Alpha-1 Anti-trypsin upregulating polynucleotides and method of use and treatment thereof

Abstract

The present invention provides a composition comprising one or more polynucleotides capable of upregulating expression of alpha- 1 anti-trypsin (Al AT). In an embodiment, the one or more polynucleotides of the present invention upregulates the expression of Al AT by downregulating antisense RNA that regulates the Al AT gene. In an embodiment, the one or more polynucleotides comprises an siRNA or an shRNA. In an embodiment, one or more polynucleotides of the present invention is delivered by a nanoparticle such as an exosome that encapsulates the one or more polynucleotides. The present invention also provides a method of treatment of Al AT deficiency related diseases or disorders using any embodiment of the one or more polynucleotides of the present invention.

Description

[0001] ALPHA-1 ANTI TRYPSIN UPREGULATING POLYNUCLEOTIDES ANDMETHOD 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, “Al AT Upregulating Polynucleotide Sequence Listing.xml” created on January 15, 2025 and having a size of 72 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 capable of upregulating expression of alpha- 1 anti-trypsin (Al AT) and method of use thereof.

[0006] BACKGROUND OF THE INVENTION

[0007] Al AT deficiency is an autosomal dominant disease that results in chronic liver disease, cirrhosis, hepatocellular carcinoma in children and an increased risk for emphysema in adults '. COPD is a major progressive pulmonary disease characterized by chronic bronchitis and / or emphysema leading to severe breathing difficulties and is found to correlate with defective expression of Al AT. It is the 3rdmost common cause of chronic morbidity and death worldwide and costs >$2 trillion per year globally, $9 billion per year in Australia. Current therapeutic approaches for COPD primarily target symptoms and aim to reduce exacerbations but do not stop the progression or reverse the disease.

[0008] The main mechanism of pathogenesis of COPD is an imbalance in protease- antiprotease function. A1AT is a 52-kDa glycoprotein that is a serum trypsin suppressor produced only liver parenchymal cells and then migrates to the lungs. It functions to inhibit neutrophil elastase and various proteases. The function of these proteases in the lungs is to clear debris and digest damaged tissues. However, when they are not properly controlled, their activity results in tissue damage that occurs, leading to abnormal deposition of extracellular matrix (ECM) proteins. The ECM of the lungs is a dynamic structure composed of a diverse set of proteins, glycoproteins, and proteoglycans that provide architectural support and enable functional processes. Elastin and various forms of collagen are the twomain types of ECM proteins, and uncontrolled degradation of these ECM proteins by proteases results in lung damage, leading to emphysema2. Excess fibrosis (airway remodeling) and tissue damage, and emphysema combine to impair lung function and gas exchange and cause severe breathing difficulties. Al AT blocks the activity of proteases and protects against the over-expression of these elastases and lung damage. Patients who have deficiencies in the Al AT gene develop severe early-onset emphysema and COPD. This group of patients does not express or have significantly reduced levels or non-functional Al AT, which is generated in the liver and released into the blood, which leads to the early loss of control of elastases that damage lung tissue and results in early emphysema and COPD. A1AT augmentation therapy, whereby patients receive intravenous infusions of recombinant Al AT protein, is a lifelong treatment, which is inefficient at best and only slows the lung damage as the injected Al AT is quickly degraded3. In essence, there is no cure for A1AT deficiency. Thus, a major treatment goal is to permanently restore functional Al AT gene expression in these patients.

[0009] RNA interference4is a mechanism of action imbued in mammalian cells that can specifically turn off the production of proteins in cells in a sequence- specific and potent manner. It works via the introduction of siRNAs (15-30bp of double stranded RNA) that specifically target Argonaute-2 (AGO2) to mRNAs via sequence complementarity, causing their subsequent degradation5. There are now 5 RNAi FDA approved drugs. RNAi can function to repress a genes expression either transiently using post-transcriptional gene silencing (PTGS), or long-term, using transcriptional gene silencing (TGS). The difference between PTGS and TGS is the target of the siRNA, when the mRNA body is targeted, the result is PTGS and when the promoter in the genome for the mRNA is targeted the result is TGS.

[0010] Long non-coding RNAs (IncRNAs) are a form of RNA that is important in regulating gene transcription as well as to be involved in a myriad of other cellular processes

[0018] . We, and others, have learned that by repressing those IncRNAs that are repressive regulators of protein-coding genes with RNAi, that it is possible to activate gene expression. This form of siRNA mediated gene activation is the result of “repressing the repressor” [20, 21], Several genes are reported to be regulated by the action of IncRNAs and we have discovered that one IncRNA (ENSG00000289541, Figure 1), which is antisense to the Al AT promoter, acts to repress Al AT expression. Therefore, there is a need for an effective composition and method that utilizes RNA interference to treat Al AT deficiency related diseases via regulation of the IncRNAs that repress Al AT expression.

[0011] SUMMARY OF THE INVENTION

[0012] A pharmaceutical composition comprising one or more alpha- 1 antitrypsin (Al AT) upregulating polynucleotides capable of upregulating expression of Al AT protein encoded by A1AT gene wherein the one or more Al AT upregulating polynucleotides targets at least a portion of promoter and / or 5’ UTR region of antisense RNA to the Al AT gene.

[0013] A method of upregulating expression of Al AT protein in a subject comprising the step of targeting at least a portion of promoter and / or 5’ UTR region of antisense RNA to the Al AT gene using RNA interference in the subject.

[0014] A method of upregulating expression of A1AT protein in a subject comprising the step of targeting at least a portion of promoter and / or 5’ UTR region of antisense RNA to the Al AT gene using RNA interference in the subject.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 illustrates the A1AT genomic locus in humans. Al AT is shown, with the antisense A1AT IncRNA (ENSG00000289541) and those siRNAs found to efficiently target the asAl AT promoter. The target susceptible region in the asAlAT promoter is also shown.

[0017] Figure 2 illustrates the siRNA mediated activation of Al AT in Huh7 cells. Duplicate screen of siRNAs in Huh7 cells was carried out with siRNAs targeted to repress ENSG00000289541. The averages of transfected cells (lOOnM siRNA with Lipofectamine 2000 (L2K)) are shown from 48hrs post-transfection with the standard error of the mean and a p value from a paired T-test.

[0018] Figure 3 illustrates the siRNA mediated activation of A1AT in HepG2 cells. Quadruplicate screen of siRNAs in HepG2 cells. The averages of Al AT expression in the HepG2 cells (100 nM, L2K transfected) are shown from 48hrs post-transfection with the standard error of the mean and p values from a paired T-test.

[0019] Figure 4 illustrates the effects of TSA treatment on siRNA activation of A1AT. HEPG2 cells transfected with siRNAs (lOOnM, L2K) were treated with TSA (0.05pM on day 1) and assessed for Al AT expression 48hrs later. The averages of triplicate treated cells are shown with the standard error of the means.

[0020] Figure 5A-C illustrate the vector maps of the various plasmids used for the exosome delivery of shRNAs targeting asAl AT of the present invention. Figure 5 A illustrates the vector map of plasmid pLV_shGFP_B 220606- 1427pzm which expresses the control shGFP, Ago-2 and Cnx43 allowing for exosome delivery of the shRNAs. Figure 5B illustrates the vector map of plasmid pLV_shAl AT_P1 _VB 221027-1180pww which expresses shPl, Ago-2 and Cnx43 allowing for exosome delivery of the shRNAs. Figure 5C illustrates the vector map of plasmid pLV_asAlAt_shP4_VB221027-1182fwc which expresses shP4, Ago-2 and Cnx43 allowing for exosome delivery of the shRNAs.

[0021] Figure 6 illustrates the EV transfer of shRNAs targeting asAl AT to the liver cells. HEK producer cells were transfected with Ago-2, connexin 43 over-expressing and shRNA expressing plasmids and co-cultured with HepG2 liver cells for 48hrs. The cultures, both producer HEK293 and recipient HepG2 cells, were then assessed for changes in Al AT expression relative to beta actin. The averages of triplicate treated cultures are shown with the standard deviations and p values from a paired T-test.

[0022] Figure 7 illustrates the effects of EV transfer of shRNAs targeting asAl AT to the liver cells on antisense Al AT expression. HEK producer cells were transfected with Ago- 2, connexin 43 over-expressing and shRNA expressing plasmids and co-cultured with HepG2 liver cells for 48hrs. The cultures, both producer HEK293 and recipient HepG2 cells, were then assessed for changes in asAlAT expression relative to beta actin.

[0023] Figure 8 illustrates the vector map of dual shRNA 7bp hairpin loop vectors. The vector pLV[Exp]-Puro-Rev(hU6>asAlAt-Pl_7bp Loop)-Hl>asAlATP4_7bp was generated to express both shRNA asAlAt-Pl and shRNA asAl ATP4, found previously to induce the activation of Al AT expression.

[0024] Figure 9 illustrates the A1AT activation by 7bp-dual shRNAs. HEK293 cells were transfected with shRNA expressing plasmids (lOOng shRNA plasmid, L3K) and assessed for A1AT expression 72hrs later. The averages are shown with standard deviations and p value from a paired T-test.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] 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.

[0027] 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.

[0028] 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 to form 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.

[0029] 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 producetertiary 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.

[0030] 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.|00031 | 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.

[0032] 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.

[0033] 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.

[0034] 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 RNAs that regulate 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 RNAs that regulate the polynucleotide being targeted. In an embodiment, such destruction or breakup is 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.

[0035] The present invention provides one or more alpha-1 antitrypsin (A1AT) upregulating polynucleotides capable of upregulating expression of alpha- 1 antitrypsin (Al AT) and a pharmaceutical composition thereof. In an embodiment, the one or more A1AT upregulating polynucleotides of the present invention increase or de-repress the expression level of Al AT by suppressing the expression level of one or more antisense RNA regulators of Al AT. In an embodiment, the antisense RNA regulators of Al AT is antisense to the Al AT promoter. In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention targets a portion of the promoter, 5’UTR, or a combination thereof of the one or more antisense RNA regulators of Al AT to suppress the expression level of said one or more antisense RNA regulators of Al AT. In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention targets about 10to about 40 nucleotides (nt) in length such as about 10, about 12, about 14, about 16, about 18, about 20, about 22, about 24, about 26, about 28, about 30, about 32, about 34, about 36, about 38 or about 40 nt including any lengths or length ranges falling within these values of a portion of the promoter, 5’UTR, or a combination thereof of the one or more antisense RNA regulators of Al AT to suppress the expression level of said one or more antisense RNA regulators of Al AT. In an embodiment, the nucleotide sequence of the promoter, 5’UTR, or a combination thereof of the one or more antisense RNA regulators of Al AT targeted by the Al AT upregulating polynucleotides of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 1.

[0036] SEQ ID NO. 1 (ENST00000693506.1 promoter and 5’ UTR):ACCCAGGAAGTAGACTTCGGGTGGAGGCAGTAGGCTGGGGAGGGGCGGGGAGC TTGGACAGGAAGGAGCCTTGCTCATTGCCCGGCAGACACAAGACTGGGCCCTCA TAAACTCAGACAGCCCGGCATGTCACCTGTTGTACCTGCCCTTTCAGCTCTGTGA CCCGGGACAAGTCACCCTCTCCCTTTGAGTTGCCGCAAGA

[0037] In an embodiment, the one or more antisense RNA regulators of A1AT is transcribed from a genomic region located within about chrl4:94,390,771 to 94,420,211 (GRCh38 / hg38). In an embodiment, the one or more antisense RNA regulators of Al AT is transcribed from a genomic region comprising a nucleotide sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of ENSG00000289541 deposited in the Ensembl database (https: / / asia.ensembl.org / index.html). In an embodiment, the nucleotide sequence of the one or more antisense RNA regulators of A1AT is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2) deposited in the Ensembl database (https: / / asia.ensembl.org / index.html).

[0038] SEQ ID NO. 2 (ENST00000693506. 1):

[0039] GCCCGGCAGACACAAGACTGGGCCCTCATAAACTCAGACAGCCCGGCATGTCACCTGTTGTACCTGCCCTTTCAGCTCTGTGACCCGGGACAAGTCACCC TCTCCCTTTGAGTTGCCGCAAGAGGCTCATAGGTGGAAGGGACTTGCCTTGTCTC AGATGAAACATTGGACTTGGACTTTTGGATTAATGCTGGAATGAGTTAAGACTTT GGGGGACTGTTGGAAGAGCATGATCGTGTTTTGAAATGTGACGACGTGAAATTT GGAAGGGGCCAGGGGCAGAAATATATGGTTTGGCCCTGTGTCTCCACCCAAATC TCACTTTGAATTGTAATAATCCCCACATGTCCAGGGCAGGGTCAGGCAGAGTTAA TGGAATCATGAGGGTGGTTTCCCCCATGCTGTTCTTGTGGTAGTGAATAAGTCTC ATGAGAGCTGATGGTTTTATAAATTGGAGTCCCCTTGCACAAGCTCTCTTGCTTGCCACCATGTAAGATGTGACTTTGCTCCTCATTCTCCATCCACCATGATTGGGAGG CCTCCCCAGCCTTGTGGAACTGTGAGTCCATTAAACCTCTTTCCTGTATAAATTA

[0040] In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention is about 10 to about 100 nucleotides (nt) in length such as about 10, about 20, about 30, about 40 about 50 about 60 about 70, about 80, about 90, or about 100 nt in length including any lengths or length ranges falling within these values. In an embodiment, the Al AT upregulating 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 Al AT upregulating polynucleotide of the present invention is double stranded or single stranded. In an embodiment, the A1AT upregulating polynucleotide of the present invention is blunt ended or comprise overhanging ends. In an embodiment, the A1AT upregulating polynucleotide of the present invention is chemically synthesized or recombinantly produced. In an embodiment, one strand of the A1AT upregulating polynucleotide of the present invention comprises nucleotide sequence having sufficient complementarity to the one or more antisense RNA regulators of Al AT for the polynucleotide to direct cleavage of the antisense RNA regulator of Al AT via RNA interference.

[0041] In an embodiment, the one or more Al AT upregulating polynucleotides 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. In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention comprise siRNA. In an embodiment, the siRNAs 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. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. In an embodiment, the siRNAs of the present invention comprise a 3’ deoxythymidine dinucleotide (dTdT) overhang to increase nuclease resistance.

[0042] In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention comprise one or more of a first siRNA and one or more of a second siRNA wherein the nucleotide sequence of the one or more first siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6 and the nucleotide sequence of the one or more of a second siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6, wherein the nucleotide sequence of the one or more of the secondsiRNA is distinct from the nucleotide sequence of the one or more of the first siRNA or wherein the nucleotide sequence of the one or more of the second siRNA is distinct from the nucleotide sequence of the one or more of the first siRNA by more than 60%.

[0043] In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention comprise one or more of a first siRNA and one or more of a second siRNA wherein the nucleotide sequence of the one or more of first siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 3 and the nucleotide sequence of the one or more of second siRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 6.

[0044] SEQ ID NO. 3 (asAlAt_Pl): GUAGGCUGGGGAGGGGCGGG

[0045] SEQ ID NO. 4 (asAlAt_P2): UUGGACAGGAAGGAGCCUUG

[0046] SEQ ID NO. 5 (asA!At_P3): ACCCAGGAAGUAGACUUCGGG

[0047] SEQ ID NO. 6 (asAlAt_P4): CGGGGAGCUUGGACAGGAAGG

[0048] In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention comprise one or more shRNA. In an embodiment, each of the one or more 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, 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 SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. 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 shRNA 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. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, or SEQ ID NO. 11. In an embodiment, the loop may be cleaved off in RNA interference pathway.

[0049] SEQ ID NO. 7: UUGC

[0050] SEQ ID NO. 8: CCUGACCCA

[0051] SEQ ID NO. 9: AAGCAC A

[0052] SEQ ID NO. 10: UGUGCUU

[0053] SEQ ID NO. 11 : UUG

[0054] In an embodiment, the one or more Al AT upregulating polynucleotides of the present invention comprise one or more of first shRNA and one or more of second shRNA wherein each of the one or more of first or one or more of second shRNA comprises a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence is reverse complementary to the first nucleotide sequence and wherein the first nucleotide sequence of the one or more of first shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 3 and the first nucleotide sequence of the one or more of second shRNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 6.

[0055] In an embodiment, any embodiment of the Al AT upregulating polynucleotides of the present invention further comprises N-acetylgalactosamine (GalNAc) wherein the one or more Al AT upregulating polynucleotides of the present invention is conjugated to the GalNAc. In an embodiment, any embodiment of the siRNA of the present invention further comprises GalNAc wherein the one or more siRNA of the present invention is conjugated to the GalNAc. In an embodiment, any embodiments of the shRNA of the present invention further comprises GalNAc wherein the one or more shRNA of the present invention is conjugated to the GalNAc.

[0056] The present invention also provides an Al AT upregulating pharmaceutical composition comprising a therapeutically effective amount of any embodiment of the one or more Al AT upregulating polynucleotides of the present invention. In an embodiment, the Al AT upregulating pharmaceutical composition of the present invention upregulates the expression of the Al AT gene of a subject 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%. In an embodiment, the Al AT upregulating pharmaceutical composition of the present invention restores the expression of the Al AT gene of a subject to 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% or about 100% of the normal Al AT expression level of the subject when healthy. In an embodiment, the Al AT upregulating pharmaceutical composition of the present invention restores the expression of the A1AT gene of a subject to 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%, about90% or about 100% of normal Al AT expression level of an average healthy human being. In an embodiment, the Al AT upregulating pharmaceutical composition of the present invention restores the expression of the Al AT gene of a subject to 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% or about 100% of normal Al AT expression level of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.

[0057] The present invention also provides a Al AT upregulating pharmaceutical composition comprising a nanoparticle encapsulating a therapeutically effective amount of any embodiment of the one or more Al AT upregulating polynucleotides 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, lipidpolymer hybrid nanoparticle and biological nanoparticles such as but not limited to liposome, exosome, virus, virus-like particle.

[0058] In an embodiment, the nanoparticle of the present invention 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 telomerase upregulating polynucleotide of the present invention. 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 an RNA-binding protein capable of binding to the packaging domain such that the cargo RNA binds to the fusion protein via the RNA-binding protein and the packaging domain.

[0059] In an embodiment, the nanoparticle of the present invention comprises an exosome prepared from an exosome-based RNA packaging and delivery system. In an embodiment, the system comprises a low immunogenic exosome-based RNA packaging and delivery system. In an embodiment, the exosome-based RNA package and delivery system of the present invention 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 Al AT upregulating polynucleotides of the present invention and a packaging domain capable of binding to the packaging protein of thefusion protein encoded by the one or more fusion protein plasmids. 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 capable of binding to the packing domain. 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 their entireties. In an embodiment, the packaging protein comprises Ula protein.

[0060] In an embodiment the one or more fusion proteins comprise CD63-Ula protein. In an embodiment, 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. 12. In an embodiment, the amino acid sequence of the CD63-Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 13. 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. 14. 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. 15. In an embodiment, the one or more fusion proteins comprise PTGFRN-U la protein. In an embodiment, nucleotide sequence of the PTGFRN-U la 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. 16. In an embodiment, the amino the 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. 17.

[0061] SEQ ID NO. 12 (nt CD63-Ula):ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGC TGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCT TGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTG GTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGGGGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTA TCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGT GATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAA CAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGG GCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTC CCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGA AGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAA AATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGG GAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGATGgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTA TATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCT GTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGG AGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGC GCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTA TGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAA |00062| Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0063] SEQ ID NO. 13 (aa CD63-Ula):MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEF NNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCI NVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQIL DILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAK MK

[0064] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0065] SEQ ID NO. 14 (nt CD81-Ula):

[0066] ATGTCCGGACTCAGATCTCGAGCTCAAGCTTCCGGAGTGGAGGGCTGCACCAAGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCT GGAGGCGTGATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTGTATCTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAG GCATCTACATCCTCATCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGC TGCTACGGGGCCATCCAGGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCCTGGTCATCCTGTTTGCCTGTGAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGATCGCCAAGGATGTGAAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGATGATGACGCCAACAACGCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGACTGCTGTGGCTCCAGCACACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAATTTGTGTCCCTCGGGCAGCAACATCATCAGCAACCTCTTCAAGGAGGACTGCCACCAGAAGATCGATGACCTCTTCTCCGGGAAGCTGTACCTCATCGGCATTGCTGCCATCGTGGTCGCTGTGATCATGATCTTCGAGATGATCCTGAGCATGGTGCTGTGCTGTGGCATCCGGAACAGCTCCGTGTACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAATAA

[0067] Uppercase sequence denotes CD81, lower case is linker sequence, and bold sequence is the 1 small nuclear ribonucleoprotein polypeptide A (U 1 snRNP A).

[0068] SEQ ID NO. 15 (aa CD81-Ula):

[0069] MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTALTTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAIVVAVIMIFEMILSMVLCCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0070] SEQ ID NO. 16 (nt PTGFRN-Ula):

[0071] ATGGGGCGCCTGGCCTCCAGGCCGCTGCTGCTGGCGCTCCTGTCGTTGGCTCTTTGCCGAGGGCGTGTGGTGAGAGTCCCCACAGCGACCCTGGTTCGAGTGGTGGGCACTGAGCTGGTCATCCCCTGCAACGTCAGTGACTATGATGGCCCCAGCGAGCAAAACTTTGACTGGAGCTTCTCATCTTTGGGGAGCAGCTTTGTGGAGCTTGCAAGCACCTGGGAGGTGGGGTTCCCAGCCCAGCTGTACCAGGAGCGGCTGCAGAGGGGCGAGATCCTGTTAAGGCGGACTGCCAACGACGCCGTGGAGCTCCACATAAAGAACGTCCAGCCTTCAGACCAAGGCCACTACAAATGTTCAACCCCCAGCACAGATGCCACTGTCCAGGGAAACTATGAGGACACAGTGCAGGTTAAAGTGCTGGCCGACTCCCTGCACGTGGGCCCCAGCGCGCGGCCCCCGCCGAGCCTGAGCCTGCGGGAGGGGGAGCCCTTCGAGCTGCGCTGCACCGCCGCCTCCGCCTCGCCGCTGCACACGCACCTGGCGCTGCTGTGGGAGGTGCACCGCGGCCCGGCCAGGCGGAGCGTCCTCGCCCTGACCCACGAGGGCAGGTTCCACCCGGGCCTGGGGTACGAGCAGCGCTACCACAGTGGGGACGTGCGCCTCGACACCGTGGGCAGCGACGCCTACCGCCTCTCAGTGTCCCGGGCTCTGTCTGCCGACCAGGGCTCCTACAGGTGTATCGTCAGCGAGTGGATCGCCGAGCAGGGCAACTGGCAGGAAATCCAAGAAAAGGCCGTGGAAGTTGCCACCGTGGTGATCCAGCCATCAGTTCTGCGAGCAGCTGTGCCCAAGAATGTGTCTGTGGCTGAAGGAAAGGAACTGGACCTGACCTGTAACATCACAACAGACCGAGCCGATGACGTCCGGCCCGAGGTGACGTGGTCCTTCAGCAGGATGCCTGACAGCACCCTACCTGGCTCCCGCGTGTTGGCGCGGCTTGACCGTGATTCCCTGGTGCACAGCTCGCCTCATGTTGCTTTGAGTCATGTGGATGCACGCTCCTACCATTTACTGGTTCGGGATGTTAGCAAAGAAAACTCTGGCTACTATTACTGCCACGTGTCCCTGTGGGCACCCGGACACAACAGGAGCTGGCACAAAGTGGCAGAGGCCGTGTCTTCCCCAGCTGGTGTGGGTGTGACCTGGCTAGAACCAGACTACCAGGTGTACCTGAATGCTTCCAAGGTCCCCGGGTTTGCGGATGACCCCACAGAGCTGGCATGCCGGGTGGTGGACACGAAGAGTGGGGAGGCGAATGTCCGATTCACGGTTTCGTGGTACTACAGGATGAACCGGCGCAGCGACAATGTGGTGACCAGCGAGCTGCTTGCAGTCATGGACGGGGACTGGACGCTAAAATATGGAGAGAGGAGCAAGCAGCGGGCCCAGGATGGAGACTTTATTTTTTCTAAGGAACATACAGACACGTTCAATTTCCGGATCCAAAGGACTACAGAGGAAGACAGAGGCAATTATTACTGTGTTGTGTCTGCCTGGACCAAACAGCGGAACAACAGCTGGGTGAAAAGCAAGGATGTCTTCTCCAAGCCTGTTAACATATTTTGGGCATTAGAAGATTCCGTGCTTGTGGTGAAGGCGAGGCAGCCAAAGCCTTTCTTTGCTGCCGGAAATACATTTGAGATGACTTGCAAAGTATCTTCCAAGAATATTAAGTCGCCACGCTACTCTGTTCTCATCATGGCTGAGAAGCCTGTCGGCGACCTCTCCAGTCCCAATGAAACGAAGTACATCATCTCTCTGGACCAGGATTCTGTGGTGAAGCTGGAGAATTGGACAGATGCATCACGGGTGGATGGCGTTGTTTTAGAAAAAGTGCAGGAGGATGAGTTCCGCTATCGAATGTACCAGACTCAGGTCTCAGACGCAGGGCTGTACCGCTGCATGGTGACAGCCTGGTCTCCTGTCAGGGGCAGCCTTTGGCGAGAAGCAGCAACCAGTCTCTCCAATCCTATTGAGATAGACTTCCAAACCTCAGGTCCTATATTTAATGCTTCTGTGCATTCAGACACACCATCAGTAATTCGGGGAGATCTGATCAAATTGTTCTGTATCATCACTGTCGAGGGAGCAGCACTGGATCCAGATGACATGGCCTTTGATGTGTCCTGGTTTGCGGTGCACTCTTTTGGCCTGGACAAGGCTCCTGTGCTCCTGTCTTCCCTGGATCGGAAGGGCATCGTGACCACCTCCCGGAGGGACTGGAAGAGCGACCTCAGCCTGGAGCGCGTGAGTGTGCTGGAATTCTTGCTGCAAGTGCATGGCTCCGAGGACCAGGACTTTGGCAACTACTACTGTTCCGTGACTCCATGGGTGAAGTCACCAACAGGTTCCTGGCAGAAGGAGGCAGAGATCCACTCCAAGCCCGTTTTTATAACTGTGAAGATGGATGTGCTGAACGCCTTCAAGTATCCCTTGCTGATCGGCGTCGGTCTGTCCACGGTCATCGGGCTCCTGTCCTGTCTCATCGGGTACTGCAGCTCCCACTGGTGTTGTAAGAAGGAGGTTCAGGAGACACGGCGCGAGCGCCGCAGGCTCATGTCGATGGAGATGGACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAATAG

[0072] Uppercase sequence denotes PTGFRN, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0073] SEQ ID NO. 17 (aa PRGFRN-Ula):|00074| MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVS WYYRMNRRSDN VVTSELLAVMDGD WTLKYGERS KQRAQDGDFIFS KEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0075] In an embodiment, the cargo RNA comprises a package RNA comprising any embodiment of the one or more Al AT upregulating polynucleotides of the present invention and a packaging 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. 18 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. 19.

[0076] 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. 20 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. 21.

[0077] SEQ ID NO. 18 (UR): AATCCATTGCACTCCGGATT1000781 SEQ ID NO. 19 (L2): AATCCATTGCACTCCGGATTT

[0079] SEQ ID NO. 20 (OH): CTGCAGATATCCAGCACAGTGGC

[0080] SEQ ID NO. 21 (MorrisMotif): GCGCAGCGCGCGCAGCGC

[0081] 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. 22.

[0082] SEQ ID NO. 22 (SIRLOIN):CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA

[0083] 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.

[0084] In an embodiment, the pharmaceutical composition comprising a nanoparticle encapsulating any embodiment of the one or more Al AT upregulating polynucleotides 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 leastabout 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 an embodiment, 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. 23. 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. 24. 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. 25.

[0085] SEQ ID NO. 23 (C2F):QFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRDHYIPNTLN PVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEE YCVSGV1000861 SEQ ID NO. 24 (C2G):PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKTDV HYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDND KFSLDDYLGFLELDLRH

[0087] SEQ ID NO. 25 (transmembrane domain): PDLKAMNPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEA DERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLI LLLFVAV

[0088] 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. 26. 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. 27. 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. 28. 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. 29. 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. 30. 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. 25.

[0089] SEQ ID NO. 26 (C2A): MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRGIPL DFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATID LVIGYDPPSAPHPNDLS

[0090] SEQ ID NO. 27 (FerA):LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIRK

[0091] SEQ ID NO. 28 (FerB):

[0092] WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCGKTQTIFLKYPQEKNNGP

[0093] SEQ ID NO. 29 (DysFN-1):AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKREFF LP

[0094] SEQ ID NO. 30 (DysFN-2):DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSELTC P

[0095] 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. 31. 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. 32. 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. 33. 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. 34.

[0096] SEQ ID NO. 31 (C2B) :PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVNMT PSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP

[0097] SEQ ID NO. 32 (C2C):TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIEKN ANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVED FSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE

[0098] SEQ ID NO. 33 (C2D):TPIVSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTEIIHSTLNP TWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEM DITPKLLWHPVMNGDKA

[0099] SEQ ID NO. 34 (C2E):RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEELYM PPLVIKVIDHRQFGRKPVVG[000100] 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. 26. 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 ID NO. 31. 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 ID NO. 32. 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. 33. 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. 34. 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. 23. 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. 24. 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 isat least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 27. In an embodiment, the modified myoferlin protein of the present invention does 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. 28. 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. 29. 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. 30. 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. 25.[000101] 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. 35.[000102] SEQ ID NO. 35 (C2F-C2G):MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRD HYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFG SHCGIPEEYCVSGVNTWRDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLD EFEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVD VFPKSLGPPGPPFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPG NEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIP PRLIIQIWDNDKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKAKTAS LFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMN PKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLILLLFVAVLLYSLPNYL SMKIVKPNVYPYDVPDYA[000103] 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 C2Adomain 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. 36. 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. 37. In an embodiment, the C2C 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. 38. 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. 39. 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. 40. 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. 41. 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. 42. 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. 43. 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. 44. 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. 45. 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. 46. 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. 47.[000104] SEQ ID NO. 36 (nt C2A): ATGCTGCGAGTGATTGTGGAATCTGCCAGCAATATCCCTAAAACGAAATTTGGCA AGCCGGATCCTATTGTTTCTGTCATTTTTAAGGATGAGAAAAAGAAAACAAAGA AAGTTGATAATGAATTGAACCCTGTCTGGAATGAGATTTTGGAGTTTGACTTGAGGGGTATACCACTGGACTTTTCATCTTCCCTTGGGATTATTGTGAAAGATTTTGAGACAATTGGACAAAATAAATTAATTGGCACGGCGACTGTAGCCCTGAAGGACCTGACTGGTGACCAGAGCAGATCCCTGCCGTACAAGCTGATCTCCCTGCTAAATGAAAGAGGGCAAGATACTGGGGCCACCATTGACTTGGTGATCGGCTATGATCCGCCTTCTGCTCCACATCCAAATGACCTGAGC[000105] SEQ ID NO. 37 (nt C2B):CCACAGGACTTCCAGATCCGCGTCCGAGTGATTGAGGGCCGACAGTTAAGTGGCAACAACATAAGGCCTGTGGTCAAAGTTCACGTCTGTGGCCAGACACACCGAACAAGAATCAAGAGAGGAAACAACCCTTTTTTTGATGAGTTGTTTTTCTACAATGTCAACATGACCCCTTCTGAATTGATGGATGAGATCATCAGCATCCGGGTTTATAATTCTCACTCTCTGCGGGCAGATTGTCTGATGGGGGAATTTAAGATTGATGTTGGATTTGTTTATGATGAACCTGGCCATGCTGTCATGAGAAAGTGGCTTCTTCTCAATGACC CG[000106] SEQ ID NO. 38 (nt C2C):ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGCCTTCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCGTGGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAAAAAGGTTTGTACAAACATAATTGAGAAGAATGCAAACCCAGAGTGGAATCAGGTCGTCAATCTTCAGATCAAGTTTCCTTCAGTGTGTGAAAAAATAAAACTAACAATATATGACTGGGACCGTCTTACTAAAAATGATGTAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTGGTGGGGAAGTGGAAGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGTAAACACAGGAGAAACAGAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTGAATCTTTATGGAAGCCCCAGAGAGTACACGGGATTCCCAGACCCCTATGATGAG[000107] SEQ ID NO. 39 (nt C2D):ACCCCCATTGTTTCCTGCAATTTTGACAGAGTCTACATCTACCATCTGCGCTGCTATGTCTATCAAGCCAGAAACCTCTTGGCTTTAGATAAGGATAGCTTTTCAGATCCATATGCTCATATCTGTTTCCTCCATCGGAGCAAAACCACTGAGATCATCCATTCAACCCTGAATCCCACGTGGGACCAAACAATTATATTCGATGAAGTTGAAATCTATGGGGAACCCCAAACAGTTCTACAGAATCCACCCAAAGTTATCATGGAACTTTTTGACAATGACCAAGTGGGCAAAGATGAATTTTTAGGACGAAGCATTTTCTCTCCTGTGGTGAAACTGAACTCAGAAATGGACATCACACCCAAACTTCTCTGGCACCCAGTAATGAATGGAGACAAAGCC[000108] SEQ ID NO. 40 (nt C2E):AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTGGAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACA CCCAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTACATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAA GCCTGTCGTCGGC[000109] SEQ ID NO. 41 (nt C2F):CAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGG GTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTC[000110] SEQ ID NO. 42 (nt C2G):CCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACT TGACTTGCGTCAC[000111] SEQ ID NO. 43 (nt FerA):CTGCAAACAAATATAGAAGCTCTAAAATCAGGGATACAAGGTAAAATTCCTGCAAACCAGCTGGCTGAATTGTGGCTGAAGCTGATAGATGAAGTTATAGAAGACACGAGATACACGTTGCCTCTCACAGAAGGAAAAGCCAACGTCACAGTTCTCGATACTC AGATCCGAAAG[000112] SEQ ID NO. 44 (nt FerB):TGGCTTGATAAATTAATGCAGCTGACTGAAGAGCCACAGAACAGCATGCCTGACATCATCATCTGGATGATCCGGGGAGAGAAGAGACTGGCCTATGCACGAATTCCC GCACATCAGGTCTTGTACTCCACCAGTGGTGAGAATGCATCTGGAAAATACTGTGGGAAAACCCAAACCATCTTTCTGAAGTATCCACAGGAGAAAAACAACGGGCCA [000113] SEQ ID NO. 45 (nt DysFN-1):GCTGTGGAGAAGAAGTTTAACAGCTTCGCAGAAGGAACTTTCACCGTCTTTGCTGAAATGTATGAAAATCAAGCTCTCATGTTTGGAAAATGGGGTACTTCTGGATTAGTAGGACGTCATAAGTTTTCTGATGTCACAGGAAAAATAAAACTCAAGAGGGAATTTTTTCTGCCT[000114] SEQ ID NO. 46 (nt DysFN-2): GATCCTGAAAGAAGCTTGCTGACTGAGGCAGATGCAGGTCACACGGAGTTCACT GATGAAGTCTACCAGAACGAGAGCCGCTACCCCGGGGGCGACTGGAAGCCGGCC GAGGACACCTACACGGATGCGAACGGCGATAAAGCAGCATCACCCAGCGAGTTG ACTTGTCCT[000115] SEQ ID NO. 47 (nt TM):CCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGC AGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCG TAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCG ACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTG GACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGA CCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTT CCTGCTTATCCTGCTGCTCTTCGTGGCCGTG1000116] In an embodiment, the modified myoferlin protein of the present invention further comprises a connexin 43 protein, wherein said dual protein comprising the modified myoferlin protein and the connexin 43 protein 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, amino acid sequence of the connexin 43 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. 48.[000117] SEQ ID NO. 48 (connexin 43):MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSA FRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEEKLNK KEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFE VAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNII ELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSP PGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFP DDNQNSKKLAAGHELQPLAIVDQRPSSRAASRASSRPRPDDLEI[000118] The present invention further provides a dual protein 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 dual proteinpolynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 49.[000119] SEQ ID NO. 49:ATGGTGCCAGCCCCTCCCAGACAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTCAATACCTGGCGAGATCAACTGAGACCAACACAGCTGCTTCAAAATGTCGCCAGATTCAAAGGCTTCCCACAACCCATCCTTTCCGAAGATGGGAGTAGAATCAGATATGGAGGACGAGACTACAGCTTGGATGAATTTGAAGCCAACAAAATCCTGCACCAGCACCTCGGGGCCCCTGAAGAGCGGCTTGCTCTTCACATCCTCAGGACTCAGGGGCTGGTCCCTGAGCACGTGGAAACAAGGACTTTGCACAGCACCTTCCAGCCCAACATTTCCCAGGGAAAACTTCAGATGTGGGTGGATGTTTTCCCCAAGAGTTTGGGGCCACCAGGCCCTCCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCACACGATCATTCCTGCAAAATCACCAGAGAAATGCAGGTTGGACATGATTCCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGCAGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCGTAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCGACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTGGACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGACCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTTCCTGCTTATCCTGCTGCTCTTCGTGGCCGTGCTCCTCTACTCTTTGCCGAACTATTTGTCAATGAAGATTGTAAAGCCAAATGTGTACCCATACGACGTCCCAGACTACGCTTAGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTG TCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGG TCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACA ACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGC CTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCC CAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAA GCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGAT CTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAA ACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGA TAATATGGCCACAACCATGGGTGACTGGAGCGCCTTAGGCAAACTCCTTGACAA GGTTCAAGCCTACTCAACTGCTGGAGGGAAGGTGTGGCTGTCAGTACTTTTCATT TTCCGAATCCTGCTGCTGGGGACAGCGGTTGAGTCAGCCTGGGGAGATGAGCAG TCTGCCTTTCGTTGTAACACTCAGCAACCTGGTTGTGAAAATGTCTGCTATGACA AGTCTTTCCCAATCTCTCATGTGCGCTTCTGGGTCCTGCAGATCATATTTGTGTCT GTACCCACACTCTTGTACCTGGCTCATGTGTTCTATGTGATGCGAAAGGAAGAGA AACTGAACAAGAAAGAGGAAGAACTCAAGGTTGCCCAAACTGATGGTGTCAATG TGGACATGCACTTGAAGCAGATTGAGATAAAGAAGTTCAAGTACGGTATTGAAG AGCATGGTAAGGTGAAAATGCGAGGGGGGTTGCTGCGAACCTACATCATCAGTA TCCTCTTCAAGTCTATCTTTGAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTAT GGATTCAGCTTGAGTGCTGTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCTCTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGCTG GTGGTGTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTT CAAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGACCAGTGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCTTATTTCAAT GGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCTCCTGGGTACAAGCT GGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAATTACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAACAAAATCGAATGGGGCAGGCGGG AAGCACCATCTCTAACTCCCATGCACAGCCTTTTGATTTCCCCGATGATAACCAG AATTCAAAAAAACTAGCTGCTGGACATGAATTACAGCCACTAGCCATTGTGGACCAGCGACCTTCAAGCAGAGCCGCCAGTCGTGCCAGCAGCAGACCTCGGCCTGAT GACCTGGAGATCTGA[000120] 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 presentinvention, 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides 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 Al AT upregulating polynucleotides targeting are 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.[000121] In an embodiment, any embodiment of the nanoparticle encapsulating the one or more Al AT upregulating polynucleotides of the present invention upregulates the expression of the Al AT gene of a subject by at least about 1%, about 2%, about 3%, about4%, 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%. In an embodiment, any embodiment of the nanoparticle encapsulating the one or more Al AT upregulating polynucloetides of the present invention restores the expression of the Al AT gene of a subject to 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% or about 100% of the normal Al AT expression level of the subject when healthy. In an embodiment, any embodiment of the nanoparticle encapsulating the one or more Al AT upregulating polynucleotides of the present invention restores the expression of the Al AT gene of a subject to 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% or about 100% of normal Al AT expression level of an average healthy human being. In an embodiment, any embodiment of the nanoparticle encapsulating the one or more Al AT upregulating polynucleotides of the present invention restores the expression of the Al AT gene of a subject to 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% or about 100% of normal Al AT expression level of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof. [000122] The present invention provides a method of upregulating expression of Al AT in a subject comprising the administration of a therapeutically effective amount of any embodiment of the one or more Al AT upregulating polynucleotides of the present invention to the subject. In an embodiment, the subject is diagnosed with AlAT-associated disease. In an embodiment, the AlAT-associated disease comprises AlAT-deficiency-associated lung diseases or AlAT-deficiency-associated liver disease. In an embodiment, the AlAT- associated disease comprises chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, or panniculitis. [000123] The present invention also provides a method of treatment of a AlAT- associated disease of a subject comprising the step of altering the Al AT expression of the subject. In an embodiment, the step of altering the A1AT expression comprises the administration of a therapeutically effective amount of any embodiment of the one or more A1AT upregulating polynucleotides of the present invention to the subject. In anembodiment, the step of altering Al AT expression comprises the administration of any embodiment of the nanoparticle encapsulating one or more Al AT upregulating polynucleotides of the present invention to the subject.[000124] The present invention also provides a method of treatment of a Al AT- associated disease of a subject comprising the step of targeting the promoter portion, the 5’UTR portion, or a combination thereof of the antisense regulator of Al AT gene. In an embodiment, the step of targeting the promoter portion, the 5’UTR portion, or a combination thereof of the antisense regulator of A 1 AT gene is performed by administration of any embodiment of the Al AT upregulating polynucleotides or any embodiment of the nanoparticle comprising the Al AT upregulating polynucleotides of the present invention to the subject.[000125] In an embodiment, the AlAT-associated disease comprises AlAT-deficiency- associated lung diseases or Al AT-deficiency-associated liver disease. In an embodiment, the AlAT-associated disease comprises chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, panniculitis, or liver failure.[000126] In an embodiment, the method of treatment of a AlAT-associated disease comprises the step of administering a therapeutic effective amount of any embodiment of the pharmaceutical composition comprising one or more Al AT upregulating polynucleotides of the present invention to a subject. In an embodiment, the method of treatment of a AlAT- associated disease 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 Al AT upregulating polynucleotides of the present invention to the subject. In an embodiment, the method of treating a AlAT-associated disease comprises the step of administering about 1 to about 100 billion of any embodiment of the exosomes, LNP, or a combination thereof encapsulating the one or more Al AT upregulating 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 1000000000, or any numbers or ranges of numbers falling within these values of exosomes, LNP, or a combination thereof encapsulating the one or more Al AT upregulating polynucleotides of the present invention.In an embodiment, the method of treatment of a AlAT-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 Al AT upregulating 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. 1000127] 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 late endosome 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. [000128] 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.[000129] 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. [000130] 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. [ 000131 ] 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 pollll promoter. In embodiments, the promoter is 116 or Hl promoter.[000132] 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.[000133] 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. [000134] 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 administeredto 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 0.05 mg / kg body weight per day, 0.01 to 0.02 mg / kg body weight per day, and 0.005 to 0.01 mg / kg body weight per day.[000135] 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.[000136] 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.[000137] 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. [000138] 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.[000139] 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.[000140] 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. [000141 ] 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.[000142] 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. [000143] EXAMPLES[000144] Materials and methods[000145] Small interfering RNAs (siRNAs) were ordered from SynGenis (Perth Australia) and screened on human cells, HEK293, HepG2, Hep3G cells. The siRNAs weretransfected into cells in 12 well plates at lOOnM final concentration using Lipofectamine 2000 (L2K). Cell RNAs were collected at 48 or 72 hrs post-transfection using Qiagen RNAeasy kit with DNAse treatment. The resultant RNAs were converted to cDNA using Thermo fisher Reverse Transcription kit. The cDNAs were then assessed for differential gene expression relative to beta actin using the primers from Table 1 by quantitative PCR. [000146] Table 1 - PCT primers used for RNA detection000147] Results[000148] Example 1 - Al AT- activating siRNAs[000149] Long non-coding RNAs (IncRNAs) are a form of RNA that is important in regulating gene transcription as well as to be involved in a myriad of other cellular processes6. We, and others, have learned that by repressing those IncRNAs that are repressive regulators of protein-coding genes with RNAi, that it is possible to activate gene expression. This form of siRNA mediated gene activation is the result of “repressing the repressor”7 8.Several genes are reported to be regulated by the action of IncRNAs and we have discovered that one IncRNA (ENSG00000289541, Figure 1), which is antisense to the Al AT promoter, acts to repress Al AT expression. When we target the promoter of this IncRNA with siRNAs (Table 2), we find a significant increase 2-4 fold expression of Al AT in Huh7 (Figure 2) and HepG2 (Figure 3) liver cells. Both siAl AT-P1 and siAlAT-P4, siRNAs are targeted to induce TGS of the ENSG00000289541 promoter (Figure 1). To confirm that the observedactivation of Al AT from treatment of the cells with siAlAT-Pl and siAlAT-P4 siRNAs is the result of epigenetic modes of regulation, we assessed the ability to activate Al AT expression in the presence of Trichostatin A (TSA), a known inhibitor of siRNA directed TGS. We find that TSA represses the observed activation of Al AT (Figure 4), suggesting that both siAlAT-Pl and siAlAT-P4 siRNAs operate in a TGS manner that utilizes epigenetic process which can result in long-term stable activation of Al AT. Collectively we describe here two siRNAs that can activate Al AT (Serpina 1) expression in human liver cells in an epigenetic manner by targeting the promoter of a the Al AT regulatory IncRNA ENSG00000289541. We also define the susceptible site in the Al AT promoter for targeting the TGS and regulation of IncRNA ENSG00000289541 whilst not affecting Al AT expression (Figure 1).[000150] Table 2 siRNAs screened targeted to IncRNA ENSG00000289541 or it’s promoter.[000151] siRNA target sites in the genome[000152] >asAlAT_Pl: GTAGGCTGGGGAGGGGCGGG[000153] >asAlAT_P2: TTGGACAGGAAGGAGCCTTG[000154] >asAlAT_P3: ACCCAGGAAGTAGACTTCGGG[000155] >asAl AT_P4: CGGGGAGCTTGGACAGGAAGG[000156] Example 2 - Exosome delivery of Al AT- activating siRNAs[000157] Exosomes are extracellular vessicles (EVs) that are shed from all cells, and those from stem or induced pluripotent stem cell (iPSC) systems can be imbued with endogenous anti-inflammatory properties, and have been found to be remarkably immunologically inert in vivo9. EVs are nano-sized (50-150 nm) and are constitutively shedand then taken up by neighbouring cells. There is an emerging understanding in the field that EVs are functional entities that they can be harnessed to deliver RNA and protein therapeutic agents to modulate cellular functions10and even control infectious diseasesn. While EVs naturally contain proteins and RNA secreted from the parent producer cell, they can also be engineered to be packaged with particular RNA10 11or protein12as therapeutic cargo. Thus, they can be exploited as natural nanoparticles for in vivo deliveryH. EVs therefore are an ideal delivery vehicle for genetic therapeutics, such as messenger RNAs and non-coding RNAs (ncRNA), recombinant proteins, and even CRTSPR13. EVs are relatively inert, non- immunogenic, anti-inflammatory14 15, biodegradable, biocompatible and safe16. Unlike other types of nanoparticles, EVs derived from stem cells (MSCs) are imbued with innate antiinflammatory properties17. The possibility of using iPSC-derived EVs has only recently started to be explored, but is a feasible therapeutic approach18. Building on the previous works of others as well as those from our research group11 12 19-21) wehave now developed a modular platform technology (RNex) whereby we can engineer cells to package therapeutic RNAs of interest. We have also developed a system, shRex that can package shRNAs into exosomes. These approaches allow for cells to be engineered in such a way that they become high producers of exosomes packaged with particular mRNAs, IncRNAs, circular RNAs, antisense RNAs (for RNex) or shRNAs (for shRex).[000158] We have readily packaged other gene regulating proteins (zinc finger proteins) into exosomes11 12and found this approach can deliver these gene regulatory proteins to the brainn. We propose here to generate stable exosome producing cells containing shRNAs able to selectively activate Al AT expression. To date we have delineated those si / shRNAs that can specifically target and activate CFTR22and more recently Al AT (Figure 2). We have discovered two candidate siRNAs targeted to repress the negative Al AT regulating antisense A1AT (asAlAT) IncRNA (ENSG00000289541), which results in the activation (depression) of Al AT (Figure 2). To date several groups have published methods to package shRNAs into exosomes, ranging from direct transfection to the embedding of particular micro-(mi)RNA scaffolds into the shRNA23. We found that direct transfection of siRNAs into exosomes greatly distorts the particles and the miRNA scaffolding approach has not proven reproducible in our hands. Thus, we turned to an approach to use Ago-dependent shRNA and over-expression of Ago2, which is naturally enriched in exosomes24, to enhance shRNA packaging into exosomes. We call this system shRex.[000159] To determine the ability to passage the activating effect of si4 on A1AT we carried out a transwell assay. In this assay we transfect the Argonaute 2, connexion 43 andshRNA expressing plasmids (Figure 5) into HEK293 producer cells. These transfected cells are then grown in a trans well plate with recipient HepG2 liver cells, as Al AT is expressed in liver cells and not HEK293 fibroblasts. After 48hrs the producer and recipient cells are collected and assessed by qRTPCR for Al AT expression. Using this assay, we validate the ability of the shRex system to transfer sh-asAlAt_P4 to activate Al AT expression (Figure 6). Notably, sh-asAlAt_P4 induced a repression of asAlAT in the recipient cells (Figure 7), suggesting that sh-asA!At_P4 is a bona fide repressor of asAlAT and that this repression correlates with activation of A 1 AT.[000160] Lastly, published works25suggest that enhanced knockdown properties can be instilled by the use of a 7bp hairpin loop for small hairpin RNAs (shRNAs). To determine if this is feasible with shRNAs targeted to activate COPD, e.g. asAlAT_Pl and asAlAT_P4 (Table 2). To test the ability of a dual shRNA expressing 7 bp hairpin loop to activate A1AT we generated a new clone containing both shRNAs asAlAT_Pl and asAlAT_P4 (Figure 8). This vector can be used to engineer any cell into a stable shRNA EV producing cells. We find that this new vector (Figure 8) can also significantly induce Al AT expression (Figure 9) and will use this vector in generating stable Al AT activating EV producing cell systems. These data support the goals of this disclosure, which is to develop these two candidate A1AT activating shRNAs and package these along with other top-candidate lung disease relevant shRNAs (Table 2) into EVs.[000161] In conclusion we report here si / shRNAs targeted to IncRNA ENSG00000289541 can induce the activation of A1AT expression and that this form of activation can be long-term as it relies on siRNA induced TGS. Collectively, these data suggest that sh-asAl At_P4 is a good candidate to be used for nanoparticle (LNP or exosome) delivered sustainable activation of Al AT expression to ameliorate disease such as COPD, Emphysema and idiopathic lung fibrosis.[000162] It can be appreciated by those skilled in the art that changes could be made to the examples described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular examples disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.[000163] References which are hereby each incorporated in its entirety1. Nelson, D.R., Teckman, J., Di Bisceglie, A.M., and Brenner, D.A. (2012). Diagnosis and management of patients with alphal -antitrypsin (A1AT) deficiency. Clin Gastroenterol Hepatol 10, 575-580. 10.1016 / j.cgh.2011.12.028.2. Liu, G., Cooley, M.A., Jamicki, A.G., Borghui, T., Nair, P.M., Tjin, G., Hsu, A.C., Haw, T.J., Fricker, M., Harrison, C.L., Jones, B., et al. (2019). Fibulin-lc regulates transforming growth factor-P activation in pulmonary tissue fibrosis JCI Insight.3. Baijaktarevic, I., and Campos, M. (2021). Management of lung disease in alpha-1 antitrypsin deficiency: what we do and what we do not know. Ther Adv Chronic Dis 12_suppl, 20406223211010172. 10.1177 / 20406223211010172.4. Casucci, M., Falcone, L., Camisa, B., Norelli, M., Porcellini, S., Stornaiuolo, A., Ciceri, F., Traversari, C., Bordignon, C., Bonini, C., and Bondanza, A. (2018). Extracellular NGFR Spacers Allow Efficient Tracking and Enrichment of Fully Functional CAR-T Cells CoExpressing a Suicide Gene. Front Immunol 9, 507. 10.3389 / fimmu.2018.00507.5. Chi, X., Gatti, P., and Papoian, T. (2017). Safety of antisense oligonucleotide and siRNA-based therapeutics. Drug Discov Today 22, 823-833. 10.1016 / j.drudis.2017.01.013.6. Morris, K.V., and Mattick, J.S. (2014). The rise of regulatory RNA. Nat Rev Genet 15, 423-437. 10.1038 / nrg3722.7. Johnsson, P., Ackley, A., Vidarsdottir, L., Lui, W.O., Corcoran, M., Grander, D., and Morris, K.V. (2013). A pseudogene long-noncoding-RNA network regulates PTEN transcription and translation in human cells. Nat Struct Mol Biol. 10.1038 / nsmb.2516.8. Morris, K.V., Santoso, S., Turner, A.M., Pastori, C., and Hawkins, P.G. (2008). Bidirectional transcription directs both transcriptional gene activation and suppression in human cells. PLoS Genet 4, el000258. 10. 1371 / journal.pgen.1000258.9. Kim, S.U. (201 1). Neural stem cell-based gene therapy for brain tumors. Stem Cell Rev Rep 7, 130-140. 10.1007 / sl2015-010-9154-l.10. Kojima, R., Bojar, D., Rizzi, G., Hamri, G.C., El-Baba, M.D., Saxena, P., Auslander, S., Tan, K.R., and Fussenegger, M. (2018). Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment. Nat Commun 9, 1305. 10.1038 / s41467-018-03733-8.11. Shrivastava, S., Ray, R.M., Holguin, L., Echavarria, L., Grepo, N., Scott, T.A., Burnett, J., and Morris, K.V. (2021). Exosome-mediated stable epigenetic repression of HIV-1. Nat Commun 12, 5541. 10.1038 / s41467-021-25839-2.12. Villamizar, O., Waters, S.A., Scott, T., Grepo, N., Jaffe, A., and Morris, K.V. (2021). Mesenchymal Stem Cell exosome delivered Zinc Finger Protein activation of cystic fibrosis transmembrane conductance regulator. J Extracell Vesicles 10, el2053. 10.1002 / jev2.12053.13. Cecchin, R., Troyer, Z., Witwer, K., and Morris, K.V. (2023). Extracellular vesicles: The next generation in gene therapy delivery. Mol Ther 31, 1225-1230. 10.1016 / j.ymthe.2023.01.021.14. Portnow, J., Synold, T.W., Badie, B., Tirughana, R., Lacey, S.F., D'Apuzzo, M., Metz, M.Z., Najbauer, J., Bedell, V., Vo, T., Gutova, M., et al. (2017). Neural Stem Cell-Based Anticancer Gene Therapy: A First-in-Human Study in Recurrent High-Grade Glioma Patients. Clin Cancer Res 23, 2951-2960. 10.1158 / 1078-0432.CCR-16-1518.15. Jeon, J.Y., An, J.H., Kim, S.U., Park, H.G., and Lee, M.A. (2008). Migration of human neural stem cells toward an intracranial glioma. Exp Mol Med 40, 84-91. 10.3858 / emm.2008.40.1.84.16. Sun, L„ Xu, R„ Sun, X., Duan, Y„ Han, Y„ Zhao, Y„ Qian, H„ Zhu, W„ and Xu, W. (2016). Safety evaluation of exosomes derived from human umbilical cord mesenchymal stromal cell. Cytotherapy 18, 413-422. 10.1016 / j.jcyt.2015.11.018.17. Long, Q., Upadhya, D., Hattiangady, B., Kim, D.K., An, S.Y., Shuai, B., Prockop, D.J., and Shetty, A.K. (2017). Intranasal MSC-derived Al-exosomes ease inflammation, and prevent abnormal neurogenesis and memory dysfunction after status epilepticus. Proc Natl Acad Sci U S A 114, E3536-E3545. 10.1073 / pnas.l703920114.18. Tu, Y.K., and Hsueh, Y.H. (2020). Extracellular vesicles isolated from human olfactory ensheathing cells enhance the viability of neural progenitor cells. Neurol Res 42, 959-967. 10.1080 / 01616412.2020.1794371.19. Idris, A., Davis, A., Supramaniam, A., Acharya, D., Kelly, G., Tayyar, Y., West, N., Zhang, P., McMillan, C.L.D., Soemardy, C., Ray, R., et al. (2021). A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19. Mol Ther. 10.1016 / j.ymthe.2021.05.004.20. Scott, T.A., Supramaniam, A., Idris, A., Cardoso, A.A., Shrivastava, S., Kelly, G., Grepo, N.A., Soemardy, C., Ray, R.M., McMillan, N.A.J., and Morris, K.V. (2022). Engineered extracellular vesicles directed to the spike protein inhibit SARS-CoV-2. Mol Ther Methods Clin Dev 24, 355-366. 10.1016 / j.omtm.2022.01.015.21. Davis, A., Morris, K.V., and Shevchenko, G. (2022). Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy using lipid nanoparticles. Mol Ther Methods Clin Dev 25, 158-169. 10.1016 / j.omtm.2022.03.008.22. Villamizar, O., Waters, S.A., Scott, T., Saayman, S., Grepo, N., Urak, R., Davis, A., Jaffe, A., and Morris, K.V. (2019). Targeted Activation of Cystic Fibrosis Transmembrane Conductance Regulator. Mol Ther 27, 1737-1748. 10.1016 / j.ymthe.2019.07.002.23. Reshke, R., Taylor, J.A., Savard, A., Guo, H., Rhym, L.H., Kowalski, P.S., Trung, M.T., Campbell, C., Little, W., Anderson, D.G., and Gibbings, D. (2020). Reduction of the therapeutic dose of silencing RNA by packaging it in extracellular vesicles via a pre-microRNA backbone. Nat Biomed Eng 4, 52-68. 10.1038 / s41551-019-0502-4. 24. Li, Y„ Xiang, G.M., Liu, L.L., Liu, C„ Liu, F„ Jiang, D.N., and Pu, X.Y. (2015).Assessment of endogenous reference gene suitability for serum exosomal microRNA expression analysis in liver carcinoma resection studies. Mol Med Rep 12, 4683-4691. 10.3892 / mmr.20L5.3919.25. Jensen, S.M., Schmitz, A., Pedersen, F.S., Kjems, J., and Bramsen, J.B. (2012). Functional selection of shRNA loops from randomized retroviral libraries. PLoS One 7, e43095. 10. 137 l / joumal.pone.0043095.26. Argemi, J., Latasa, M.U., Atkinson, S.R., Blokhin, I.O., Massey, V., Gue, J.P., Cabezas, J., Lozano, J.J., Van Booven, D., Bell, A., Cao, S., et al. (2019). Defective HNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis. Nat Commun 10, 3126. 10.1038 / s41467-019- 11004-3.

Claims

1. A pharmaceutical composition comprising one or more alpha-1 antitrypsin (Al AT) upregulating polynucleotides capable of upregulating expression of Al AT protein encoded by Al AT gene wherein the one or more Al AT upregulating polynucleotides targets at least a portion of promoter and / or 5 ’ UTR region of antisense RNA to the Al AT gene.

2. The pharmaceutical composition of claim 1, wherein nucleotide sequence of the antisense RNA to the Al AT gene targeted by the one or more Al AT upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2).

3. The pharmaceutical composition of claim 1, wherein nucleotide sequence of the at least a portion of the promoter and / or 5’ UTR region of the antisense RNA to the A1AT gene is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence SEQ ID NO. 1.

4. The pharmaceutical composition of claim 1, wherein nucleotide sequence of each of the one or more Al AT upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, or a combination thereof.

5. The pharmaceutical composition of claim 1, wherein the one or more Al AT upregulating polynucleotides upregulates expression of Al AT by downregulating the expression of an antisense RNA wherein the antisense RNA down regulates the expression of Al AT and wherein the nucleotide sequence of the antisense RNA is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2).

6. The pharmaceutical composition of claim 1, wherein each of the one or more Al AT upregulating polynucleotides comprise small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof.

7. The pharmaceutical composition of claim 1, wherein the one or more A1AT upregulating polynucleotides comprise one or more of a first Al AT upregulating polynucleotides and one or more of a second Al AT upregulating polynucleotides wherein the nucleotide sequence of the one or more of the first Al AT upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 3 and the nucleotide sequence of the one or more of the secondAl AT upregulating polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 6.

8. The pharmaceutical composition of claim 1 further comprising a nanoparticle wherein the nanoparticle encapsulates the one or more Al AT upregulating polynucleotides.

9. The pharmaceutical composition of claim 8, wherein the nanoparticle comprises lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle, liposome, exosome, virus or virus-like particle.

10. 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 Al AT upregulating polynucleotides 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.

11. The pharmaceutical composition of claim 10, wherein the one or more Al AT upregulating polynucleotides is further fused to a nuclear localization sequence SIRLOIN.

12. The pharmaceutical composition of claim 10, wherein the fusion protein comprises CD63-Ula, CD81-Ula, PTGFRN-Ula, or a combination thereof.

13. The pharmaceutical composition of claim 10, wherein the exosome further comprises an Ago2 protein or a S387A mutant thereof.

14. The pharmaceutical composition of claim 10, wherein the exosome further comprises a modified myoferlin protein.

15. The pharmaceutical composition of claim 14, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain of the myoferlin protein, or a combination thereof.

16. The pharmaceutical composition of claim 14, wherein the modified myoferlin protein further comprises a connexin 43 protein or a S3 68A mutant thereof.

17. The pharmaceutical composition of claim 10, 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 Al AT of claim 1 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.

18. The pharmaceutical composition of claim 17, 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.

19. A method of treatment of Al AT-associated disease of a subject comprising the step of administration of a therapeutically effective amount of the pharmaceutical composition of claim 1 to the subject.

20. The method of claim 19, wherein the AlAT-associated disease comprises A1AT-associated lung diseases or Al AT-associated liver disease.

21. The method of claim 19, wherein the AlAT-associated disease comprises chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, panniculitis, or liver failure.

22. A method of upregulating expression of Al AT protein in a subject comprising the step of targeting at least a portion of promoter and / or 5’ UTR region of antisense RNA to the Al AT gene using RNA interference in the subject.

23. The method of claim 22, wherein the step of targeting at least a portion of promoter and / or 5’ UTR region of antisense RNA to the A1AT gene using RNA interference is performed by administration of the pharmaceutical composition of claim 1 to the subject.

24. The method of claim 22, wherein nucleotide sequence of the antisense RNA to the A1AT gene is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2).

25. The method of claim 22, wherein nucleotide sequence of the at least a portion of the promoter and / or 5’ UTR region of the antisense RNA to the Al AT gene being targeted is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence SEQ ID NO. 1.

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