human, and swine.
  • siRNAs e.g., siPBl-GC-1 to siPBl- GC-5, siPBl-GC-8, siPBl-GC-16, siPB l-GC-17, siPBl-GC-21, and siPB l-GC-22
  • FIG. 11 shows the relative locations of the siRNAs (e.g., siPB2-GC-l to siPB2- GC-4, siPB2-GC-7, and siPB2-GC-9) and the conserved and ultra-conserved sites in PB2 gene of avian, human, and swine.
  • siRNAs e.g., siPB2-GC-l to siPB2- GC-4, siPB2-GC-7, and siPB2-GC-9
  • FIG. 12 shows the repression of PB1 gene expression by siRNAs.
  • FIG. 13 shows the repression of PB1 gene expression by siRNAs in a different experiment.
  • FIG. 14 shows the repression of PB2 gene expression by siRNAs.
  • FIG. 15 shows the vector map of antisense RNA reporter plasmid psiCheck- PBl_asRNATargets_VB230831-1500kvh (p8; plasmids used in screening antisense RNA repression of reporter gene expression).
  • FIG. 16 shows the vector map of control plasmid (pl 8; pLV[Exp]-Hygro- EF1A> ⁇ GFP-CD-UR ⁇ ) used as a negative control for antisense RNA studies.
  • This plasmid expresses the GFP transgene.
  • FIG. 17 shows the repression of PB1 gene expression by asRNAs.
  • FIG. 18 shows the repression of PB 1 and PB2 gene expressions by siRNA combinations.
  • FIG. 19 shows the vector map of the Ago-2 expressing plasmids (p24; pRP[Exp]- Bsd-EF1A> ⁇ Ago-2 ⁇ -(Blastocydin)) used for EV packaging of shRNAs.
  • FIG. 20 shows the vector map of the enhancer plasmids (p54; pDB68 (Conx43)(Enhancer)(Neomycin)) used to enhance packaging and release of shRNAs from the exosomes.
  • FIG. 21 shows the repression of PB1 gene expression by exosome-mediated transfer of shRNAs at 48 hours.
  • FIG. 22 shows the repression of PB 1 gene expression by exosome-mediated transfer of shRNAs at 72 hours.
  • FIG. 23 shows the repression of PB1 and PB2 gene expressions by RNA combinations including siRNAs and asRNA.
  • FIG. 24 shows the vector map of shRNA expressing plasmid expressing shPBl- GC-1 and shPBl-GC-8 shRNAs.
  • FIG. 25 shows the vector map of shRNA expressing plasmid expressing shPBl- GC-3 and shPBl-GC-5 shRNAs.
  • FIG. 26 shows the repression of PB1 gene expression by siRNAs in another independent experiment.
  • FIG. 28 shows the repression of PB 1 and PB2 gene expressions by siRNA combinations (20 nM) in a different experiment.
  • FIG. 29 shows the repression of PB1 and PB2 gene expressions by siRNA combinations (50 nM) in a different experiment.
  • FIG. 30 shows the repression of PB1 gene expressions by siRNA in stable reporter cell line.
  • FIG. 31 shows the repression of PB2 gene expressions by siRNA in stable reporter cell line.
  • FIG. 32 shows the repression of Flu M protein of H INI virus by siRNAs.
  • FIG. 33 shows the repression of Flu M protein of H3N2 virus by siRNAs.
  • FIG. 34 shows the repression of Flu M protein of H1N1 virus by siRNA combinations.
  • FIG. 35 shows the repression of Flu M protein of H3N2 virus by siRNA combinations.
  • FIG. 36 shows the repression of PB1 and PB2 gene expressions by exosome- mediated transfer of shRNAs at 48 hours.
  • FIG. 37 shows the repression of PB1 and PB2 gene expressions by exosome- mediated transfer of shRNAs at 72 hours.
  • RNAi is a mechanism of action that can specifically turn off the production of proteins in cells in a sequence-specific and potent manner. It works via the introduction of double-stranded RNAs (dsRNAs) of 15 to 30 base pairs that specifically target argonaute-2 (AGO2) to mRNAs via sequence complementarity, causing their subsequent degradation. RNAi can function to repress a gene expression either transiently by using post-transcriptional gene silencing (PTGS) or long-term by using transcriptional gene silencing (TGS).
  • PTGS post-transcriptional gene silencing
  • TLS transcriptional gene silencing
  • a short hairpin RNA (shRNA) molecule comprises paired RNA sequences and a loop portion positioned between the paired RNA sequences so as to form the hairpin.
  • the loop can vary in length. In some embodiments, the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the loop is 18 nucleotides in length.
  • the hairpin structure can also contain 3’ and/or 5’ overhang portions. In some embodiments, the overhang is a 3’ overhang and/or a 5’ overhang with 1 , 2, 3, 4, or 5 nucleotides in length.
  • the nucleotide sequence of the loop region may vary and could be, for example, 5’-GCAA-3’, 5’- GCGC-3’, 5’-TTGC-3’, or other sequences as will be well understood by a skilled person in the art.
  • exosome refers to a cell-derived small vesicle (between 20 nm to 300 nm in diameter, e.g., 40 nm to 200 nm in diameter), which comprises a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane.
  • the exosome comprises lipids or fatty acids and polypeptides and further comprises the inhibitory nucleic acids described herein as a payload.
  • exosomes can be derived from a producer cell and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. Exosomes can be directly loaded with exogenous nucleic acids or drugs by electroporation, lipofection, sonication, or contact with calcium chloride. Alternatively, purified exosomes may be loaded ex vivo by, for example, electroporation.
  • exosomes of the present disclosure can be produced from a cell grown in vitro or a body fluid of a subject.
  • various producer cells e.g., HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs)
  • HEK293 cells HEK293 cells
  • CHO cells Chinese hamster ovary (CHO) cells
  • MSCs mesenchymal stem cells
  • compositions of the present disclosure may also be formulated by incorporation of the RNA molecules described herein into adenoviruses or adeno- associated viruses (AAVs), formulated with cell-penetrating peptides, lentiviral vectors, polymers, dendrimers, or prepared as small interference RNA (siRNA) bioconjugates such as the N-acetylgalactosamine (GalNAc)-siRNA conjugate delivery platform.
  • siRNA small interference RNA
  • the candidate siRNAs can be delivered as short hairpin RNAs (shRNAs). Both siRNAs and shRNAs can target and repress viruses and are functionally equivalent.
  • shRNAs short hairpin RNAs
  • the candidate siRNAs are delivered as shRNAs, they are derived from a cell system and packaged into exosomes or a vector (an AAV or lentiviral vector) as described above.
  • an shRNA may be provided in an expression cassette containing a promoter contiguously linked to an siRNA as described herein.
  • the promoter is a pol II promoter or a pol III promoter, such as a U6 promoter (e.g., a mouse U6 promoter) or an Hl promoter.
  • the expression cassette further contains a marker gene.
  • the promoter is a pol II promoter.
  • the promoter is a tissue-specific promoter.
  • the promoter is an inducible promoter.
  • the promoter is a pol III promoter.
  • the promoter is a U6 promoter or an Hl promoter.
  • vector containing an expression cassette described herein examples include adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based virus vectors.
  • AAV adeno-associated virus
  • HSV herpes simplex virus
  • Maloney-based virus vectors examples include adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based virus vectors.
  • the pharmaceutical compositions described herein may be administered in dosages sufficient to inhibit the expression of the target gene or the biological activity of nontranslated target sequences (e.g., regulatory sequences) in a cell, tissue, or organism under treatment.
  • the specific dosages of the inhibitory nucleic acids described herein administered to a given subject will depend on factors such as the route of administration and physical characteristics of the subject (including health status) and so forth.
  • the appropriate dosage of a given pharmaceutical composition comprising the inhibitory nucleic acids described herein may depend on a variety of factors including, but not limited to, a subject’s physical characteristics (e.g., age, weight, and gender), the progression (i.e., pathological state) of a disease, and other factors that will be readily recognized by one skilled in the art.
  • Non-limiting examples of suitable dosages of the inhibitory nucleic acids described herein include those in the range of 0.01 to 200 milligrams per kilogram body weight of the recipient per day, e.g., 1 to 50 mg/kg body weight per day, 1 to 40 mg/kg body weight per day, 1 to 30 mg/kg body weight per day, 1 to 20 mg/kg body weight per day, 1 to 10 mg/kg body weight per day, 1 to 5 mg/kg body weight per day, 1 to 3 mg/kg body weight per day, 1 to 2 mg/kg body weight per day, 0.1 to 1 mg/kg body weight per day, 0.1 to 0.9 mg/kg body weight per day, 0.1 to 0.8 mg/kg body weight per day, 0.1 to 0.7 mg/kg body weight per day, 0.1 to 0.6 mg/kg body weight per day, 0.1 to 0.5 mg/kg body weight per day, 0.1 to 0.4 mg/kg body weight per day, 0.1 to 0.3 mg/kg body weight per day, 0.1 to 0.2 mg/kg body weight per day, 0.
  • the treatment would be for the duration of the disease state or condition.
  • the optimal quantity and interval of individual dosages will be determined by the nature and extent of the disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimal conditions can also be determined using conventional techniques.
  • a pharmaceutical composition described herein may be administered 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times.
  • the administrations may be from about one to about twelve-week intervals, e.g., from about one to about four- week intervals.
  • Suitable techniques for introduction of the inhibitory nucleic acids described herein into cells, tissues, and organisms include various carrier systems, vectors, and reagents.
  • Non-limiting examples include lipid nanoparticles (LNP), micelles, nucleic-acid-lipid particles, lipoplexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus-like particles (VLPs), and any mixtures thereof.
  • compositions of the present disclosure may be administered in any suitable way, for example, intravenously, buccally, parenterally, intranasally, orally, sublingually, or topically. Accordingly, the administration may be topical, pulmonary (e.g., by inhalation or insufflation of aerosols or powders with a nebulizer), intranasal, intratracheal, epidermal, transdermal, oral, or parenteral.
  • Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, or intracranial (e.g., intraparenchymal, intrathecal, or intraventricular) administration.
  • the pharmaceutical composition is adapted for intranasal administration.
  • the pharmaceutical composition of the present disclosure is formulated as a direct-acting nasal spray.
  • the nasal spray can be self-administered at point-of-care.
  • a reference for a disease state may be a normal, healthy state; a reference for a mutated protein may be a non-mutated protein; a reference for a disease treatment may be no treatment or may be a standard of care treatment.
  • the reference is the activity and/or expression of the target molecule in the absence of the agent.
  • a reference is based on a predetermined level, e.g., based on functional expression or empirical assays.
  • a reference obtained from one cell, sample, or subject e.g., a cell or sample from a healthy subject, a subject without having a particular disease; a healthy subject, a subject without having the particular disease).
  • a reference is obtained from more than one (e.g., a population of) cell, sample, or subject (e.g., a cell or sample from a healthy subject, a subject without having a particular disease; a healthy subject, a subject without having a particular disease), such as 2, 3, 4, 5, 10, 20, 30, 50, 100 or more, or a statistically significant number of cells, samples, or healthy subjects.
  • a reference obtained from more than one cell, sample, or subject can be represented as a statistic (e.g., an average or median).
  • the RNA molecule is an analog or derivative thereof.
  • the polynucleotide, or the analog or derivative thereof is an inhibitor of the target molecule.
  • the RNA molecule can have sequences containing naturally occurring ribonucleotide monomers, non-naturally occurring nucleotides, or combinations thereof. Accordingly, the RNA molecule can include, for example, nucleotides comprising naturally occurring bases (e.g., A, G, C, or U) and nucleotides comprising modified bases (e.g., 7-deazaguanosine, inosine, or methylated nucleotides, such as 5- methyl dCTP and 5 -hydroxymethyl cytosine). In some embodiments, the polynucleotide comprises at least one modified nucleotide.
  • naturally occurring bases e.g., A, G, C, or U
  • modified bases e.g., 7-deazaguanosine, inosine, or methylated nucleotides, such as 5- methyl dCTP and 5 -hydroxymethyl cytosine.
  • the polynucleotide comprises at least one modified nucleot
  • Non-limiting examples of modified nucleotides include 2’-fluoro, 2’-o-methyl, 2’-deoxy, unlocked nucleic acid, 2’-hydroxy, phosphorothioate, 2’ -thiouridine, 4’ -thiouridine, and 2 ’-deoxy uridine.
  • the modification increases nuclease resistance, increases serum stability, decrease immunogenicity, or a combination of the foregoing.
  • the RNA molecule is included in a vector, e.g., expression vector or plasmid.
  • the RNA molecule comprises an analog or a derivative of a polynucleotide.
  • the analog or derivative is a peptide nucleic acid (PNA).
  • the analog or derivative is a locked nucleic acid (LNA).
  • the analog or derivative is a morpholino oligonucleotide.
  • the analog or derivative comprises one or more phosphorothioate-linkages.
  • the RNA molecule is a ribonucleic guanidine (RNG) nucleotide.
  • the RNA molecule modulates the expression and/or activity of a nucleic acid, or a portion thereof (e.g., a biologically active portion or fragment thereof).
  • the RNA molecule can be single stranded (ss) or double stranded (ds).
  • the polynucleotide is double stranded (ds).
  • the length of the double-stranded polynucleotide is about 15 to 50 base pairs, e.g., about: 15 to 45, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 20 to 50, 20 to 45, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 50, 25 to 45, 25 to 40, 25 to 35, 25 to 30, 30 to 50, 30 to 45, 30 to 40, 30 to 35, 35 to 50, 35 to 45, 35 to 40, or 40 to 50 base pairs.
  • the length of the polynucleotide is about 19 to 23 base pairs. In some embodiments, the length of the polynucleotide is about 21 base pairs.
  • the RNA molecule is single stranded (ss).
  • the length of the single stranded RNA molecule is about 50 to 400 nucleotides, e.g., about: 50 to 380, 50 to 350, 50 to 325, 50 to 300, 50 to 250, 75 to 400, 75 to 350, 75 to 300, 75 to 250, 75 to 225, 75 to 200, 75 to 210, 75 to 190, 75 to 180, 100 to 400, 100 to 350, 100 to 300, 100 to 250, 100 to 225, 100 to 200, 120 to 400, 120 to 350, 120 to 300, 120 to 280, 120 to 250, 120 to 225, 130 to 400, 130 to 350, 130 to 300, 130 to 250, 1 0 to 225, 130 to 200, or 150 to 400 nucleotides.
  • the RNA molecule (e.g., an antisense oligonucleotide) can hybridize to an mRNA encoding the target molecule (e.g., under physiological conditions).
  • the length of the RNA molecule is at least about 10 nucleotides, e.g., at least about: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides or about: 10 to 30, 15 to 30, 15 to 25, or 20 to 25 nucleotides.
  • the polynucleotide is at least 75% identical to an antisense sequence of the targeted transcript, e.g., at least about: 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%.
  • the RNA molecule further comprises an overhang sequence (e.g., unpaired, overhanging nucleotides which are not directly involved in the formation of the double helical structure by the core sequences).
  • the RNA molecule comprises a 3’ overhang, a 5’ overhang, or both.
  • the overhang is about 1 to 5 nucleotides.
  • the overhang comprises a modified ribonucleotide.
  • RNA molecules suitable for use in the compositions, kits, and methods described herein include a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antagomir, an antisense DNA, an antisense RNA, a morpholino nucleic acid (MNA), a locked nucleic acid (LNA), a peptide nucleic acid (PNA), an aptamer, and a guide RNA (gRNA).
  • siRNA small interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • antagomir an antisense DNA
  • an antisense RNA a morpholino nucleic acid
  • LNA locked nucleic acid
  • PNA peptide nucleic acid
  • gRNA guide RNA
  • the RNA molecule inhibits gene expression (e.g., through the biological process of RNA interference (RNAi)).
  • RNAi RNA interference
  • the RNA molecule appropriate for RNA interference can be readily designed and produced by a person of ordinary skill using techniques, assays, and reagents known in the art, including computational tools. See, e.g., Pei et al., Nat. Methods. 2006, 3(9):670-6; Reynolds et al., Nat. Biotechnol. 2004, 22(3):326-30; Khvorova et al., Cell. 2003, 115(2):209-16; Schwarz et al., Cell. 2003, 115(2): 199-208; Ui-Tei et al., Nucleic Acids Res.
  • the RNA molecule is an siRNA.
  • the siRNA comprises a nucleotide sequence that is identical to about a 15 to 25 contiguous mRNA sequence encoding the target protein.
  • the siRNA is a double-stranded RNA molecule having about 19 to 25 base pairs.
  • the siRNA commences with the dinucleotide AA.
  • the siRNA has a GC-content of about 30% to 70%, e.g., about: 30% to 65%, 30% to 60%, 30% to 55%, 30% to 50%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 45% to 70%, 45% to 65%, 45% to 60%, or 45% to 55%.
  • the RNA molecule is an shRNA.
  • the shRNA is an RNA molecule including a hairpin turn that decreases expression of a target gene via RNAi.
  • the shRNAs can be delivered to cells in the form of plasmids, e.g., viral or bacterial vectors, e.g., by transfection, electroporation, or transduction.
  • siRNAs and shRNAs resemble intermediates in the processing pathway of the endogenous microRNA (miRNA) genes (see, e.g., Bartel, Cell 116:281-97 (2004)).
  • the siRNA functions as a miRNA; in other embodiments, the miRNA functions as an siRNA (see, e.g., Zeng et al., Mol. Cell 9:1327-33 (2002); Doench et al., Genes Dev. 17:438-42 (2003)).
  • the RNA molecule is chemically synthesized. In some embodiments, the RNA molecule is expressed recombinantly. In some embodiments, the RNA is transcribed in vitro.
  • RNA therapeutics are known in the art. See, for example, RNA Therapeutics: Function, Design, and Delivery (Mouldy Sioud Eds., 2010) and Kaczmarek et al., Advances in the delivery of RNA therapeutics: from concept to clinical reality, Genome Medicine 9:60 (2017).
  • the RNA molecule is an aptamer. In some embodiments, the aptamer binds to a target molecule described herein. In some embodiments, the aptamer binds to a binding partner of a target molecule described herein.
  • the RNA molecule is linked (e.g., covalently) to a delivery polymer. In some embodiments, the link between the RNA molecule and the delivery polymer is reversible. In some embodiments, the RNA molecule is linked to the delivery polymer via a physiologically labile linker. In some embodiments, the physiologically labile linker is a disulfide bond.
  • part when a part “includes” or “comprises” a component or a step, unless there is a particular description contrary thereto, the part can further include other components or other steps, not excluding the others.
  • the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element listed within the list of elements and not excluding any combinations of elements in the list of elements.
  • This definition also allows that elements may optionally be present other than the elements identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements identified.
  • a “percentage of sequence identity” may be calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, I) or the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys, and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e. , the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical nucleic acid base e.g., A, T, C, G, U, I
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Tr
  • nucleotides and polypeptides having at least about 80%, about 83%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 92%, about 95%, about 97%, about 98%, about 99%, or about 100% sequence identity to any of the reference sequences described herein (see, e.g., Sequence Listing), typically where the polypeptide variant maintains at least one biological activity or function of the reference polypeptide.
  • treatment refers to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein.
  • treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed.
  • treatment may be administered in the absence of signs or symptoms of the disease.
  • treatment may be administered to a susceptible subject prior to the onset of symptoms (e.g., in light of a history of symptoms) to delay or prevent disease occurrence. Treatment may also be continued after symptoms have resolved, for example, to delay or prevent recurrence.
  • compositions can be prepared according to any method known in the art for the manufacture of pharmaceuticals. Such composition or combination may contain sweetening agents, flavoring agents, coloring agents, and preserving agents.
  • a formulation can be admixed with nontoxic and pharmaceutically acceptable excipients which are suitable for manufacture. Non-limiting formulations may comprise one or more diluents, emulsifiers, preservatives, buffers, excipients, etc.
  • pharmaceutically acceptable carriers including buffers
  • the PB 1 reporter was generated by cloning PB 1 ultra-conserved sites into enhanced green fluorescent protein (EGFP) which was generated to be transcribed in fusion with hRluc (Renilla reniformis').
  • EGFP enhanced green fluorescent protein
  • hRluc Renilla reniformis'
  • HSV-TK herpes simplex virus thymidine kinase
  • Table 1 lists the names and corresponding vector maps of the reporter plasmids used.
  • shRNAs small hairpin RNAs
  • Table 3 lists the information regarding the shRNAs, including the shRNA names, corresponding siRNAs, plasmid names and number, and vector maps of the plasmids.
  • Example 1 siRNAs to repress influenza virus genes
  • siRNAs were designed to target to ultra-conserved sites in PB 1 (Table 4) or PB2 (Table 5) genes of avian, human, and swine influenza viruses. These siRNAs were designed a priori targeted to ultra-conserved sites curated from the National Center for Biotechnology Information (NCBI) influenza database.
  • FIGs. 10 and 11 show the relative locations of the siRNAs and the conserved and ultra-conserved sites in PB1 and PB2 genes of avian, human, and swine influenza viruses. Table 4.
  • RNAeasy kit Qiagen, Venlo, Netherlands
  • DNase treatment DNase treatment
  • RNA reverse Transcription kit Thermo Fisher Scientific, Waltham, MA, USA
  • hFluc, hRluc, and beta actin expressions were assessed using gene specific primers listed in Table 6.
  • the hFluc expression contains the PB1 or PB2 target transcripts and is standardized to beta actin expression as well as hRluc standardized to beta actin expression.
  • the standardization of hFluc/beta actin to hRluc/beta actin allows for control of transfection efficiency as transient transfections were carried out. This is because hRluc is included in the plasmid and thus can be used as a reference for transfection efficiency. These procedures were repeated 3 times for each siRNA. Table 6. Primers for qRT-PCR analysis
  • FIGs. 12 and 13 show that transfections of siRNAs targeting at PB1 as listed in Table 4 repress PB 1 expression in different experiments compared to cells transfected with the control siRNA, indicating that these siRNAs target at PB 1 and inhibit expression of PB1.
  • siRNAs targeting at PB2 as listed in Table 5 repress PB2 expression compared to cells transfected with the control siRNA, suggesting that these siRNAs target at PB2 and inhibit expression of PB2.
  • HEK293 cells were plated at 200,000 cells per well in a 12- well plate.
  • the PB 1 or PB2 reporter plasmids p2 or p3 (0.1 pg/well), siRNAs from Table 4 and Table 5 (20 nM/well), and Lipofectamine 3000 (L3000015; Invitrogen) were gently mixed in serum- free Opti-MEM I Reduced Serum Medium (Opti-MEM; 31985070; Gibco).
  • siRNA-DNA-Lipid complexes were then added to the wells containing cells and fresh complete DMEM (cDMEM). After 48 hours of incubation at 37°C, the cells were collected for assessment of PB1 or PB2 gene expressions by total RNA extraction and reverse transcription, followed by quantitative qRT-PCR, as described above.
  • FIG. 26 shows the result of the PB 1 repression by transfecting siRNAs targeting at PB 1, including siPBl-GC-1, siPBl-GC-2, siPBl-GC-3, and siPBl-GC-22 (labeled as PBl-sil, PBl-si2, PBl-si3, and PBl-si22, respectively).
  • the reporter gene expression was significantly reduced to only 40% to 50% compared to that transfected with miN367 RNA (labeled as siControl in FIG. 26).
  • FIG. 27 shows the result of the PB2 repression by transfecting siRNAs targeting at PB2, including siPB2-GC-l, siPB2-GC-2, siPB2-GC-3, siPB2-GC-4, siPB2-GC-5, and siPB2-GC-7 (labeled as PB2-sil, PB2-si2, PB2-si3, PB2-si4, PB2-si5, and PB2-si7 in FIG. 27).
  • the reporter gene expression was significantly reduced to only 40% to 60% compared to that transfected with miN367 RNA (labeled as siControl in FIG. 27).
  • Example 2 Antisense RNAs to repress influenza virus genes
  • asRNAs long antisense RNAs
  • Table 2 Information of these plasmids was tabulated in Table 2 above, and FIGs. 3 to 6 show the vector maps of these plasmids.
  • HEK293 cells were co-transfected with plasmids in Table 2 along with reporter plasmid psiCheck-PB l_asRNATargets_VB230831-1500kvh (p8, vector map shown in FIG. 15) using 50 ng of each plasmid with an equal amount per cell by using Eipofectamine 3000 (E3K, Thermo Fisher Scientific, Waltham, MA, USA).
  • a control plasmid (p!8, pLV[Exp]-Hygro-EFlA> ⁇ GFP-CD-UR ⁇ , vector map shown in FIG. 16) was used as a negative control.
  • Table 7 lists the exemplary combinations of siRNAs and asRNAs of the present disclosure to treat influenza virus infections in swine, avian, and/or human.
  • siRNAs packaged in lipid nanoparticles
  • shRNAs having the same sequences of siRNAs but packaged in exosomes
  • noncoding antisense RNAs packaged in exosomes
  • LNPs lipid nanoparticles
  • siRNA combinations to repress influenza genes PB1 and PB2 expression
  • various combinations of PB1 and PB2 repressive siRNAs were used. Briefly, multiple siRNAs were transfected into cells simultaneously and assessed for PB1 and PB2 gene repression following the same transfection method and qRT-PCR analysis as described in Example 1. Table 8 below shows the combinations tested.
  • siRNA combinations used to repress PB1 and PB2 in FIG. 18 It is shown that all these combinations of the PB1 and PB2 siRNAs potently repressed PB 1 and PB2 gene expression and that both PB 1 and PB2 siRNAs can work in concert with one another, as shown in FIG. 18. hi another set of experiments, more siRNA combinations were tested to evaluate the effectiveness of combinational siRNAs. These combinations of siRNAs against PB1 and PB2 genes were transfected with reporter plasmids, and RNA levels were assessed 72 hours later for Flue vs. Rluc expression to determine repression of the Rluc target standardized to the Flue expressed transgene, so as to measure the reporter mRNA suppression levels.
  • FIG. 1 siRNA combinations used to repress PB1 and PB2 in FIG. 18 It is shown that all these combinations of the PB1 and PB2 siRNAs potently repressed PB 1 and PB2 gene expression and that both PB 1 and PB2 siRNAs can
  • FIG. 28 shows the results of siRNA combinations (20 nM) against PB 1 and PB2 genes, respectively, and all combinations were able to repress the reporter expression.
  • FIG. 29 shows the results of combinations containing siRNAs (50 nM) targeting both PB 1 and PB2, and it was also found that all combinations can repress the reporter expression.
  • siRNAs may more potently repress PB1 and PB2 target gene expression than single siRNA treatments.
  • multiple conserved sites in the PB1 and PB2 viral genes are targeted with siRNA combinations to repress viral escape and can be used to target influenza viruses generally across different species, e.g., human, swine, and avian influenza viruses.
  • Exosomes are nano-sized (50 to 150 nm) extracellular vesicles (EVs) that are shed from cells and then taken up by neighboring cells. EVs have been found to be immunologically inert in vivo and are also anti-inflammatory, biodegradable, biocompatible, and safe. Thus, they can be exploited as natural nanoparticles for in vivo delivery of therapeutic agents such as RNAs and proteins to modulate cellular functions. Exosomes have been found to package small hairpin RNAs (shRNAs), which are the cellular equivalent to siRNAs.
  • shRNAs small hairpin RNAs
  • a transient transfection transwell assay was carried out. This assay used a transwell plate and HEK293 cells.
  • shRNA expressing plasmids based on the siRNAs observed to repress PB 1 and PB2 gene expression were generated and obtained from VectorBuilder Inc. (Chicago, IL, USA). Information of these shRNA expressing plasmids, shl, sh2, sh3, sh4, and sh5, are compiled in Table 3, and their vector maps are shown in FIGs. 7 to 9 and FIGs. 24 to 25.
  • HEK293 cells 0.5 x 10 6 /well were plated for both producer and recipient cells.
  • HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS) (GeminiBio) and incubated at 37°C and 5% CO2.
  • DMEM Dulbecco’s modified Eagle’s medium
  • FBS fetal bovine serum
  • the producer cells were transfected (a total of 300 ng/0.5 x 10 6 cells) with the plasmid cocktails including the shRNA expressing plasmids listed in Table 3 along with p24 (pRP[Exp]-Bsd- EF1 A> ⁇ Ago-2 ⁇ -(Blastocydin), vector map shown in FIG.
  • Ago-2 and Cnx43 are the machinery required to package shRNAs and to enhance endosomal release upon uptake of the exosomes into target cells.
  • the recipient cells were transfected with p8 (psiCheck-PBl_asRNATargets_VB230831-1500kvh plasmid) (200 ng), and when both PB1 and PB2 were targeted, p3 (VB230710-1643ubv pRP
  • exosome producer cells were seeded in the basolateral chamber, and exosome recipient cells were seeded on the apical chamber on the top of insert with 0.4 pm pore size.
  • Nearly 300,000 HEK293T cells were seeded in the basolateral chamber per well of 24-well plate (Catalog No.: 140620, Thermo Scientific).
  • transwell inserts were arranged in unused wells and seeded with 50,000 exosome recipient cells in 100 pF complete DMEM and transfected if necessary. In the unused basolateral chamber with inserts, 500 pF complete DMEM was added.
  • the cells were collected, and mRNAs were isolated from the recipient cells followed by qRT-PCR analysis with the primers listed in Table 6 for assessment of PB1 and PB2 gene repression, following the same procedures as described above. Therefore, the exosome transfer experiments were carried out such that the producer cells are transfected with plasmid combinations as shown in Table 9, which engineer these cells into shRNA-EV producing factories.
  • the recipient cells are those cells that are separated from the producer cells by the transwell and transfected with the reporter plasmid (p8, psiCheck- PB l_asRNATargets_VB230831-1500kvh), or when both FBI and PB2 targeting was assessed, then both reporter plasmids p8 (psiCheck-PBl_asRNATargets_VB 230831- 1500kvh) and p3 (VB230710-1643ubv pRP[Exp]-Puro-CMV+intron> ⁇ Luc-PB2- Ffluc ⁇ ) were used. In this assay, the recipient cells received the exosome-transferred shRNAs from the producer cells through the transmembrane in the culture medium.
  • exosomes can deliver two shRNAs corresponding to siPBl-GC-2 and siPBl-GC22, i.e., sh3, to repress PB 1 expression at 48 hours, as shown in FIG. 21, and be able to repress PB1 expression while transfection of only siPBl-GC- 2 corresponding to shl or only siPB l-GC-22 corresponding to sh2 does not significantly repress PB1 expression at 48 hours.
  • shRNA combination transferred in exosomes was shown to further repress the PB1 expression to 55% of the control, as shown in FIG. 22.
  • sh4 containing shRNAs corresponding to siPBl-GC-1 and siPBl-GC-8 (labeled as PB1 shl&8 in FIGs. 36 and 37), as well as sh5 containing shRNAs corresponding to siPB2-GC-3 and siPB2-GC-5 (labeled as PB2 sh3&5 in FIGs. 36 and 37) were also shown to be delivered by exosomes and to repress PB1 and PB2 expressions at 48 hours and 72 hours, as shown in FIGs. 36 and 37, respectively.
  • RNA combinations including both multiple shRNAs and long antisense RNAs were assessed for their suppressive effects on PB1 and PB2 expression.
  • siRNA combinations targeted to PB1 and PB2 e.g., siPBl-GC-2 and siPBl-GC-22 for PB1, siPB2-GC-l and siPB2-GC-2 or siPB2-GC-2 and siPB2-GC-7 for PB2 were cotransfected into cells with the long antisense RNA, e.g., as2 (pLV[ncRNA]-Hygro- CMV> ⁇ asRNAPBl-l-CD-Ula ⁇ ) shown in Table 2.
  • PB1 and PB2 reporter cell lines were generated with HEK293 cells that constantly express the FBI and PB2 reporter genes.
  • HEK293 cells were plated at 50,000 cells per well in a 24-well plate and were transfected with the FBI or PB2 plasmid (50 ng/well) using Lipofectamine 3000 reagent on the following day. After 24 hours of incubation at 37°C, the transfection medium was replaced with fresh medium containing 0.5 g/mL of puromycin (BA-PJ593-0025; Protech) for drug selection. After one month of selection, stable cell lines were validated by qRT-PCR to measure PB1 or PB2 gene expression.
  • the stable HEK293 PB1 or PB2 reporter cells were plated at 50,000 cells per well in a 24-well plate.
  • siRNAs 50 or 100 nM/well
  • Lipofectamine RNAiMax 13778150; Invitrogen
  • Opti-MEM serum-free Opti-MEM
  • the siRNA and Lipofectamine RNAiMax in Opti-MEM formed siRNA-Lipid complexes. These complexes were then added to the wells containing cells and fresh cDMEM. After 72 hours of incubation at 37°C, the cells were collected for gene expression assays.
  • the stable cell lines were validated with previously screened siRNAs targeting PB 1 , including siPB l-GC-2, siPBl-GC-22, and the combination of siPB l-GC-2 and siPBl- GC-22, and were further used to show repression of Flu genes in siPBl-GC-1 (labeled as PBl-si2, PB l-si22, PBl-si2&22, and PBl-sil in FIG. 30, respectively).
  • the stable cell lines were also validated with previously screened combinations of siRNAs targeting PB2, including siPB2-GC-l and siPB2-GC-2 as well as siPB2-GC-5 and siPB2-GC-7, and were further used to show repression of Flu genes in siPB2-GC- 3 and siPB2-GC-5 (labeled as PB2-sil&2, PB2-si5&7, PB2-si3, and PB2-si5 in FIG. 31, respectively).
  • siRNAs alone or in combination, were able to repress PB1 or PB2 reporter gene expression in the stable cell lines similar to the results observed in Example 1.
  • real-world influenza viruses were used to test the ability of siRNAs in inhibiting viral replication and influenza infection in Madin-Darby Canine Kidney (MDCK) cells.
  • MDCK cells were plated at 0.5 to 2 x 10 5 cells per well in a 24-well plate. On the following day, the cells were transfected with 50 nM or 100 nM of individual siRNA or siRNA combinations using Lipofectamine 3000 reagent.
  • H1N1 A/swine/Changhua/415 -7/2009
  • H3N2 A/swine/Taiwan ex USA/28-9/2010 strains at a multiplicity of infection (MOI) of 0.1
  • VGM virus growth medium
  • H1N1 A/swine/Changhua/415 -7/2009
  • H3N2 A/swine/Taiwan ex USA/28-9/2010
  • siPBl-GC-1, siPB2-GC-l, siPB2-GC-3, and siPB2- GC-5 (labeled as PBl-sil, PB2-sil, PB2-si3, and PB2-si5 in FIGs. 32 and 33, respectively) reduced Flu M protein gene expression in H1N1 and H3N2 by more than 98% comparing to the siControl-treated group.
  • both siRNA combinations e.g., siPB2-GC-3 in combination with siPB2-GC-5 and siPBl-GC-2 in combination with siPBl-GC-22 (labeled as PB2-si3, PB2-si5, PB2-si3+5, PBl-si2, PBl-si22, and PBl-si2&22 in FIGs. 34 and 35, respectively), effectively inhibited Flu M protein expression of both flu virus strains, significantly reducing H1N1 and H3N2 Flu M protein gene expression by more than 98%.

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Abstract

L'invention concerne des compositions et des méthodes permettant de réprimer l'expression d'un gène du virus de la grippe. La composition comprend une pluralité de molécules d'ARN, telles qu'un ARNsi, un shARN ou un asARN. L'invention concerne également des méthodes de prévention ou de traitement de la grippe.
PCT/US2025/013848 2024-01-31 2025-01-30 Composition et méthode de prévention ou de traitement de la grippe Pending WO2025166047A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007064802A1 (fr) * 2005-12-02 2007-06-07 The Mount Sinai Medical Center Of New York University Virus chimeriques presentant des proteines de surface non natives et leurs utilisations
WO2014011512A1 (fr) * 2012-07-08 2014-01-16 Sirnaomics, Inc. Compositions et procédés pour des produits thérapeutiques arnsi « résistants » pour la grippe

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007064802A1 (fr) * 2005-12-02 2007-06-07 The Mount Sinai Medical Center Of New York University Virus chimeriques presentant des proteines de surface non natives et leurs utilisations
WO2014011512A1 (fr) * 2012-07-08 2014-01-16 Sirnaomics, Inc. Compositions et procédés pour des produits thérapeutiques arnsi « résistants » pour la grippe

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