Polynucleotide targeting RNA polymerase primary σ 70 and method of use and treatment thereof

Abstract

The present invention provides a pharmaceutical composition comprising one or more RNA polymerase primary σ70 (encoded by gene rpoD) targeting polynucleotides capable of targeting rpoD. In an embodiment, the one or more rpoD targeting polynucleotides is encapsulated in a nanoparticle such as exosomes or LNP. The present invention also provides a method of treatment of rpoD-associated disease of a subject comprising administration of a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject.

Description

[0001] POLYNUCLEOTIDE TARGETING RNA POLYMERASE PRIMARY <j7° AND METHOD OF USE AND TREATMENT THEREOF

[0002] INCORPORATION BY REFERENCE OF A SEQUENCE LISTING XML

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “rpoD Targeting Polynucelotide ” created on January 23, 2025 and having a size of 80 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0004] FIELD OF THE INVENTION

[0005] The present invention provides a composition comprising one or more polynucleotide targeting RNA polymerase primary o70(encoded by gene rpoD) and method of use and treatment thereof.

[0006] BACKGROUND OF THE INVENTION

[0007] Methicillin-resistant Staphylococcus aureus (MRSA) causes life threatening infection- related mortality worldwide with a particular ability to affect aged and hospital populations. The emergence of multi-drug resistant MRSA is an unmet medical need that requires new anti-MRSA therapeutics. RNA polymerase primary o70(encoded by gene rpoD) is a highly conserved prokaryotic factor involved in RNA polymerase (RNAP) activities essential for transcription initiation in exponentially growing cells of diverse S. aureus, implying a conserved functional target for effective siRNA inhibition. Notably, peptide nucleic acids have been found to potently repress rpoD expression (PMID: 22253815), suggesting this gene is good lead druggable target. Small interfering RNAs have been observed to be functional against MRSA in human cells and in vivo using mouse models (PMID:16344286). RNA interference technology such as siRNAs and shRNAs have the advantage to being easily formulated and are amendable to nanoparticle means of delivery. Therefore, there is a need for a composition and method to target RNAP using RNA interference technology to repress MRSA infection.

[0008] SUMMARY OF THE INVENTION

[0009] A pharmaceutical composition comprising one or more RNA polymerase primary o70(encoded by gene rpoD) targeting polynucleotides capable of targeting rpoD, wherein the nucleotide sequence of each of the one or more rpoD targeting polynucleotides is at leastabout 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or a combination thereof.

[0010] A method of treatment of a rpoD-associated disease of a subject comprising administration of a therapeutically effective amount of the pharmaceutical composition comprising one or more polynucleotides targeting rpoD of the present invention to the subject.

[0011] BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 illustrates MRS A siRNA screen targeted to Staph Aureus RNA polymerase 2. Sil -5 were screened in combinations of MRSA targeted siRNAs (40nM total siRNA / well) with the target reporter plasmid (lOOng). After 72hrs later collect the cell RNAs and assess luciferase (Rluc vs Flue and beta actin by qRTPCR. All value represent fraction of siControl (+SEM, n=3). ** p value< 0.01, **** p< 0.0001, ****** p< 0.000001.

[0013] Figure 2 illustrates the effect of combination siRNA treatment on MRSA RNAP1I targeting. Triplicate treated siRNA and reporter plasmid transfected cells HEK cells were assessed for changes in Rluc (MRSA RNAPII reporter) relative to internal control FLuc. The averages are shown with standard errors of the mean. P-values from a paired T-test are shown (** p< 0.01, **** p< 0.0001, and ****** p< 0.000001).

[0014] Figure 3 illustrates the reporter plasmid, pRP[Exp]-Puro- CMV+intron>{hRluc-RNAPII_StaphTarget] VB23091 l-1685xts used in assessing siRNA screen. siRNAs 1 -5 were screened when co-transfected with this reporter plasmid. The relative ratios of siRNAs to plasmid are shown (Table 3)

[0015] Figure 4 illustrates the effect of single or combination siRNA treatment on MRSA RNAPII targeting. Producer cells transfected with plasmid combinations were cocultured in a transwell assay with stable reporter recipient cells. After 72hrs the recipient cells are collected and assessed by qRTPCR for Rluc / Fluc mRNA expression. All value represents fraction of reporter cell alone (±SEM, n=3) and p values from a paired T-test are also shown.

[0016] Figure 5 illustrates the effect of single or combination siRNA treatment on MRSA RNAPII targeting. Producer cells transfected with plasmid combinations were cocultured in a transwell assay with stable reporter recipient cells. After 72hrs the recipient cells are collected and assessed by qRTPCR for Rluc / Fluc mRNA expression. All value represents fraction of reporter cell alone (+SEM, n=3).

[0017] Figure 6 illustrates the target sites identification in multiple MRSA strains. In total, 16 MRSA strains (SI -SI 6), including 7 strains (S1-S4, S9-S10) that show additional resistance to clindamycin (CC), ciprofloxacin (CIP), erythromycin (E), flusidic acid (FA), gentamycin (GM), levofloxacin (LVX), sulfamethoxazole-trimethoprim (SXT), tetracycline (TE), or a combination thereof were analyzed. OX, oxacillin (methicillin); VA, vancomycin; LZD, linezolid; R, resistant; S, sensitve.

[0018] DETAILED DESCRIPTION OF THE INVENTION

[0019] As used in this specification and in claims which follow, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes mixtures of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and the like.

[0020] As used herein, the term “about” as a modifier to a quantity is intended to mean + or - 5%, + or - 10%, + or - 15%, or + or -20%, inclusive of the quantity being modified.

[0021] As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms "polynucleotide," "oligonucleotide," "oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term "nucleotide" refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of nucleic acids contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acids contemplated herein include any types of RNA (e.g., antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, dicer substrate RNA, dicer substrate siRNAs (dsiRNAs) (dsiRNA are cleaved by the RNase I class endoribonuclease dicer into 21-23 base duplexes having 2-base 3'-overhangs siRNA), and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "duplex" in the context of nucleic acids refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides or the nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched toform higher ordered structures such as dendrimers and the like. In an embodiment, the nucleotide sequence is provided using symbols ATCG (adenine (A), cytosine (C), guanine (G), and thymine (T)) for a DNA molecule, and provided with codes using symbols AUCG (adenine (A), cytosine (C), guanine (G), and uracil (U)) for a RNA molecule. In an embodiment, the symbols T and U are used interchangeably in a nucleotide sequence to illustrate the DNA and RNA molecule respectively made according to the nucleotide sequence.

[0022] As used herein, the terms "polypeptide," "peptide" and "protein" generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally occurring amino acids or are unnatural amino acids. The term "protein", as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and / or quaternary structure. In an embodiment, the "polypeptide," "peptide" or "protein" of the present invention is prepared from a plasmid encoding said "polypeptide," "peptide" or "protein". Therefore, the "polypeptide," "peptide" or "protein" of the present invention further comprises a nucleotide sequence encoding said "polypeptide," "peptide" or "protein" that could be converted using a genetic code such as but not limited to the standard genetic code.

[0023] As used herein, “sequence identity” and “% identity,” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions as compared to the reference sequence for optimal alignment of the two sequences. The number of positions at which identical amino acid residues occur in both sequences is determined, yielding the number of matched positions, which is divided by the total number of positions in the window of comparison and the result multiplied by 100 to yield the percentage of sequence identity. The comparison window is the entire length of the sequence being referred to unless indicated otherwise.

[0024] As used herein, “% similarity” is calculated as described for “% identity,” with the exception that the hydrophobic residues Ala, Vai, Phe, Pro, Leu, He, Trp, Met, and Cys are similar; the basic residues Lys, Arg, and His are similar; the acidic residues Glu and Asp are similar; and the hydrophilic, uncharged residues Gin, Asn, Ser, Thr, and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

[0025] As used herein, the term “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0026] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug, compound, pharmacologically active agent or a pharmaceutical composition comprises administering an amount thereof necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular active agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a drug, compound, pharmacologically active agent or a pharmaceutical composition is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In certain embodiments, the drug, compound, pharmacologically active agent or a pharmaceutical composition inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, at least about 90% or at least about 100%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a drug, compound, pharmacologically active agent or a pharmaceutical composition sufficient to cause reversal of disease. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any numbers and number ranges falling within these values.

[0027] As used herein, the term “target” or “targeting” a polynucleotide comprises directly or indirectly regulating the expression level, biological function, or a combination thereof, of said polynucleotide. The indirect regulation of a polynucleotide comprises indirectly regulating the expression level, biological function, or a combination thereof, of a polynucleotide by regulation of one or more antisense RNA that regulates the polynucleotide being targeted. In an embodiment, in the case of direct regulation, down regulating a polynucleotide may comprise destruction or breakup of the polynucleotide being targeted or regulated. In an embodiment, in the case of indirect regulation, down regulating a polynucleotide may comprise destruction or breakup of one or more antisense RNA that regulates the polynucleotide being targeted. In an embodiment, such destruction or breakupis done using a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof targeting or regulating the polynucleotide.

[0028] The present invention provides one or more polynucleotide targeting rpoD and a pharmaceutical composition thereof. In an embodiment, the polynucleotide of the present invention is about 10 to about 1500 nucleotides (nt) in length such as about 10, about 20, about 30, about 40 about 50 about 60 about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 nt in length including any lengths or length ranges falling within these values. In an embodiment, the rpoD targeting polynucleotide of the present invention is about 15 to about 55 nt in length, about 20 to about 30 nt in length or about 21 to about 23 nt in length. In an embodiment, the rpoD targeting polynucleotide of the present invention is double stranded or single stranded. In an embodiment, the rpoD targeting polynucleotide of the present invention is blunt ended or comprise overhanging ends. In an embodiment, the rpoD targeting polynucleotide of the present invention is chemically synthesized or recombinantly produced. In an embodiment, one strand of the rpoD targeting polynucleotide of the present invention comprises nucleotide sequence having sufficient complementarity to rpoD for the polynucleotide to direct cleavage of the rpoD via RNA interference. In an embodiment, the one or more rpoD targeting polynucleotide of the present invention comprise a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof.

[0029] In an embodiment, the one or more rpoD targeting polynucleotide of the present invention comprise siRNA. In an embodiment, the rpoD targeting siRNAs of the present invention comprise nucleotide sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5. In an embodiment, the rpoD targeting siRNAs of the present invention comprise a 3’ deoxy thymidine dinucleotide (dTdT) overhang to increase nuclease resistance. In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a first rpoD targeting siRNA and a second rpoD targeting siRNA wherein the nucleotide sequence of the first rpoD targeting siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 1 and the nucleotide sequence of the second rpoD targeting siRNA is at least about 80%, about85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5.

[0030] In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a first rpoD targeting siRNA, a second rpoD targeting siRNA, and a third rpoD targeting siRNA wherein the nucleotide sequence of the first rpoD targeting siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. I, the nucleotide sequence of the second rpoD targeting siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5, and the nucleotide sequence of the third rpoD targeting siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5 wherein the nucleotide sequence of the third rpoD targeting siRNA is distinct from the nucleotide sequence of the second rpoD targeting siRNA or wherein the nucleotide sequence of the third rpoD targeting siRNA is distinct from the nucleotide sequence of the second rpoD targeting siRNA by more than 60%, 70% or 80%.1000311 In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a shRNA. In an embodiment, each of the one or more rpoD targeting shRNA comprises a first nucleotide sequence and a second nucleotide sequence wherein the second nucleotide sequence is reverse complementary to the first nucleotide sequence. In an embodiment, the nucleotide sequence of the first nucleotide sequence of each of the one or more shRNAs of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5. In an embodiment, the shRNA of the present invention further comprises a loop connecting the first nucleotide sequence and the second nucleotide sequence. In an embodiment, the loop of the shRNA of the present invention comprises nucleotide sequence of about 2-9 bp such as about 2, 3, 4, 5, 6, 7, 8, or 9 bp. In an embodiment, the nucleotide sequence of the loop of the rpoD targeting shRNA of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, or SEQ ID No.10. In an embodiment, the loop may be cleaved off in RNA interference pathway. In an embodiment, the nucleotide sequence of the rpoD targeting shRNA of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO.16, SEQ ID NO. 17, SEQ ID NO. 18, or SEQ ID NO. 19.

[0032] In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a first rpoD targeting shRNA and a second rpoD targeting shRNA wherein the nucleotide sequence of the first rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 11 and the nucleotide sequence of the second rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15.

[0033] In an embodiment, the one or more rpoD targeting polynucleotides comprise a first rpoD targeting shRNA, a second rpoD targeting shRNA, and a third rpoD targeting shRNA wherein the nucleotide sequence of the first rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 11, the nucleotide sequence of the second rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15, and the nucleotide sequence of the third rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, or SEQ ID NO. 15 wherein the nucleotide sequence of the third rpoD targeting shRNA is distinct from the nucleotide sequence of the second rpoD targeting shRNA or wherein the nucleotide sequence of the third rpoD targeting shRNA is distinct from the nucleotide sequence of the second rpoD targeting shRNA by more than 60%, 70% or 80%.

[0034] In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a first rpoD targeting shRNA and a second rpoD targeting shRNA wherein the nucleotide sequence of the first rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 16 and the nucleotide sequence of the second rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 17, SEQ ID NO. 18, or SEQ ID NO. 19.

[0035] In an embodiment, the one or more rpoD targeting polynucleotides of the present invention comprise a first rpoD targeting shRNA, a second rpoD targeting shRNA, and a third rpoD targeting shRNA wherein the nucleotide sequence of the first rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 16, the nucleotide sequence of the second rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100%identical to the nucleotide sequence of SEQ ID NO. 17, SEQ ID NO. 18, or SEQ ID NO. 19, and the nucleotide sequence of the third rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 17, SEQ ID NO. 18 or SEQ ID NO. 19 wherein the nucleotide sequence of the third shRNA is distinct from the nucleotide sequence of the second shRNA or wherein the nucleotide sequence of the third shRNA is distinct from the nucleotide sequence of the second shRNA by more than 60%, 70% or 80%.

[0036] In an embodiment, the one or more polynucleotides comprise a first rpoD targeting shRNA and a second rpoD targeting shRNA wherein the nucleotide sequence of the first rpoD targeting shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 11 and the nucleotide sequence of the second rpoD targeting hRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 15.

[0037] In an embodiment, the one or more rpoD targeting polynucleotide of the present invention comprises antisense RNA (asRNA). In an embodiment, the rpoD targeting asRNAs of the present invention comprise nucleotide sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 20, SEQ ID NO. 21, or SEQ ID NO. 22.

[0038] SEQ ID NO. 1 (siStaphRNAP2-sil): CAAUUGGUGAAGAAGAUGA

[0039] SEQ ID NO. 2 (siStaphRNAP2-si2): GAUACUGACGAGAAACUGA

[0040] SEQ ID NO. 3 (siStaphRNAP2-si3: GUGCACAAGAAGAAAUCGA

[0041] SEQ ID NO. 4 (siStaphRNAP2-si4): UUAAUUGAAAAAGGUAAAA

[0042] SEQ ID NO. 5 (siStaphRNAP2-si5): GUCAUGAAGAAAUUGCUGA

[0043] SEQ ID NO. 6: UUGC

[0044] SEQ ID NO. 7: CCUGACCCA

[0045] SEQ ID NO. 8: AAGCACA

[0046] SEQ ID NO. 9: UGUGCUU

[0047] SEQ ID NO. 10: UUG

[0048] SEQ ID NO. 11 (shStaphRNAP2-shl):CAAUUGGUGAAGAAGAUGAuugcUCAUCUUCUUCACCAAUUG

[0049] SEQ ID NO. 12 (shStaphRNAP2-sh2):GAUACUGACGAGAAACUGAuugcUCAGUUUCUCGUCAGUAUC

[0050] SEQ ID NO. 13 (shStaphRNAP2-sh3): GUGCACAAGAAGAAAUCGAuugcUCGAUUUCUUCUUGUGCAC

[0051] SEQ ID NO. 14 (shStaphRNAP2-sh4):UUAAUUGAAAAAGGUAAAAuugcUUUUACCUUUUUCAAUUAA

[0052] SEQ ID NO. 15 (shStaphRNAP2-sh5):GUCAUGAAGAAAUUGCUGAuugcUCAGCAAUUUCUUCAUGAC

[0053] SEQ ID NO. 16 (staphPol2-sil):CAAUUGGUGAAGAAGAUGAugugcuuUCAUCUUCUUCACCAAUUG

[0054] SEQ ID NO. 17 (staphPol2-si2):GAUACUGACGAGAAACUGAugugcuuUCAGUUUCUCGUCAGUAUC

[0055] SEQ ID NO. 18 (staphPol2-si3):GUGCACAAGAAGAAAUCGAaagcacaUCGAUUUCUUCUUGUGCAC

[0056] SEQ ID NO. 19 (staphPol2-si5):GUCAUGAAGAAAUUGCUGAaagcacaUCAGCAAUUUCUUCAUGAC

[0057] SEQ ID NO. 20 (asStaphRNAP2-asl):

[0058] AAATAAGCGCTTATTCATGTCTTGGTATCAATCATTAAAATTGATATAAATTAATCCATAAAGTCTTTCAAACGTTTACTACGACTTGGATGTCTTAATTTTCTAAGTGCTTTTGCTTCAATTTGTCGAATACGTTCACGTGTAACACCGAAAACTTTACCAACTTCTTCAAGTGTTCTTGTTCTGCCGTCATCAAGACCAAATCTTAATCGTAATACATTTTCTTCTCTATCAGTTAATGTATCAAGCACATCTTCTAATTGCTCTTTTAATAATTCATAAGCAGCATGATCTGAAGGACTTTGTGCTTCCTGATCCTCAATAAAGTCTCCTAAATGACTATCATCTTCTTCACCAATTGGTGTTTCTAATGAAACAGGTTCTTGCGCAATTTTTAAAACTTCACGAACTTTTTCTGCTGGTAAATCCATTTCTTCACCAATTTCTTCTGGTGCTGGATCTCGACCTAAGTCCTGTAATAATTGACGTTGAACACGAATTAATTTATTAATTGTTTCTACCATATGCACAGGGATACGAATCGTACGTGCTTGGTCAGCAATTGCACGAGTGATTGCTTGTCTAATCCACCATGTTGCATATGTTGAAAACTTAAATCCTTTGTTAAAGTCAAATTTTTCAACAGCTTTAATAAGACCCATATTACCTTCTTGGATTAAATCAAGGAATAACATACCACGACCTACGTATCTTTTAGCAATACTTACAACTAAACGTAAGTTCGCTTCTGCAAGTCTTGATTTTGCTACTTCATCACCTTGTTCAATACGTTTGGCTAATTCGATTTCTTCTTGTGCACTTAATAAGTTAACACGCCCAATTTCTTTAAGGTACATACGAACTGGGTCATTTATTTTAACACCTGGAGGGGCACTAAGATCACTTGGATTCAGTTTCTCGTCAGTATCTGAACTATCTTTTTCATTAACTAGTGAAATATCATTATCATTTAATTGATCAAAGAAATCATCCATTTGATCAGAGTCGATATCAAAATTCTGAAGTTTTTCAGCAATTTCTTCATGACTTAAATGACCCTCTTTTTTACCTTTTTCAATTAATTGCTTCTTAACATCTTCTAATGTTAATGTCGGATCAATTGTTTGTTTTTTAATTTTAACTGTGTTATCAGACAT

[0059] SEQ ID NO. 21 (asStaphRNAP2-as2):

[0060] CTGGGTCATTTATTTTAACACCTGGAGGGGCACTAAGATCACTTGGATTCAGTTTCTCGTCAGTATCTGAACTATCTTTTTCATTAACTAGTGAAATATCAT TATCATTTAATTGATCAAAGAAATCATCCATTTGATCAGAGTCGATATCAAAATT CTGAAGTTTTTCAGCAATTTCTTCATGACTTAAATGACCCTCTTTTTTACCTTTTTC AATTAATTGCTTCTTAACATCTTCTAATGTTAATGTCGGATCAATTGTTTGTTTTTT AATT

[0061] SEQ ID NO. 22 (asStaphRNAP2-as3):

[0062] TTAATAATTCATAAGCAGCATGATCTGAAGGACTTTGTGCTTCCTGATCCTCAATAAAGTCTCCTAAATGACTATCATCTTCTTCACCAATTGGTGTTTCTA ATGAAACAGGTTCTTGCGCAATTTTTAAAACTTCACGAACTTTTTCTGCTGGTAA ATCCATTTCTTCACCAATTTCTTCTGGTGCTGGATCTCGACCTAAGTCCTGTAATA ATTGACGTTGAACACGAATTAATTTATTAATTGTTTCTACCATATGCACAGGGAT ACGAATCGTACGTGCTTGGTCAGCAATTGCACGAGTGATTGCTTGTCTAATCCAC CATGTTGCATATGTTGAAAACTTAAATCCTTTGTTAAAGTCAAATTTTTCAACAGC TTTAATAAGACCCATATTACCTTCTTGGATTAAATCAAGGAATAACATACCACGA CCTACGTATCTTTTAGCAATACTTACAACTAAACGTAAGTTCGCTTCTGCAAGTCT TGATTTTGCTACTTCATCACCTTGTTCAATACGTTTGGCTAATTCGATTTCTTCTTG TGCACTTAATAAGTTAACACGCCCAATTTCTTTAAGGTACATAC

[0063] In an embodiment, the rpoD comprises Staphylococcus aureus (.S'. aureus) rpoD or a variant thereof. In an embodiment, the S. aureus is resistant to at least an antibiotic such as but not limited to methicillin, clindamycin, ciprofloxacin, erythromycin, flusidic acid, gentamycin, levofloxacin, sulfamethoxazole-trimethoprim, tetracycline, vancomycin, linezolid, or a combination thereof. In an embodiment, the 5. aureus comprises methicillin- resistant S. aureus. In another embodiment, the rpoD comprises a rpoD exon, a rpoD intron, a rpoD regulator element, or a combination thereof. In an embodiment, the regulatory element comprises a promoter, a transcriptional enhance, a transcriptional repressor, or a combination thereof. In an embodiment, the nucleotide sequence of the portion of rpoD targeted by the rpoD targeting polynucleotide of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 23.

[0064] SEQ ID NO. 23 (rpoD):

[0065] ATGTCTGATAACACAGTTAAAATTAAAAAACAAACAATTGATCCGACATTAACATTAGAAGATGTTAAGAAGCAATTAATTGAAAAAGGTAAAAAAGAGG GTCATTTAAGTCATGAAGAAATTGCTGAAAAACTTCAGAATTTTGATATCGACTCTGATCAAATGGATGATTTCTTTGATCAATTAAATGATAATGATATTTCACTAGTTA ATGAAAAAGATAGTTCAGATACTGACGAGAAACTGAATCCAAGTGATCTTAGTG CCCCTCCAGGTGTTAAAATAAATGACCCAGTTCGTATGTACCTTAAAGAAATTGG GCGTGTTAACTTATTAAGTGCACAAGAAGAAATCGAATTAGCCAAACGTATTGA ACAAGGTGATGAAGTAGCAAAATCAAGACTTGCAGAAGCGAACTTACGTTTAGT TGTAAGTATTGCTAAAAGATACGTAGGTCGTGGTATGTTATTCCTTGATTTAATCC AAGAAGGTAATATGGGTCTTATTAAAGCTGTTGAAAAATTTGACTTTAACAAAGG ATTTAAGTTTTCAACATATGCAACATGGTGGATTAGACAAGCAATCACTCGTGCA ATTGCTGACCAAGCACGTACGATTCGTATCCCTGTGCATATGGTAGAAACAATTA ATAAATTAATTCGTGTTCAACGTCAATTATTACAGGACTTAGGTCGAGATCCAGC ACCAGAAGAAATTGGTGAAGAAATGGATTTACCAGCAGAAAAAGTTCGTGAAGT TTTAAAAATTGCGCAAGAACCTGTTTCATTAGAAACACCAATTGGTGAAGAAGAT GATAGTCATTTAGGAGACTTTATTGAGGATCAGGAAGCACAAAGTCCTTCAGATC ATGCTGCTTATGAATTATTAAAAGAGCAATTAGAAGATGTGCTTGATACATTAAC TGATAGAGAAGAAAATGTATTACGATTAAGATTTGGTCTTGATGACGGCAGAAC AAGAACACTTGAAGAAGTTGGTAAAGTTTTCGGTGTTACACGTGAACGTATTCGA CAAATTGAAGCAAAAGCACTTAGAAAATTAAGACATCCAAGTCGTAGTAAACGT TTGAAAGACTTTATGGATTAATTTATATCAATTTTAATGATTGATACCAAGACAT GAATAAGCGCTTATTT

[0066] In an embodiment, the rpoD comprises rpoD of Str. JKD6008, TW20, ED98, JH1, NCTC 8325, USA300_TCH1516, USA300_FPR3757, ST398, MRSA252, MSSA476, JKD6159, ED133, Staphylococcus epidermidis RP62A, ATCC 12228, Staphylococcus lugdunensis HKU09-01, N920143, Staphylococcus haemolyticus JCSC1435, Staphylococcus carnosus subsp. Carnosus TM300, Staphylococcus pseudintermedius ED99, HKU10-03 or Staphylococcus saprophyticus subsp. Saprophyticus ATCC 15305.

[0067] In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising N-acetylgalactosamine (GalNAc) wherein the one or more polynucleotide targeting rpoD of the present invention is conjugated to the GalNAc. In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising GalNAc wherein the one or more siRNA of the present invention is conjugated to the GalNAc. In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising GalNAc wherein the one or more shRNA of the present invention is conjugated to the GalNAc.

[0068] In an embodiment, any embodiment of the rpoD targeting polynucleotide of the present invention downregulates the expression of the rpoD gene in any bacteria capable of infecting a subject such as S. aureus by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%.

[0069] The present invention also provides a pharmaceutical composition comprising a nanoparticle encapsulating any embodiment of the one or more rpoD targeting polynucleotide of the present invention. In an embodiment, the nanoparticle of the present invention comprises chemical nanoparticles such as but not limited to lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle and biological nanoparticles such as but not limited to liposome, exosome, virus, virus-like particle.

[0070] In an embodiment, the nanoparticle comprises an exosome. In an embodiment, the exosome encapsulates one or more fusion proteins and one or more cargo RNA. In an embodiment, the cargo RNA comprises a package RNA and a packaging domain wherein the package RNA comprises any embodiment of the one or more rpoD targeting polynucleotide of the present invention and wherein the packaging domain is capable of binding to the packaging protein of the fusion protein. In an embodiment, the fusion protein comprises an exosome associated transmembrane protein fused to a packaging protein. In an embodiment, the exosome associated transmembrane protein comprises CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactadherin, or PTGFRN. In an embodiment, the packaging protein comprises U1 a protein. In an embodiment, the packaging protein comprises a RNA-binding protein capable of binding tot the packaging domain such that the cargo RNA binds to the fusion protein via the RNA-binding protein and the packaging domain.

[0071] In an embodiment, the nanoparticle comprises an exosome prepared from an exosome-based packaging and delivery system. In an embodiment, the system comprises a low immunogenic exosome-based packaging and delivery system. In an embodiment, the exosome-based RNA package and delivery system comprises an exosome producing cell, a cargo RNA plasmid and one or more fusion protein plasmids, wherein the cargo RNA plasmid encodes a package RNA comprising any embodiment of the one or more rpoD targeting polynucleotide of the present invention and a packaging domain capable of binding to the packaging protein of the fusion protein encoded by the one or more fusion proteinplasmids. In an embodiment, the fusion protein expressed by the exosome producing cell based on the fusion protein plasmid comprises an exosome associated transmembrane protein fused to a packaging protein. In an embodiment, the exosome associated transmembrane protein comprises CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactadherin, or PTGFRN. In an embodiment, the packaging protein comprises an RNA-binding protein. In an embodiment, the packaging protein is endogenous to a subject. Various embodiments for the recombinant fusion protein comprising an exosome associated transmembrane protein and a packaging protein were disclosed in PCT application no. PCT / US2021 / 026892 filed 12 April, 2021, the contents of which are incorporated by reference herein in its entirety. In an embodiment, the packaging protein comprises Ula protein.

[0072] In an embodiment the one or more fusion proteins comprise CD63-U 1 a protein. In an embodiment, the nucleotide sequence of the CD63-Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 24. In an embodiment, the amino acid sequence of the CD63-U la fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 25. In an embodiment the one or more fusion proteins comprise CD81 -Ula protein. In an embodiment, nucleotide sequence of the CD81-Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 26. In an embodiment, the amino the acid sequence of the CD81-Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 27. In an embodiment, the one or more fusion proteins comprise PTGFRN-Ula protein. In an embodiment, nucleotide sequence of the PTGFRN-Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 28. In an embodiment, the amino acid sequence of the PTGFRN-Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 29.

[0073] SEQ ID NO. 24 (nt CD63-Ula):ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGC TGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCT TGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTG GTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGG GGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTATCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGT GATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAA CAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGG GCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTC CCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGA AGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAA AATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGG GAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGAT GgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTA TATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCT GTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGC GCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTA TGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAA

[0074] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U 1 snRNP A).

[0075] SEQ ID NO. 25 (aa CD63-Ula):MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEF NNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCI NVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLV KSIRSGYEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQIL DILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAK MK

[0076] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0077] SEQ ID NO. 26 (nt CD81-Ula):

[0078] ATGTCCGGACTCAGATCTCGAGCTCAAGCTTCCGGAGTGGAGGGCTGCACCAAGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCT GGAGGCGTGATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTGTATCTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAG GCATCTACATCCTCATCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGC TGCTACGGGGCCATCCAGGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCC TGGTCATCCTGTTTGCCTGTGAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGATCGCCAAGGATGTGAAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGATGATGACGCCAACAACGCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGACTGCTGTGGCTCCAGCACACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAATTTGTGTCCCTCGGGCAGCAACATCATCAGCAACCTCTTCAAGGAGGACTGCCACCAGAAGATCGATGACCTCTTCTCCGGGAAGCTGTACCTCATCGGCATTGCTGCCATCGTGGTCGCTGTGATCATGATCTTCGAGATGATCCTGAGCATGGTGCTGTGCTGTGGCATCCGGAACAGCTCCGTGTACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTC CATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAG ACCGACTCAGATATCATTGCCAAGATGAAATAA

[0079] Uppercase sequence denotes CD81 , lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).1000801 SEQ 1D NO. 27 (aa CD81-Ula):

[0081] MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTALTTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAI VVAVIMIFEMILSMVLCCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNLNEKIKKDE LKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0082] SEQ ID NO. 28 (nt PTGFRN-Ula):

[0083] ATGGGGCGCCTGGCCTCCAGGCCGCTGCTGCTGGCGCTCCTGTCGTTGGCTCTTTGCCGAGGGCGTGTGGTGAGAGTCCCCACAGCGACCCTGGTTCGAGTGGTGGGCACTGAGCTGGTCATCCCCTGCAACGTCAGTGACTATGATGGCCCCAGCGAGCAAAACTTTGACTGGAGCTTCTCATCTTTGGGGAGCAGCTTTGTGGAGCTTGCAAGCACCTGGGAGGTGGGGTTCCCAGCCCAGCTGTACCAGGAGCGGCTGCAGAGGGGCGAGATCCTGTTAAGGCGGACTGCCAACGACGCCGTGGAGCTCCACATAAAGAACGTCCAGCCTTCAGACCAAGGCCACTACAAATGTTCAACCCCCAGCACAGATGCCACTGTCCAGGGAAACTATGAGGACACAGTGCAGGTTAAAGTGCTGGCCGACTCCCTGCACGTGGGCCCCAGCGCGCGGCCCCCGCCGAGCCTGAGCCTGCGGG AGGGGGAGCCCTTCGAGCTGCGCTGCACCGCCGCCTCCGCCTCGCCGCTGCACACGCACCTGGCGCTGCTGTGGGAGGTGCACCGCGGCCCGGCCAGGCGGAGCGTCCTCGCCCTGACCCACGAGGGCAGGTTCCACCCGGGCCTGGGGTACGAGCAGCGCTACCACAGTGGGGACGTGCGCCTCGACACCGTGGGCAGCGACGCCTACCGCCTCTCAGTGTCCCGGGCTCTGTCTGCCGACCAGGGCTCCTACAGGTGTATCGTCAGCGAGTGGATCGCCGAGCAGGGCAACTGGCAGGAAATCCAAGAAAAGGCCGTGGAAGTTGCCACCGTGGTGATCCAGCCATCAGTTCTGCGAGCAGCTGTGCCCAAGAATGTGTCTGTGGCTGAAGGAAAGGAACTGGACCTGACCTGTAACATCACAACAGACCGAGCCGATGACGTCCGGCCCGAGGTGACGTGGTCCTTCAGCAGGATGCCTGACAGCACCCTACCTGGCTCCCGCGTGTTGGCGCGGCTTGACCGTGATTCCCTGGTGCACAGCTCGCCTCATGTTGCTTTGAGTCATGTGGATGCACGCTCCTACCATTTACTGGTTCGGGATGTTAGCAAAGAAAACTCTGGCTACTATTACTGCCACGTGTCCCTGTGGGCACCCGGACACAACAGGAGCTGGCACAAAGTGGCAGAGGCCGTGTCTTCCCCAGCTGGTGTGGGTGTGACCTGGCTAGAACCAGACTACCAGGTGTACCTGAATGCTTCCAAGGTCCCCGGGTTTGCGGATGACCCCACAGAGCTGGCATGCCGGGTGGTGGACACGAAGAGTGGGGAGGCGAATGTCCGATTCACGGTTTCGTGGTACTACAGGATGAACCGGCGCAGCGACAATGTGGTGACCAGCGAGCTGCTTGCAGTCATGGACGGGGACTGGACGCTAAAATATGGAGAGAGGAGCAAGCAGCGGGCCCAGGATGGAGACTTTATTTTTTCTAAGGAACATACAGACACGTTCAATTTCCGGATCCAAAGGACTACAGAGGAAGACAGAGGCAATTATTACTGTGTTGTGTCTGCCTGGACCAAACAGCGGAACAACAGCTGGGTGAAAAGCAAGGATGTCTTCTCCAAGCCTGTTAACATATTTTGGGCATTAGAAGATTCCGTGCTTGTGGTGAAGGCGAGGCAGCCAAAGCCTTTCTTTGCTGCCGGAAATACATTTGAGATGACTTGCAAAGTATCTTCCAAGAATATTAAGTCGCCACGCTACTCTGTTCTCATCATGGCTGAGAAGCCTGTCGGCGACCTCTCCAGTCCCAATGAAACGAAGTACATCATCTCTCTGGACCAGGATTCTGTGGTGAAGCTGGAGAATTGGACAGATGCATCACGGGTGGATGGCGTTGTTTTAGAAAAAGTGCAGGAGGATGAGTTCCGCTATCGAATGTACCAGACTCAGGTCTCAGACGCAGGGCTGTACCGCTGCATGGTGACAGCCTGGTCTCCTGTCAGGGGCAGCCTTTGGCGAGAAGCAGCAACCAGTCTCTCCAATCCTATTGAGATAGACTTCCAAACCTCAGGTCCTATATTTAATGCTTCTGTGCATTCAGACACACCATCAGTAATTCGGGGAGATCTGATCAAATTGTTCTGTATCATCACTGTCGAGGGAGCAGCACTGGATCCAGATGACATGGCCTTTGATGTGTCCTGGTTTGCGGTGCACTCTTTTGGCCTGGACAAGGCTCCTGTGCTCCTGTCTTCCCTGGATCGGAAGGGCATCGTGACCACCTCCCGGAGGGACTGGAAGAGCGACCTCAGCCTGGAGCGCGTGAGTGTGCTGGAATTCTTGCTGCAAGTGCATGGCTCCGAGGACCAGGACTTTGGCAACTACTACTGTTCCGTGACTCCATGGGTGAAGTCACCAACAGGTTCCTGGCAGAAGGAGGCAGAGATCCACTCCAAG CCCGTTTTTATAACTGTGAAGATGGATGTGCTGAACGCCTTCAAGTATCCCTTGCT GATCGGCGTCGGTCTGTCCACGGTCATCGGGCTCCTGTCCTGTCTCATCGGGTAC TGCAGCTCCCACTGGTGTTGTAAGAAGGAGGTTCAGGAGACACGGCGCGAGCGC CGCAGGCTCATGTCGATGGAGATGGACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAA GAAGGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAG ATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTT GTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAG GGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACT CAGATATCATTGCCAAGATGAAATAG

[0084] Uppercase sequence denotes PTGFRN, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0085] SEQ ID NO. 29 (aa PRGFRN-Ula):

[0086] MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGE1LLRRTANDAVELHI KNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREG EPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGD VRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQP SVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLAR LDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHK VAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRF TVS WYYRMNRRSDNVVTSELLAVMDGD WTLKYGERS KQRAQDGDFIFS KEHTDTF NFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVK ARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDS VVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMA FDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGS EDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLS TVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGGGGSMAVPETRPNH TIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALR SMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0087] In an embodiment, the cargo RNA comprises a package RNA comprising any embodiment of the one or more polynucleotide targeting rpoD of the present invention and apackaging domain capable of binding to the packaging protein of the fusion protein. In an embodiment, the packaging domain comprises UR domain or L2 domain. In an embodiment the nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 30 and the nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID No. 31.

[0088] In an embodiment, the packaging domain further comprises stabilizing domains. In an embodiment, the stabilizing domain comprises OH domain and / or MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 32 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 33.

[0089] SEQ ID NO. 30 (UR): AATCCATTGCACTCCGGATT

[0090] SEQ ID NO. 31 (L2): AATCCATTGCACTCCGGATTT

[0091] SEQ ID NO. 32 (OH): CTGCAGATATCCAGCACAGTGGC|00092| SEQ ID NO. 33 (MorrisMotif): GCGCAGCGCGCGCAGCGC

[0093] In an embodiment, the cargo RNA further comprises a SIRLOIN (SINE- derived nuclear RNA LOcalizatloN) nuclear localization sequences wherein the SIRLOIN is upstream of the UR or L2 sequence of the cargo RNA. In an embodiment, the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 34.

[0094] SEQ ID NO. 34 (SIRLOIN):CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA

[0095] In an embodiment, the exosome-based packaging and delivery system further comprises an argonaute 2 (Ago2)-encoding plasmid. In an embodiment, the Ago2 comprises S387A mutation. Overexpression of Ago2 or the S387A mutant thereof increases the packaging efficiency of the cargo RNA into the exosomes in an exosome-producing cell.

[0096] In an embodiment, the pharmaceutical composition comprising a nanoparticle encapsulating any embodiment of the one or more rpoD targeting polynucleotide of the present invention further comprises a nanoparticle payload release enhancer. In an embodiment, the nanoparticle release enhancer enhances the release of payload from the nanoparticle for at least about 2-fold, at least about 5 -fold, at least about 10-fold, at least about 15 -fold, at least about 20-fold, or at least about 25 -fold. In an embodiment, the nanoparticle payload release enhancer comprises a modified myoferlin protein. In anembodiment, the modified myoferlin protein comprises C2F, C2G, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein consists of C2F, C2G, transmembrane domain, or a combination thereof. In an embodiment, the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 35. In an embodiment, the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 36. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 37.

[0097] SEQ ID NO. 35 (C2F):QFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRDHYIPNTLN PVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEE YCVSGV

[0098] SEQ ID NO. 36 (C2G):PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKTDV HYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWS1DQTEFRIPPRL1IQ1WDND KFSLDDYLGFLELDLRH

[0099] SEQ ID NO. 37 (transmembrane domain): PDLKAMNPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEA DERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLI LLLFVAV[000100] In an embodiment, the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein consists of C2A, FerA, FerB, DysFN, transmembrane domain. In an embodiment, the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 38. In an embodiment, the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 39. In an embodiment, the amino acid sequence of the FerB is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 40. In an embodiment, the DysFN domain comprises DysFN- 1, DysFN-2, or a combination thereof. In an embodiment, the amino acid sequence of the DysFN-1 is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 41. In an embodiment, the amino acid sequence of the DysFN-2 is at least about 80%, about 85%,about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 42. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 37. [000101] SEQ ID NO. 38 (C2A): MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRGIPL DFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATID LVIGYDPPSAPHPNDLS[000102] SEQ ID NO. 39 (FerA):LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIRK [000103] SEQ ID NO. 40 (FerB):[000104] WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCGKTQTIFLKYPQEKNNGP[000105] SEQ ID NO. 41 (DysFN-1):AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKREFF LP1000106] SEQ ID NO. 42 (DysFN-2):DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSELTC P[000107] In an embodiment, any embodiment of the modified myoferlin protein of the present invention further comprises C2B, C2C, C2D, C2E domains, or a combination thereof. In an embodiment, the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 43. In an embodiment, the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 44. In an embodiment, the amino acid sequence of the C2D is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 45. In an embodiment, the amino acid sequence of the C2E is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 46.[000108] SEQ ID NO. 43 (C2B):PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVNMT PSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP [000109] SEQ ID NO. 44 (C2C): TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIEKNANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVED FSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE[000110] SEQ ID NO. 45 (C2D):TPIVSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTEIIHSTLNP TWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEM DITPKLLWHPVMNGDKA[000111] SEQ ID NO. 46 (C2E):RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEELYM PPLVIKVIDHRQFGRKPVVG[000112] In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2A domain wherein the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 38. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2B domain wherein the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ DI NO. 43. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2C domain wherein the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ DI NO. 44. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2D domain wherein the amino acid sequence of the C2D domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 45. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2E domain wherein the amino acid sequence of the C2E domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 46. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2F domain wherein the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 35. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2G domain wherein the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 36. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerA domain wherein the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 39. In an embodiment, the modified myoferlin protein of the present inventiondoes not comprise the FerB domain wherein the amino acid sequence of the FerB domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 40. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-1 domain wherein the amino acid sequence of the DysFN-1 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 41. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-2 domain wherein the amino acid sequence of the DysFN-2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 42. In an embodiment, the modified myoferlin protein of the present invention does not comprise the transmembrane domain wherein the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 37.[000113] In an embodiment, amino acid sequence of the modified myoferlin protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 47.1000114 ] SEQ ID NO. 47 (C2F-C2G) :MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRD HYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFG SHCGIPEEYCVSGVNTWRDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLD EFEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVD VFPKSLGPPGPPFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPG NEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIP PRLIIQIWDNDKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKAKTAS LFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMN PKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLILLLFVAVLLYSLPNYL SMKIVKPNVYPYDVPDYA[000115] The present invention also provides a modified myoferlin protein encoding polynucleotide encoding any embodiment of the modified myoferlin protein of the present invention. In an embodiment, the modified myoferlin protein encoding polynucleotide encodes a C2A domain, a C2B domain, a C2C domain, a C2D domain, a C2E domain, a C2F domain, a C2G domain, a FerA domain, a FerB domain, a DysFN-1 domain, a DysFN-2 domain, a transmembrane domain, or a combination thereof. In an embodiment, the C2A domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ IDNO. 48. In an embodiment, the C2B domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 49. In an embodiment, the C2C domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 50. In an embodiment, the C2D domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 51 . In an embodiment, the C2E domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 52. In an embodiment, the C2F domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 53. In an embodiment, the C2G domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 54. In an embodiment, the FerA domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 55. In an embodiment, the FerB domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 56. In an embodiment, the DysFN-1 domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 57. In an embodiment, the DysFN-2 domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 58. In an embodiment, the transmembrane domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 59. [000116] SEQ ID NO. 48 (nt C2A):ATGCTGCGAGTGATTGTGGAATCTGCCAGCAATATCCCTAAAACGAAATTTGGCA AGCCGGATCCTATTGTTTCTGTCATTTTTAAGGATGAGAAAAAGAAAACAAAGA AAGTTGATAATGAATTGAACCCTGTCTGGAATGAGATTTTGGAGTTTGACTTGAG GGGTATACCACTGGACTTTTCATCTTCCCTTGGGATTATTGTGAAAGATTTTGAGA CAATTGGACAAAATAAATTAATTGGCACGGCGACTGTAGCCCTGAAGGACCTGACTGGTGACCAGAGCAGATCCCTGCCGTACAAGCTGATCTCCCTGCTAAATGAAAGAGGGCAAGATACTGGGGCCACCATTGACTTGGTGATCGGCTATGATCCGCCTTCTGCTCCACATCCAAATGACCTGAGC[000117] SEQ ID NO. 49 (nt C2B):CCACAGGACTTCCAGATCCGCGTCCGAGTGATTGAGGGCCGACAGTTAAGTGGCAACAACATAAGGCCTGTGGTCAAAGTTCACGTCTGTGGCCAGACACACCGAACAAGAATCAAGAGAGGAAACAACCCTTTTTTTGATGAGTTGTTTTTCTACAATGTCAACATGACCCCTTCTGAATTGATGGATGAGATCATCAGCATCCGGGTTTATAATTCTCACTCTCTGCGGGCAGATTGTCTGATGGGGGAATTTAAGATTGATGTTGGATTTGTTTATGATGAACCTGGCCATGCTGTCATGAGAAAGTGGCTTCTTCTCAATGACC CG[000118] SEQ ID NO. 50 (nt C2C):ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGCCTTCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCGTGGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAAAAAGGTTTGTACAAACATAATTGAGAAGAATGCAAACCCAGAGTGGAATCAGGTCGTCAATCTTCAGATCAAGTTTCCTTCAGTGTGTGAAAAAATAAAACTAACAATATATGACTGGGACCGTCTTACTAAAAATGATGTAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTGGTGGGGAAGTGGAAGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGTAAACACAGGAGAAACAGAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTGAATCTTTATGGAAGCCCCAGAGAGTACACGGGATTCCCAGACCCCTATGATGAG[000119] SEQ ID NO. 51 (nt C2D):ACCCCCATTGTTTCCTGCAATTTTGACAGAGTCTACATCTACCATCTGCGCTGCTATGTCTATCAAGCCAGAAACCTCTTGGCTTTAGATAAGGATAGCTTTTCAGATCCATATGCTCATATCTGTTTCCTCCATCGGAGCAAAACCACTGAGATCATCCATTCAACCCTGAATCCCACGTGGGACCAAACAATTATATTCGATGAAGTTGAAATCTATGGGGAACCCCAAACAGTTCTACAGAATCCACCCAAAGTTATCATGGAACTTTTTGACAATGACCAAGTGGGCAAAGATGAATTTTTAGGACGAAGCATTTTCTCTCCTGTGGTGAAACTGAACTCAGAAATGGACATCACACCCAAACTTCTCTGGCACCCAGTAATGAATGGAGACAAAGCC[000120] SEQ ID NO. 52 (nt C2E):AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTGGAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACACCCAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTACATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAA GCCTGTCGTCGGC[000121] SEQ ID NO. 53 (nt C2F):CAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGG GTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTC[000122] SEQ ID NO. 54 (nt C2G):CCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACT TGACTTGCGTCAC[000123] SEQ ID NO. 55 (nt FerA):CTGCAAACAAATATAGAAGCTCTAAAATCAGGGATACAAGGTAAAATTCCTGCAAACCAGCTGGCTGAATTGTGGCTGAAGCTGATAGATGAAGTTATAGAAGACACGAGATACACGTTGCCTCTCACAGAAGGAAAAGCCAACGTCACAGTTCTCGATACTC AGATCCGAAAG[000124] SEQ ID NO. 56 (nt FerB):TGGCTTGATAAATTAATGCAGCTGACTGAAGAGCCACAGAACAGCATGCCTGACATCATCATCTGGATGATCCGGGGAGAGAAGAGACTGGCCTATGCACGAATTCCC GCACATCAGGTCTTGTACTCCACCAGTGGTGAGAATGCATCTGGAAAATACTGTGGGAAAACCCAAACCATCTTTCTGAAGTATCCACAGGAGAAAAACAACGGGCCA [000125] SEQ ID NO. 57 (nt DysFN-1):GCTGTGGAGAAGAAGTTTAACAGCTTCGCAGAAGGAACTTTCACCGTCTTTGCTGAAATGTATGAAAATCAAGCTCTCATGTTTGGAAAATGGGGTACTTCTGGATTAGTAGGACGTCATAAGTTTTCTGATGTCACAGGAAAAATAAAACTCAAGAGGGAATT TTTTCTGCCT[000126] SEQ ID NO. 58 (nt DysFN-2): GATCCTGAAAGAAGCTTGCTGACTGAGGCAGATGCAGGTCACACGGAGTTCACT GATGAAGTCTACCAGAACGAGAGCCGCTACCCCGGGGGCGACTGGAAGCCGGCC GAGGACACCTACACGGATGCGAACGGCGATAAAGCAGCATCACCCAGCGAGTTG ACTTGTCCT[000127] SEQ ID NO. 59 (nt TM):CCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGC AGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCG TAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCG ACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTG GACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGA CCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTT CCTGCTTATCCTGCTGCTCTTCGTGGCCGTG[000128] In an embodiment, the modified myoferlin protein of the present invention further comprises a connexin 43 protein to form a dual protein. The dual protein comprising the modified myoferlin protein and the connexin 43 protein of the present invention can be used to greatly enhance the efficacy and delivery of the RNA or nucleic acid payloads. In an embodiment, the connexin 43 protein comprises a S368A mutation. In an embodiment, the connexin 43 protein of the present invention comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 60.[000129] SEQ ID NO. 60 (connexin 43):MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSA FRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEEKLNK KEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFE VAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNII ELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSP PGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFP DDNQNSKKLAAGHELQPLAIVDQRPSSRAASRASSRPRPDDLEI[000130] The present invention further provides a polynucleotide encoding the dual protein comprising a modified myoferlin protein of the present invention fused to a connexin 43 protein S368A mutant wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 61.[000131] SEQ ID NO. 61:ATGGTGCCAGCCCCTCCCAGACAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTCAATACCTGGCGAGATCAACTGAGACCAACACAGCTGCTTCAAAATGTCGCCAGATTCAAAGGCTTCCCACAACCCATCCTTTCCGAAGATGGGAGTAGAATCAGATATGGAGGACGAGACTACAGCTTGGATGAATTTGAAGCCAACAAAATCCTGCACCAGCACCTCGGGGCCCCTGAAGAGCGGCTTGCTCTTCACATCCTCAGGACTCAGGGGCTGGTCCCTGAGCACGTGGAAACAAGGACTTTGCACAGCACCTTCCAGCCCAACATTTCCCAGGGAAAACTTCAGATGTGGGTGGATGTTTTCCCCAAGAGTTTGGGGCCACCAGGCCCTCCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCACACGATCATTCCTGCAAAATCACCAGAGAAATGCAGGTTGGACATGATTCCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGCAGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCGTAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCGACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTGGACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGACCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTTCCTGCTTATCCTGCTGCTCTTCGTGGCCGTGCTCCTCTACTCTTTGCCGAACTATTTGTCAATGAAGATTGTAAAGCCAAATGTGTACCCATACGACGTCCCAGACTACGCTTAGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACA ACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGC CTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCC CAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAA GCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGAT CTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAA ACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGA TAATATGGCCACAACCATGGGTGACTGGAGCGCCTTAGGCAAACTCCTTGACAA GGTTCAAGCCTACTCAACTGCTGGAGGGAAGGTGTGGCTGTCAGTACTTTTCATT TTCCGAATCCTGCTGCTGGGGACAGCGGTTGAGTCAGCCTGGGGAGATGAGCAG TCTGCCTTTCGTTGTAACACTCAGCAACCTGGTTGTGAAAATGTCTGCTATGACA AGTCTTTCCCAATCTCTCATGTGCGCTTCTGGGTCCTGCAGATCATATTTGTGTCT GTACCCACACTCTTGTACCTGGCTCATGTGTTCTATGTGATGCGAAAGGAAGAGAAACTGAACAAGAAAGAGGAAGAACTCAAGGTTGCCCAAACTGATGGTGTCAATG TGGACATGCACTTGAAGCAGATTGAGATAAAGAAGTTCAAGTACGGTATTGAAG AGCATGGTAAGGTGAAAATGCGAGGGGGGTTGCTGCGAACCTACATCATCAGTA TCCTCTTCAAGTCTATCTTTGAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTAT GGATTCAGCTTGAGTGCTGTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCTCTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGCTG GTGGTGTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTT CAAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGACCAG TGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCTTATTTCAAT GGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCTCCTGGGTACAAGCT GGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAATTACAACAAGCAAGCAAG TGAGCAAAACTGGGCTAATTACAGTGCAGAACAAAATCGAATGGGGCAGGCGGG AAGCACCATCTCTAACTCCCATGCACAGCCTTTTGATTTCCCCGATGATAACCAG AATTCAAAAAAACTAGCTGCTGGACATGAATTACAGCCACTAGCCATTGTGGAC CAGCGACCTTCAAGCAGAGCCGCCAGTCGTGCCAGCAGCAGACCTCGGCCTGAT GACCTGGAGATCTGA[000132] In an embodiment, the various components comprising the fusion protein or any variant thereof of the present invention, the Ago2 protein or any variant thereof of the present invention, the modified myoferlin protein or any variant thereof of the present invention, or the connexin 43 protein or any variant thereof of the present invention that could be incorporated or encapsulated to the nanoparticle encapsulating the one or more rpoDtargeting polynucleotide of the present invention for the enhanced delivery and uptake of said nanoparticle in a subject can be incorporated or encapsulated to the nanoparticle in all possible combinations. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention further comprises a fusion protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention further comprises an Ago2 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention further comprises a modified myoferlin protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention further comprises a connexin 43 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention does not comprise a fusion protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention does not comprise an Ago2 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention does not comprise a modified myoferlin protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more rpoD targeting polynucleotide of the present invention does not comprise a connexin 43 protein or any variant thereof of the present invention. In an embodiment where the nanoparticle comprises an exosome encapsulating one or more rpoD targeting polynucleotide of the present study and a fusion protein comprising an exosome- associated transmembrane protein fused to a packaging protein of the present invention, the one or more rpoD targeting polynucleotide is fused to a packaging domain or any variant thereof of the present invention. In an embodiment, the packaging domain or any variant thereof of the present invention comprises UR, L2, the OH domain, the MorrisMotif domain, the nuclear localization sequence SIRLOIN, or a combination thereof.[000133] In an embodiment, any embodiment of the nanoparticle encapsulating the rpoD targeting polynucleotide of the present invention downregulates the expression of the rpoD gene o by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%.[000134] The present invention also provides a method of treatment of a rpoD- associated disease of a subject comprising the step of suppressing the rpoD expression in bacteria infecting the subject such as S. aureus. In an embodiment, the step of suppressing rpoD expression comprises the administration of a therapeutically effective amount of any embodiment of the one or more rpoD targeting polynucleotide of the present invention to the subject. In an embodiment, the step of suppressing rpoD expression comprises the administration of any embodiment of the nanoparticle encapsulating one or more rpoD targeting polynucleotide of the present invention to the subject.[000135] In an embodiment, the rpoD-associated disease comprises S. aureus infection. In an embodiment, the .S', aureus infection is caused by a .S', aureus strain resistant to at least an antibiotic such as but not limited to methicillin, clindamycin, ciprofloxacin, erythromycin, flusidic acid, gentamycin, levofloxacin, sulfamethoxazole-trimethoprim, tetracycline, vancomycin, linezolid, or a combination thereof. In an embodiment, the .S', aureus comprises MRSA. In an embodiment, the rpoD-associated disease comprises infections caused by Str. JKD6008, TW20, ED98, JH1, NCTC 8325, USA300_TCH1516, USA300_FPR3757, ST398, MRSA252, MSSA476, JKD6159, ED133, Staphylococcus epidermidis RP62A, ATCC 12228, Staphylococcus lugdunensis HKU09-01 , N920143, Staphylococcus haemolyticus JCSC1435, Staphylococcus camosus subsp. Camosus TM300, Staphylococcus pseudintermedius ED99, HKU10-03, Staphylococcus saprophyticus subsp. Saprophyticus ATCC 15305, or a combination thereof.[000136] In an embodiment, the method of treatment of a rpoD-associated disease in a subject comprises the step of administering a therapeutic effective amount of any embodiment of the pharmaceutical composition comprising one or more rpoD targeting polynucleotide of the present invention to the subject. In an embodiment, the method of treatment of a rpoD-associated disease in a subject comprises the step of administering a therapeutic effective amount of any embodiment of the pharmaceutical composition comprising any embodiment of the nanoparticle encapsulating one or more rpoD targeting polynucleotide of the present invention to the subject. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agentcomprising a site-specific targeting moiety to a particular location. For example, to target liver cells, intravenous injection may be used.[000137] In an embodiment, the method of treating a rpoD-associated disease comprises the step of administering about 1 to about 1000 billion of any embodiment of the exosomes, LNP, or a combination thereof encapsulating the one or more rpoD targeting polynucleotides of the present invention such as about 1, about 10, about 50, about 100, about 250, about 500, about 750, about 1000, about 1250, about 5000, about 7500, about 10000, about 12500, about 50000, about 75000, about 100000, about 125000, about 500000, about 750000, about 1000000, about 1250000, about 5000000, about 7500000, about 10000000, about 12500000, about 50000000, about 75000000, about 100000000, about 125000000, about 500000000, about 750000000, about 1 billion, about 10 billion, about 50 billion, about 100 billion, about 300 billion, about 600 billion, or about 1000 billion including any numbers or ranges of numbers falling within these values of exosomes encapsulating the one or more rpoD targeting polynucleotides of the present invention. In an embodiment, the method of treatment of a rpoD -associated disease comprises the step of administering about 0.01 to about 20 mg / kg of the body weight of the subject such as about 0.01, about 0.05, about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1 , about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20 mg / kg, or any concentration or concentration ranges falling within these values of exosomes, LNP, or a combination thereof encapsulating the one or more rpoD targeting polynucleotides of the present invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agent comprising a site-specific targeting moiety to a particular location. For example, to target liver cells, intravenous injection may be used. [000138] The pharmaceutical compositions may be administered in the form of any embodiment of the exosomes of the present invention. As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the lateendosome with the plasma membrane. The any embodiment of the exosome of the present invention comprises lipid or fatty acid and polypeptide and further comprises the inhibitory nucleic acids described herein as a payload. The any embodiment of the exosome of the present invention can be derived from a producer cell, and isolated from the producer cell based on its size, density, biochemical parameters, or a combination thereof. The any embodiment of the exosome of the present invention can be directly loaded with exogenous nucleic acids or drugs by electroporation, lipofection, sonication and contact with calcium chloride. Alternatively, purified exosomes may be loaded ex vivo by, for example, electroporation. [000139] Any embodiment of the exosome of the present invention 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 26 cells, or mesenchymal stem cells (MSCs), can be used.[000140] The pharmaceutical compositions may also be formulated by incorporation of the inhibitory nucleic acids described herein into adenoviruses or adeno-associated viruses (AAVs), formulated with cell-penetrating peptides, lentiviral vectors, polymers, dendrimers, or prepared as siRNA bioconjugates such as the GalNAc-siRNA conjugate delivery platform.[000141] If using the exosomes or a vector as a vehicle to deliver siRNA, the candidate siRNAs are delivered as 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 (AAV or Lentiviral vector) as described above.[000142] An shRNA may be provided in an expression cassette containing a promoter contiguously linked to an siRNA as described herein. In embodiments, the promoter is a polll or a polIII promoter, such as a U6 promoter (e.g., a mouse U6 promoter) or a Hl promoter. In embodiments, the expression cassette further contains a marker gene. In embodiments, the promoter is a polll promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a polIII promoter. In embodiments, the promoter is U6 or Hl promoter. [000143] Also provided is a vector containing an expression cassette described herein.Examples of appropriate vectors include adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based viral vectors. In an embodiment, the vector is an adeno-associated virus (AAV) vector.[000144] An 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 certain 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 ' and / or a 5 " overhang 0, 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’) or (5’-TTGC-3’) or other sequences as will be well understood by the skilled person. [ 000145] 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, sex), the progression (i.e. pathological state) of a given coronavirus infection, and other factors that will be readily recognised 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 such as 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 30 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 0.05mg / 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.[000146] 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 outcome.[000147] Typically, in therapeutic applications, the treatment would be for the duration of the infection, disease state or condition. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the infection, disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimum conditions can also be determined using conventional techniques.[000148] 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, and in certain embodiments from about one to about four week intervals.Periodic re-administration may be desirable in the case of recurrent exposure to a particular pathogen targeted by a pharmaceutical composition described herein.[000149] 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.[000150] Suitable techniques for introduction of the inhibitory nucleic acids described herein into cells, tissues, and organisms include various carrier systems, vectors and reagents. Nonlimiting examples include lipid nanoparticles (LNP), micelles, nucleic-acid-lipid particles, lipoplexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus like particles (VLP), and mixtures thereof.[000151] Pharmaceutical compositions of the present invention may be administered in any suitable way, such as, 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 including 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 an embodiment, the pharmaceutical composition is adapted for intranasal administration.[000152] In an embodiment, a pharmaceutical composition of the present invention is formulated as a direct-acting nasal spray. In an embodiment, a nasal spray can be selfadministered at point-of-care.[000153] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.[000154] EXAMPLES[000155] Materials and methods[000156] To determine the optimal siRNA for repression of MRSA gene expression, we ordered the siRNAs (Table 2) and screened them in co-transfection assays with pRP[Exp]-Puro-CMV+intron>{hRluc-RNAPII_StaphTarget]VB23091 l-1685xts reporter plasmid (Figure 3). The reporter plasmid contains the full-length sense RNAP2-SigA mRNA (Table 1) embedded within the hRluc reporter gene, expressed off the CMV promoter, and an internal HSV-TK promoter driving the hFluc gene, which acts as an internal control gene for standardization (Figure 3). On day 0 HEK293 cells 0.1xl0A6 cells / well were plated and then one day later these cells were co-transfected with plasmid (Table 3) and various accompanying siRNAs using lipofectamine 3000 (L3K). After 72hrs the hRluc and hFLuc transgene expression was determined by qRTPCR using primers (hRLuc and beta actin, Table 5). For screening multiple siRNA effects on RNAP2-SigA expression, combinations of MRSA targeted siRNAs (lOOnM total siRNA / well) with the target reporter plasmid (100ng)(Table 4) were co-transfected into HEK cells and 72hrs later the hRluc and hFLuc transgene expression determined by qRTPCR using primers (hRLuc and beta actin, Table 4).[000157] Table 1 RNA polymerase sigma factor targeting (based on PMID: 222538J 5). Underlined sequence is structured region that is functionally required and the bold represents the PNA target site that was found to work previously (PMID: 2225381 ). Italic highlighted indicates siRNA target sites found to repress reporter gene expression.[000158] Table 2 siRNAs targeted to RNAPII SigA from staph aureous (Table 1).[000159] Table 3 Plasmids to co-transfect with siRNAs for each treatment.[000160] Table 4 Primers used for screening repression of the reporter gene by qRTPCR.[000161] Table 5 HEK293 cells (12 well plate) siRNA combinations targeted to theMRSA target RNAPII gene were screened. Total plasmid is lOOng and total siRNAs is 120 nM.[000162] Table 6 RNA combination therapy to MRSA infections. The following combinations of siRNAs (LNP) or shRNAs (exosomes) can be developed generated and packaged into lipid nanoparticles (LNPs) or exosomes and used to treat MRSA infection by IV or intranasal administration of the RNA containing nanoparticles. Note siRNAs are shown but shRNA containing the same sequences can be used packaged into exosomes.[000163] Table 7 Plasmids to transfect into producer cells. Recipient cells are transfected with p31 pRP[Exp]-Puro-CMV+intron>[hRluc-RNAPII StaphTarget] plasmid(300ng).[000164] Table 8 Plasmid combinations used to generate shRNA packaged EVs. All shRNAs require p88 and p87 in all formulations to enhance shRNA packaging and endosomal release.[000165] Results[000166] Using our in-house siRNA algorithm we designed and tested several siRNAs to RNA polymerase sigma factor (rpoD, Table 1) and have now defined candidate therapeutic siRNAs, both alone and in combination, that can repress MRSA infection by delivery with lipid nanoparticles and / or exosomes. We found that all of our selected siRNAs had some effect on rpoD expression in co-transfected reporter assays (Figure 1). However, one siRNA, siStaphRNAP2-sil stood out for it’s pronounced repression (Figure 1). Next, we screened to what extent combinations of siRNAs can repress the rpoD MRSA reporter expression andobserved that siStaphRNAP2-sil and siStaphRNAP2-si2 can work to simultaneously target repression of rpoD (Figure 2). Collectively, these data suggest that siStaphRNAP2-sil and siStaphRNAP2-si2 are good siRNAs targeted to disrupt rpoD gene expression and repress MRSA and can repress MRSA either alone or in combination (Figures 1 and 2).[000167] We also demonstrated the targeting of rpoD using shRNAs targeted to MRSA being packaged into EVs with the various shRNA expressing vectors as well as packer p88 and enhancer p87 (Figure 4 and 5). One candidate dual shRNA-EV expressing cell treatment resulted in significant repression of the corresponding MRSA stable reporter Rluc-target expression (Figure 5). Figure 4 illustrates the effect of single or combination siRNA treatment on MRSA RNAPII targeting (Table 7). Producer cells transfected with plasmid combinations were co-cultured in a transwell assay with stable reporter recipient cells. After 72hrs the recipient cells are collected and assessed by qRTPCR for Rluc / Fluc mRNA expression (Table 4). Figure 5 illustrates the effect of single or combination siRNA treatment on MRSA RNAPII targeting (Table 8). Producer cells transfected with plasmid combinations (Table 8) were co-cultured in a transwell assay with stable reporter recipient cells. After 72hrs the recipient cells are collected and assessed by qRTPCR for Rluc / Fluc mRNA expression (Table 4). All value represents fraction of reporter cell alone (+SEM, n=3).[000168] We further analyzed whether the target sites of the siRNAs of the present invention are presented in different MRSA strains using sequence alignment. In total, 16 MRSA strains (SI -SI 6), including 7 strains (S1-S4, S9-S10) that show additional resistance to clindamycin (CC), ciprofloxacin (CIP), erythromycin (E), flusidic acid (FA), gentamycin (GM), levofloxacin (LVX), sulfamethoxazole-trimethoprim (SXT), tetracycline (TE), or a combination thereof were analyzed. Our results show that target sites of siStaphRNAP2-sil, siStaphRNAP2-si2, siStaphRNAP2-si3, siStaphRNAP2-si5 are presented in all the 16 strains (Figure 6), and the target site of siStaphRNAP2-si4 is presented in 14 strains (Figure 6), suggesting the siRNAs of the present invention alone or in combination can be used to target multiple MRSA strains.

Claims

What is claimed is:

1. A pharmaceutical composition comprising one or more RNA polymerase primary o70(rpoD) targeting polynucleotides capable of targeting rpoD, wherein the nucleotide sequence of each of the one or more rpoD targeting polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, or a combination thereof.

2. The pharmaceutical composition of claim 1 , wherein the one or more rpoD targeting polynucleotides comprise antisense RNA (asRNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof.

3. The pharmaceutical composition of claim 1, wherein the one or more rpoD targeting polynucleotides comprise one or more first polynucleotide and one or more second polynucleotide wherein the nucleotide sequence of each of the one or more first polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1 and nucleotide sequence of each of the one or more second polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5.

4. The pharmaceutical composition of claim 1 , wherein the one or more polynucleotides comprise one or more first polynucleotide, one or more second polynucleotide and one or more third polynucleotide wherein nucleotide sequence of the one or more first polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1, nucleotide sequence of the one or more second polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, or SEQ ID NO. 5, and nucleotide sequence of the one or more third polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 2, SEQ ID NO.3, SEQ ID NO. 4, or SEQ ID NO. 5 and wherein the nucleotide sequence of the third polynucleotide is distinct from the nucleotide sequence of the second polynucleotide by at least 80%.

5. The pharmaceutical composition of claim 1 , wherein nucleotide sequence of the one or more rpoD targeting polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO.18, SEQ ID NO. 19, or a combination thereof.

6. The pharmaceutical composition of claim 1 , wherein the pharmaceutical composition comprises a first shRNA and a second shRNA wherein nucleotide sequence of the first shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 11 or SEQ ID NO. 16 and nucleotide sequence of the second shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 15 or SEQ ID NO. 19.

7. The pharmaceutical composition of claim 1 , wherein the rpoD comprises Staphylococcus aureus (S. aureus) rpoD or a variant thereof.

8. The pharmaceutical composition of claim 7, wherein the S. aureus comprises methicillin-resistant 5. aureus (MRS A).

9. The pharmaceutical composition of claim 1 further comprising a nanoparticle wherein the nanoparticle encapsulates the one or more rpoD targeting polynucleotides.

10. The pharmaceutical composition of claim 9, wherein the nanoparticle comprises lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle, liposome, exosome, virus or virus-like particle.

11. The pharmaceutical composition of claim 9, further comprising a fusion protein and a packaging domain wherein the nanoparticle comprises exosome, wherein the one or more polynucleotides targeting rpoD is fused to the packaging domain, wherein the fusion protein comprises an exosome-associated transmembrane protein fused to a packaging protein, wherein the packaging domain comprises an UR domain, a L2 domain, or a combination thereof and wherein the packaging domain is capable of binding with the packaging protein.

12. The pharmaceutical composition of claim 11, wherein the one or more polynucleotides targeting rpoD is further fused to a nuclear localization sequence SIRLOIN.

13. The pharmaceutical composition of claim 11, wherein the fusion protein comprises CD63-Ula, CD81-Ula, PTGFRN-Ula, or a combination thereof.

14. The pharmaceutical composition of claim 11, wherein the exosome further comprises an Ago2 protein or a S387A mutant thereof.

15. The pharmaceutical composition of claim 11, wherein the exosome further comprises a modified myoferlin protein.

16. The pharmaceutical composition of claim 15, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain of the myoferlin protein, or a combination thereof.

17. The pharmaceutical composition of claim 15, wherein the modified myoferlin protein further comprises a connexin 43 protein or a S368 A mutant thereof.

18. The pharmaceutical composition of claim 11, wherein the exosome is prepared using an exosome-based packaging and delivery system comprising one or more cargo RNA encoding plasmid encoding the one or more polynucleotide targeting rpoD fused to the packaging domain, and one or more fusion protein encoding plasmids encoding the fusion protein comprising an exosome-associated transmembrane protein fused to a packaging protein.

19. The pharmaceutical composition of claim 18, wherein the exosome-based delivery and packaging system further comprises a plasmid encoding a modified myoferlin protein, a connexin 43 protein or a S368A mutant thereof, or a combination thereof.

20. A method of treatment of rpoD-associated disease of a subject comprising administration of a therapeutically effective amount of the pharmaceutical composition of claim 1 to the subject.

21. The method of claim 20, wherein the rpoD-associated disease comprises S. aureus infection.

22. The method of claim 21, wherein the S. aureus is resistant to at least an antibiotic comprising methicillin, clindamycin, ciprofloxacin, erythromycin, flusidic acid, gentamycin, levofloxacin, sulfamethoxazole-trimethoprim, tetracycline, vancomycin, linezolid, or a combination thereof.

23. The method of claim 21, wherein the S. aureus comprises MRSA.

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