Composition and method for preventing or treating influenza

Abstract

Provided are compositions and methods to repress an expression of an influenza gene. The composition includes a plurality of RNA molecules, such as an siRNA, an shRNA, or an asRNA. Also provided are methods of preventing or treating influenza.

Description

[0001] COMPOSITION AND METHOD FOR PREVENTING OR TREATING

[0002] INFLUENZA

[0003] BACKGROUND

[0004] Technical Field

[0005] 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.

[0006] Description of Related Art

[0007] Seasonal influenza epidemics caused by influenza A and B viruses impose significant financial and clinical burdens worldwide every year. This is because while most infected individuals experience fever and myalgia, some cases can be bedbound for at least several days or even fatal. It is estimated that a billion cases of seasonal influenza happen annually, including 3 to 5 million cases of severe illness and causing 290,000 to 650,000 respiratory deaths annually, making it the most widespread and common infection.

[0008] 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. The fact that 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.

[0009] Although there are antiviral drugs approved for treating influenza, their use, however, is limited due to concerns about side effects and compliance. In addition, there is always emergence of resistant strains that render the antiviral drugs ineffective. Therefore, there is still an unmet need for an effective treatment for influenza.

[0010] SUMMARY

[0011] 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.

[0012] In at least one embodiment of the present disclosure, 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.

[0013] In at least one embodiment of the present disclosure, 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.

[0014] In at least one embodiment of the present disclosure, 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.

[0015] In at least one embodiment of the present disclosure, 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.

[0016] In at least one embodiment of the present disclosure, 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.

[0017] 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.

[0018] 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. In at least one embodiment, the subject is a human, swine, or avian. In at least one embodiment, the present disclosure provides a use of any of the above pharmaceutical compositions for preventing or treating influenza in a subject in need thereof.

[0019] In at least one embodiment, 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.

[0020] BRIEF DESCRIPTIONS OF THE DRAWINGS

[0021] The present disclosure will become more readily appreciated by reference to the following descriptions in conjunction with the accompanying drawings.

[0022] FIG. 1 shows the vector map of the PB1 reporter plasmid used in assessing siRNA repression of influenza virus gene PB 1.

[0023] FIG. 2 shows the vector map of the PB2 reporter plasmid used in assessing siRNA repression of influenza virus gene PB2.

[0024] FIG. 3 shows the vector map of antisense RNA as 1 expressing plasmid.

[0025] FIG. 4 shows the vector map of antisense RNA as2 expressing plasmid.

[0026] FIG. 5 shows the vector map of antisense RNA as3 expressing plasmid.

[0027] FIG. 6 shows the vector map of antisense RNA as4 expressing plasmid.

[0028] FIG. 7 shows the vector map of shRNA expressing plasmid expressing shPBl-GC- 2 shRNA.

[0029] FIG. 8 shows the vector map of shRNA expressing plasmid expressing shPB 1-GC- 22 shRNA.

[0030] FIG. 9 shows the vector map of shRNA expressing plasmid expressing shPB 1-GC- 2 and shPBl-GC-22 shRNAs.

[0031] 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.

[0032] 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.

[0033] FIG. 12 shows the repression of PB1 gene expression by siRNAs.

[0034] FIG. 13 shows the repression of PB1 gene expression by siRNAs in a different experiment.

[0035] FIG. 14 shows the repression of PB2 gene expression by siRNAs.

[0036] 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).

[0037] 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.

[0038] FIG. 17 shows the repression of PB1 gene expression by asRNAs.

[0039] FIG. 18 shows the repression of PB 1 and PB2 gene expressions by siRNA combinations.

[0040] 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.

[0041] 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.

[0042] FIG. 21 shows the repression of PB1 gene expression by exosome-mediated transfer of shRNAs at 48 hours.

[0043] 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.

[0044] FIG. 24 shows the vector map of shRNA expressing plasmid expressing shPBl- GC-1 and shPBl-GC-8 shRNAs.

[0045] FIG. 25 shows the vector map of shRNA expressing plasmid expressing shPBl- GC-3 and shPBl-GC-5 shRNAs.

[0046] FIG. 26 shows the repression of PB1 gene expression by siRNAs in another independent experiment.

[0047] FIG. 27 shows the repression of PB2 gene expression by siRNAs in another independent experiment.

[0048] FIG. 28 shows the repression of PB 1 and PB2 gene expressions by siRNA combinations (20 nM) in a different experiment.

[0049] FIG. 29 shows the repression of PB1 and PB2 gene expressions by siRNA combinations (50 nM) in a different experiment.

[0050] FIG. 30 shows the repression of PB1 gene expressions by siRNA in stable reporter cell line.

[0051] FIG. 31 shows the repression of PB2 gene expressions by siRNA in stable reporter cell line.

[0052] FIG. 32 shows the repression of Flu M protein of H INI virus by siRNAs.

[0053] FIG. 33 shows the repression of Flu M protein of H3N2 virus by siRNAs.

[0054] FIG. 34 shows the repression of Flu M protein of H1N1 virus by siRNA combinations.

[0055] FIG. 35 shows the repression of Flu M protein of H3N2 virus by siRNA combinations.

[0056] 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.

[0057] DETAILED DESCRIPTIONS

[0058] In the following descriptions of the embodiments of the present disclosure, reference is made to the accompanying drawings, which are shown to illustrate the embodiments in which the present disclosure may be practiced. These embodiments are provided to enable those skilled in the art to practice the present disclosure. It is understood that other embodiments may be used and that changes can be made to the embodiments without departing from the scope of the present disclosure. The following descriptions are therefore not to be considered as limiting the scope of the present disclosure.

[0059] The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, immunohistochemistry, and immunology, which are well within the purview of a skilled artisan in the art. Such techniques are explained fully in the literature, such as “Molecular Cloning: A Laboratory Manual,” second edition (Sambrook, et al., 1989), Cold Spring Harbor Press; “Oligonucleotide Synthesis” (M. J. Gait, 1984); “Methods in Molecular Biology,” Humana Press; “Cell Biology: A Laboratory Notebook” (J. E. Cellis, Ed., 1998) Academic Press; “Animal Cell Culture” (R. I. Freshney, Ed., 1987); “Handbook of Experimental Immunology” (Weir, 1996); “Introduction to Cell and Tissue Culture” (J. P. Mather and P. E. Roberts, 1998); “Cell and Tissue Culture: Laboratory Procedures” (A. Doyle, J. B. Griffiths, and D. G. Newell, Eds., 1993-1998); “Methods in Enzymology” (Academic Press, Inc.); “Handbook of Experimental Immunology” (D. M. Weir and C. C. Blackwell, Eds.); “Gene Transfer Vectors for Mammalian Cells” (J. M. Miller and M. P. Calos, Eds., 1987); “Current Protocols in Molecular Biology” (F. M. Ausubel, et al., Eds., 1987); “PCR: The Polymerase Chain Reaction (Mullis, et al., Eds., 1994); “Current Protocols in Immunology” (J. E. Coligan et al., Eds., 1991); “Short Protocols in Molecular Biology” (Wiley and Sons, 1999); “Immunobiology” (C. A. Janeway and P. Travers, 1997); “Antibodies” (P. Finch, 1997); “Antibodies: A Practical Approach” (D. Catty, Ed., IRL Press, 1988-1989); “Monoclonal Antibodies: A Practical Approach” (P. Shepherd and C. Dean, Eds., Oxford University Press, 2000); “Using Antibodies: A Laboratory Manual” (E. Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, Eds., Harwood Academic Publishers, 1995). Particularly useful techniques for particular embodiments will be discussed in the sections that follow. Without further elaboration, it is believed that one skilled in the art can, based on the descriptions, utilize the present disclosure to its fullest extent. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referenced herein.

[0060] 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).

[0061] 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.

[0062] The pharmaceutical compositions of the present disclosure may be administered in the form that is composed of exosomes. As used herein, the term “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. The 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.

[0063] The exosomes of the present disclosure can be produced from a cell grown in vitro or a body fluid of a subject. When exosomes are produced from in vitro cell culture, various producer cells, e.g., HEK293 cells, Chinese hamster ovary (CHO) cells, or mesenchymal stem cells (MSCs), can be used.

[0064] The pharmaceutical 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.

[0065] If using the exosomes or a vector as a vehicle to deliver siRNA, the candidate siRNAs can be delivered as short hairpin RNAs (shRNAs). Both siRNAs and shRNAs can target and repress viruses and are functionally equivalent. When 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.

[0066] An shRNA may be provided in an expression cassette containing a promoter contiguously linked to an siRNA as described herein. In at least one embodiment, 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. In at least one embodiment, the expression cassette further contains a marker gene. In at least one embodiment, the promoter is a pol II promoter. In at least one embodiment, the promoter is a tissue-specific promoter. In at least one embodiment, the promoter is an inducible promoter. In at least one embodiment, the promoter is a pol III promoter. In at least one embodiment, the promoter is a U6 promoter or an Hl promoter.

[0067] Also provided herein is a vector containing an expression cassette described herein. Examples of appropriate vectors include adenovirus, lentivirus, adeno-associated virus (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based virus vectors.

[0068] 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. For example, 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. Various general considerations that may be considered when determining an appropriate dosage are described, for example, in Gennaro et al. (Eds), (1990), “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al. (Eds), (1990), “Goodman and Gilman’s: The Pharmacological Bases of Therapeutics,” Pergamon Press. 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, 0.01 to 0.1 mg / kg body weight per day, 0.01 to 0.05 mg / kg body weight per day, 0.01 to 0.02 mg / kg body weight per day, and 0.005 to 0.01 mg / kg body weight per day.

[0069] Those of ordinary skill in the art will be able, by routine experimentation, to determine an effective, non-toxic amount of the pharmaceutical compositions and / or inhibitory nucleic acids described herein to include in a dosage or in a series of dosages to achieve the desired therapeutic effect.

[0070] In therapeutic applications, the treatment would be for the duration of the disease state or condition. Further, it will be apparent to one of ordinary skill in the art that 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.

[0071] In many instances, it will be desirable to have several or multiple administrations of a pharmaceutical composition described herein. For example, they 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.

[0072] It will also be apparent to one of ordinary skill in the art that the optimal course of treatment can be ascertained using conventional course of treatment determination tests.

[0073] 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.

[0074] The pharmaceutical 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. In at least one embodiment, the pharmaceutical composition is adapted for intranasal administration. In at least one embodiment, the pharmaceutical composition of the present disclosure is formulated as a direct-acting nasal spray. In at least one embodiment, the nasal spray can be self-administered at point-of-care.

[0075] As used herein, the term “reference” refers to a standard used for comparison purpose(s). A person skilled in the art can select an appropriate reference for a particular comparison purpose(s). Thus, for example, 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. In some embodiments, as particular embodiments involving methods of identifying an agent that modulates the expression and / or activity of a target molecule, the reference is the activity and / or expression of the target molecule in the absence of the agent. In some embodiments, a reference is based on a predetermined level, e.g., based on functional expression or empirical assays. In some embodiments, 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). In some embodiments, 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).

[0076] In some embodiments, the RNA molecule is an analog or derivative thereof. In some embodiments, the polynucleotide, or the analog or derivative thereof, is an inhibitor of the target molecule.

[0077] 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. 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. In some embodiments, the modification increases nuclease resistance, increases serum stability, decrease immunogenicity, or a combination of the foregoing.

[0078] In some embodiments, the RNA molecule is included in a vector, e.g., expression vector or plasmid.

[0079] In some embodiments, the RNA molecule comprises an analog or a derivative of a polynucleotide. In some embodiments, the analog or derivative is a peptide nucleic acid (PNA). In some embodiments, the analog or derivative is a locked nucleic acid (LNA). In some embodiments, the analog or derivative is a morpholino oligonucleotide. In some embodiments, the analog or derivative comprises one or more phosphorothioate-linkages. In some embodiments, the RNA molecule is a ribonucleic guanidine (RNG) nucleotide.

[0080] In some embodiments, 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).

[0081] In some embodiments, the RNA molecule comprises a nucleotide sequence that is complementary (e.g., fully complementary or partially complementary) to at least a portion of a gene or gene transcript encoding a target molecule described herein, such that the RNA molecule is capable of hybridizing or annealing to the gene or gene transcript (e.g., under physiological conditions). In other embodiments, the RNA molecule comprises a nucleotide sequence that is complementary to at least a portion of a gene or gene transcript encoding a protein, which is capable of modulating the expression or activity of a target molecule described herein.

[0082] In at least one embodiment, the RNA molecule can be single stranded (ss) or double stranded (ds). In some embodiments, the polynucleotide is double stranded (ds). In some embodiments, 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. In some embodiments, 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.

[0083] In some embodiments, the RNA molecule is single stranded (ss). In some embodiments, 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.

[0084] In some embodiments, the RNA molecule (e.g., an antisense oligonucleotide) can hybridize to an mRNA encoding the target molecule (e.g., under physiological conditions). In some embodiments, 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. In some embodiments, 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%.

[0085] In some embodiments, 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). In some embodiments, the RNA molecule comprises a 3’ overhang, a 5’ overhang, or both. In some embodiments, the overhang is about 1 to 5 nucleotides. In some embodiments, the overhang comprises a modified ribonucleotide.

[0086] Non-limiting examples of 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).

[0087] In some embodiments, the RNA molecule inhibits gene expression (e.g., through the biological process of RNA interference (RNAi)). 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. 2004, 32(3):936- 48; Heale et al., Nucleic Acids Res. 2005, 33(3):e30; Chalk et al., Biochem. Biophys. Res. Commun. 2004, 319(l):264-74; and Amarzguioui et al., Biochem. Biophys. Res. Commun. 2004, 316(4): 1050-8.

[0088] In some embodiments, the RNA molecule is an siRNA. In some embodiments, the siRNA comprises a nucleotide sequence that is identical to about a 15 to 25 contiguous mRNA sequence encoding the target protein. In some embodiments, the siRNA is a double-stranded RNA molecule having about 19 to 25 base pairs. In some embodiments, the siRNA commences with the dinucleotide AA. In some embodiments, 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%.

[0089] In some embodiments, 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)). In some embodiments, 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)).

[0090] In some embodiments, the RNA molecule is chemically synthesized. In some embodiments, the RNA molecule is expressed recombinantly. In some embodiments, the RNA is transcribed in vitro. The making and using of 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).

[0091] In some embodiments, 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.

[0092] In some embodiments, 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.

[0093] The following examples are used for illustrating the present disclosure. A person skilled in the art can easily conceive the other advantages and effects of the present disclosure, based on the disclosure of the specification. The present disclosure can also be implemented or applied as described in different examples. It is possible to modify or alter the above examples for carrying out this disclosure without contravening its scope for different aspects and applications.

[0094] All terms including descriptive or technical terms which are used herein should be construed as having meanings that are obvious to one of ordinary skill in the art. However, the terms may have different meanings according to an intention of one of ordinary skill in the art, case precedents, or the appearance of new technologies. Also, some terms may be arbitrarily selected by the applicant, and in this case, the meaning of the selected terms will be described in detail in the descriptions of the present disclosure. Thus, the terms used herein have to be defined based on the meaning of the terms together with the descriptions throughout the specification.

[0095] It is further noted that, as used in this disclosure, the singular forms “a,” “an,” and “the” include plural referents unless expressly and unequivocally limited to one referent. The term “or” is used interchangeably with the term “and / or” unless the context clearly indicates otherwise.

[0096] Also, 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.

[0097] As used herein, 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. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently, “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements).

[0098] Numerical ranges recited herein by endpoints include all numbers and fractions subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” The term “about” means plus or minus 0.1% to 50%, 5% to 50%, 10% to 40%, 10% to 20%, or 10% to 15% of the number to which reference is being made.

[0099] As used herein, the term “sequence identity” or, for example, comprising a “sequence having 80% sequence identity with,” as used herein, refers to the extent that sequences are identical on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, 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. Included are 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.

[0100] The terms “subject,” “patient,” and “individual” are used interchangeably herein and refer to a warm-blooded animal such as a mammal that is afflicted with, or suspected of having, at risk for or being pre-disposed to, or being screened for a disease, including influenza infection. These terms include, but are not limited to, domestic animals, sports animals, primates, and humans. For example, the terms refer to a human.

[0101] As used herein, the terms “therapy” and “therapies” can refer to any protocol(s), method(s), composition(s), formulation(s), and / or agent(s) that can be used in prevention or treatment of a disease or symptom associated therewith. In at least one embodiment, the terms “therapy” and “therapies” refer to biological therapy, supportive therapy, and / or other therapies useful in prevention or treatment of a disease or symptom associated therewith known to one of ordinary skill in the art.

[0102] The terms “treatment,” “treat,” and “treating” refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease described herein. In some embodiments, treatment may be administered after one or more signs or symptoms of the disease have developed or have been observed. In other embodiments, treatment may be administered in the absence of signs or symptoms of the disease. For example, 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.

[0103] As used herein, the term “pharmaceutical composition” or “pharmaceutical combination” 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. and may be provided in such forms as liquids, powders, emulsions, lyophilized powders, sprays, creams, lotions, controlled release formulations, tablets, pills, gels, lozenges, packets, troches, elixirs, suspensions, solutions, syrups, soft and hard gelatin capsules, suppositories, sterilized injection fluid, packaged powder, on patches, in implants, etc.

[0104] As used herein, pharmaceutically acceptable carriers, including buffers, are well known in the art. and may comprise phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; amino acids; hydrophobic polymers; monosaccharides; disaccharides; and other carbohydrates; metal complexes; physiological saline; sterilized water; isotonic agents; and / or non-ionic surfactants. See, e.g., Remington: The Science and Practice of Pharmacy 20thEd. (2000) Lippincott Williams and Wilkins, Ed. K. E. Hoover.

[0105] EXAMPLES

[0106] Exemplary embodiments of the present disclosure are further described in the following examples, which should not be construed to limit the scope of the present disclosure. Plasmids

[0107] Plasmids were designed and prepared to be used in the following examples for different needs and purposes.

[0108] I. Reporter plasmid in reporter assay to assess siRNA repression of PB1 and PB2 expression

[0109] A reporter assay without employing the full-length PB1 or PB2 genes has been developed, as full PB 1 or PB2 gene expressions are considered hazardous reagents and are avoided whenever possible. To carry out the reporter assay, reporter plasmid constructs were designed to have the ultra-conserved sequence sites in both PB 1 and PB2 genes embedded and generated de novo at VectorBuilder Inc. (Chicago, IL, USA).

[0110] Briefly, 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'). A downstream herpes simplex virus thymidine kinase (HSV-TK) promoter expressing hFluc was used as an internally expressed transcript control to allow for cellular transfection efficiency standardization.

[0111] The PB2 reporter was generated by cloning PB2 ultra-conserved sites into EGFP which was generated to be transcribed in fusion with hRluc. A downstream HSV-TK promoter expressing hFluc was used as an internally expressed transcript control to allow for cellular transfection efficiency standardization.

[0112] Table 1 below lists the names and corresponding vector maps of the reporter plasmids used.

[0113] Table 1. Reporter plasmids to assess siRNA repression of PB1 and PB2 expression

[0114] II. Antisense RNA expressing plasmids

[0115] To generate the long antisense RNAs to be used in the examples for assessment of

[0116] PB1 and PB2 repression, the antisense RNA sequences were cloned into plasmids, as listed in Table 2 below.

[0117] Table 2. Antisense RNA expressing plasmids III. shRNA expressing plasmids

[0118] Plasmids were designed and generated to express small hairpin RNAs (shRNAs), which are the cellular equivalent to siRNAs, to be used in the transwell assay for assessing exosome-mediated shRNA repression of PB 1 or PB2 expression. Table 3 below lists the information regarding the shRNAs, including the shRNA names, corresponding siRNAs, plasmid names and number, and vector maps of the plasmids.

[0119] Table 3. shRNA expressing plasmids

[0120] Example 1 : siRNAs to repress influenza virus genes

[0121] To determine the ability of siRNAs to repress influenza virus genes PB1 or PB2, 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. siRNAs targeted to the PB1 gene of influenza viruses

[0122] Table 5. siRNAs targeted to the PB2 gene of influenza viruses

[0123]

[0124] These siRNAs were ordered and obtained from SynGenis (Bentley, WA, Australia). To examine their ability to repress PB 1 or PB2 genes, the reporter plasmids p2 or p3 as listed in Table 1 were transfected into HEK293 cells with 50 ng of each plasmid and Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocols. The siRNAs (20 nM) targeted to PB1 listed in Table 3 or the siRNAs (20 nM) targeted to PB2 listed in Table 4 were transfected along with an irrelevant miN367 RNA (sense: 5’-CUUUCCGCUGGGGACUUUCU-3’ (SEQ ID NO.: 39); antisense: 5’-AGAAAGUCCCCAGCGGAAAGAG-3’ (SEQ ID NO.: 40)) as control, respectively, after four hours.

[0125] After 72 hours, the cellular RNAs were collected from the transfected cells using RNAeasy kit (Qiagen, Venlo, Netherlands) with DNase treatment, and the resultant RNAs were converted to cDNA using Reverse Transcription kit (Thermo Fisher Scientific, Waltham, MA, USA) for assessment of gene expressions by conventional quantitative RT-PCR (qRT-PCR) protocols.

[0126] In this example, 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

[0127] 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. Similarly, as shown in FIG. 14, 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.

[0128] The efficiency of these siRNAs to repress PB 1 or PB2 genes were further examined in another set of experiments. This time, HEK293 cells were plated at 200,000 cells per well in a 12- well plate. On the following day, 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). After 15 minutes of incubation at room temperature, the plasmid DNA, siRNA, and Lipofectamine 3000 reagent in Opti- MEM formed siRNA-DNA-Lipid complexes. These 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.

[0129] 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).

[0130] 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).

[0131] Example 2: Antisense RNAs to repress influenza virus genes

[0132] To test the ability of long antisense RNAs (asRNAs) to repress PB1 and PB2, several long antisense RNAs were designed and cloned to be expressed from plasmids. Information of these plasmids was tabulated in Table 2 above, and FIGs. 3 to 6 show the vector maps of these plasmids.

[0133] To determine the repressive effect of the various long antisense RNAs to PB1, 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. Then, following the same procedures as in example 1 , repression of PB 1 was assessed by measuring expression of hRluc-GFP transgene after 48 to 72 hours with qRT-PCR analysis using primers GFP-F and GFP-R shown in Table 6.

[0134] As shown in FIG. 17, the asRNAs were able to repress PB1 reporter gene expression. Collectively, these data demonstrate that both siRNAs and asRNAs can repress PB 1 expression.

[0135] Example 3: Repression of influenza virus genes with multiple siRNAs

[0136] Multiple targeted anti-viral approaches are beneficial to repress viral infections by inhibiting the virus ability to evolve viral resistance and viral variant. 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.

[0137] The following combinations of siRNAs (packaged in lipid nanoparticles) or shRNAs (having the same sequences of siRNAs but packaged in exosomes) and noncoding antisense RNAs (packaged in exosomes) can be developed by generating and packaging the RNA molecules into lipid nanoparticles (LNPs) or exosomes and used to treat Flu infection by intravenous or intranasal administration of the RNA-containing nanoparticles.

[0138] Table 7. Combination of RNAs to treat influenza viruses

[0139]

[0140]

[0141]

[0142] To test the ability of 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.

[0143] Table 8. 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. 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.

[0144] These data demonstrate that the combinations of siRNAs may more potently repress PB1 and PB2 target gene expression than single siRNA treatments. Hence, 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.

[0145] Example 4: Exosome-mediated shRNA and asRNA repression of Flu gene expression

[0146] 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.

[0147] To determine if the PB 1 targeted shRNAs can be delivered to PB 1 expressing cells by exosomes and repress PB 1 gene expression, a transient transfection transwell assay was carried out. This assay used a transwell plate and HEK293 cells. First, 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.

[0148] Briefly, on day 0, HEK293 cells (0.5 x 106 / 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. After 24 hours, the producer cells were transfected (a total of 300 ng / 0.5 x 106cells) 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. 19) and p54 (pDB68- Connexin 43 S368A, vector map shown in FIG. 20), which are plasmids expressing argonaute 2 (Ago-2) and connexin 43 (Cnx43), respectively. 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|ExpJ-Puro- CMV+intron>{Euc-PB2-Ffluc}) was also transfected. These transfected HEK293 cells were then grown in a transwell plate.

[0149] In this set-up, 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). Simultaneously, 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. After 24 hours following transfection, media were changed to 500 pF of 10% exosome-depleted FBS+ DMEM. After 8 to 24 hours, the producer cells were added to the trans well containing the recipient cells. The transwell containing recipient cells was placed on the top of the producer cells, and media were changed to 100 p L of 10% exo-depleted FBS+ DMEM carefully. Contact between media from basolateral chamber and apical chamber cells was ensured at all times. The details of the preparation and transfection were disclosed in U.S. Serial No. 63 / 598,499, filed November 14, 2023, the contents of which are hereby incorporated by reference in its entirety.

[0150] After 2 to 3 days, depending on the confluency, 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.

[0151] Table 9. Plasmids transfected into producer cells for assessing shRNAs targeted to PB1

[0152] 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. These are the cells of interest for transfer of shEV function and repression of the PB 1 or PB2 reporter gene expression. At 72 hours after transfection, only the recipient cells were collected and assessed by qRT-PCR for Fluc / Rluc mRNA expression using primer sets listed in Table 6.

[0153] The same assays were also carried out to determine if multiple combinations of shRNA expressing plasmids targeted to both PB1 and PB2 could be combined and functionally repress PB1 and PB2 in recipient cells through transferred exosomes.

[0154] As a result, it is shown that 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. At 72 hours post-treatment, the shRNA combination transferred in exosomes was shown to further repress the PB1 expression to 55% of the control, as shown in FIG. 22. Also, 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. These data demonstrate that exosomes are viable means to deliver the influenza gene-targeted shRNAs to target cells.

[0155] Next, RNA combinations including both multiple shRNAs and long antisense RNAs were assessed for their suppressive effects on PB1 and PB2 expression. Briefly, 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.

[0156] As a result, it is shown that the combinations of siRNAs with long antisense RNA as2 rendered potent repression of PB 1 and PB2 expressions, as depicted in FIG. 23, repressing the expression of influenza genes to 34% and 23% of the control. These data demonstrate that multi-targeted RNAs can potently repress influenza PB 1 and PB2 gene expression and that combinations of RNA therapeutics are viable means to repress viral gene expression. In addition, shRNAs delivered to target cells by exosomes can functionally repress PB1 and PB2 gene expressions.

[0157] Example 5 : Repression of Flu gene expressions in stable reporter cell lines

[0158] In this example, a consistent and reproducible reporter system was established to examine repression of Flu gene expression. In brief, stable PB1 and PB2 reporter cell lines were generated with HEK293 cells that constantly express the FBI and PB2 reporter genes. Briefly, 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.

[0159] To examine repression of Flu gene expressions using the stable reporter cell lines, the stable HEK293 PB1 or PB2 reporter cells were plated at 50,000 cells per well in a 24-well plate. On the following day, siRNAs (50 or 100 nM / well) and Lipofectamine RNAiMax (13778150; Invitrogen) were gently mixed in serum-free Opti-MEM. After 15 minutes of incubation at room temperature, 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.

[0160] 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).

[0161] 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).

[0162] It was found that these 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.

[0163] Example 6: Inhibition of H1N1 and H3N2 replication by siRNAs

[0164] In this example, 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.

[0165] Briefly, MDCK cells were plated at 0.5 to 2 x 105cells 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. After 48 hours of transfection, the cells were infected with H1N1 (A / swine / Changhua / 415 -7 / 2009) or H3N2 (A / swine / Taiwan ex USA / 28-9 / 2010) strains at a multiplicity of infection (MOI) of 0.1, and cultured in a virus growth medium (VGM), which consists of DMEM supplemented with 25 mM 4-(2 -hydroxyethyl)- 1 -piperazineethanesulfonic acid (HEPES), 0.2% bovine serum albumin (BSA), 1 to 2 pg / mL tosyl phenylalanyl chloromethyl ketone (TPCK) -treated trypsin, and a penicillin / streptomycin solution. At 24 hours post-infection (p.i.), the cells were collected, and RNA was extracted for qRT- PCR to measure Flu M protein expression.

[0166] As shown in FIGs. 32 and 33, 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.

[0167] To examine viral replication inhibiting ability of siRNA combinations, siRNA combinations of siPB2-GC-3 and siPB2-GC-5 as well as siPBl-GC-2 and siPBl-GC- 22 were tested for their efficiency to inhibit H1N1 and H3N2 viral infections. Similarly, MDCK cells were transfected with the siRNA combinations and later infected with H1N1 or H3N2 at an MOI of 0.1. After 24 hours of infection, mRNA levels were assessed for strain-specific Flu M protein expression.

[0168] As shown in FIGs. 34 and 35, 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%.

[0169] It will be understood that the above descriptions of embodiments are given by way of examples only and that various modifications may be made by those having ordinary skill in the art. Although various embodiments of the disclosure have been described above with a certain degree of particularity, or with reference to one or more individual embodiments, those having ordinary skill in the art could make numerous alterations to the disclosed embodiments without departing from the scope of this disclosure.

Claims

CLAIMSWhat is claimed is:

1. A pharmaceutical composition comprising a plurality of RNA molecules and a pharmaceutically acceptable carrier thereof, wherein the plurality of RNA molecules comprise at least two RNA molecules targeting to an influenza gene including PB 1 , PB2, or a combination thereof.

2. The pharmaceutical composition of claim 1 , wherein the plurality of RNA molecules are selected from the group consisting of RNA molecules having at least 80% sequence identity to SEQ ID NOs: 1 to 38.

3. The pharmaceutical composition of claim 1, wherein the RNA molecule is a double stranded RNA.

4. The pharmaceutical composition of claim 3, wherein the RNA molecule is a small interference RNA (siRNA), a short hairpin RNA (shRNA), or a combination thereof.

5. The pharmaceutical composition of claim 3, wherein the RNA molecule is about 10 to 50 nucleotides long.

6. The pharmaceutical composition of claim 1 , wherein the RNA molecule is a single stranded RNA.

7. The pharmaceutical composition of claim 6, wherein the RNA molecule is an antisense RNA (asRNA).

8. The pharmaceutical composition of claim 7, wherein the RNA molecule is about 50to 400 nucleotides long.

9. The pharmaceutical composition of claim 1 , wherein the plurality of RNA molecules are a combination of siRNAs, a combination of shRNAs, or a combination of siRNAs and shRNAs.

10. The pharmaceutical composition of claim 9, wherein the plurality of RNA molecules are selected from the group consisting of siPBl-GC-1, siPBl-GC-2, siPBl- GC-3, siPBl-GC-4, siPBl-GC-5, siPBl-GC-8, siPBl-GC-16, siPBl-GC-17, siPBl- GC-21, siPB l-GC-22, siPB2-GC-l, siPB2-GC-2, siPB2-GC-3, siPB2-GC-4, siPB2- GC-5, siPB2-GC-7, and siPB2-GC-9.

11. The pharmaceutical composition of claim 9, wherein at least one of the combinations further comprises at least one asRNA.

12. The pharmaceutical composition of claim 11, wherein the at least one asRNA is selected from asl, as2, as3, and as4.

13. The pharmaceutical composition of claim 11, wherein the plurality of RNA molecules is a combination selected from the group consisting of GC-Flul to GC- Flul23.

14. The pharmaceutical composition of claim 1, wherein the pharmaceutically acceptable carrier is an extracellular vesicle or a lipid nanoparticle.

15. The pharmaceutical composition of claim 14, wherein the pharmaceuticallyacceptable carrier is an extracellular vesicle.

16. The pharmaceutical composition of claim 15, wherein the extracellular vesicle is an exosome.

17. A method for repressing an expression of an influenza gene in a cell, comprising contacting the cell with the pharmaceutical composition of any one of claims 1 to 16.

18. The method of claim 17, wherein the influenza gene is at least one of PB1 and PB2.

19. A method of preventing or treating influenza in a subject in need thereof, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 16.

20. The method of claim 19, wherein the subject is human, swine, or avian.

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