Alpha-1 antitrypsin upregulating polynucleotides and methods of use and treatment

Abstract

The present disclosure provides a composition comprising one or more polynucleotides capable of upregulating alpha-1 anti-trypsin (A1AT) expression. In one embodiment, the one or more polynucleotides of the present disclosure upregulate A1AT expression by downregulating antisense RNA that regulates the A1AT gene. In one embodiment, the one or more polynucleotides comprise siRNA or shRNA. In one embodiment, the one or more polynucleotides of the present disclosure are delivered by a nanoparticle (e.g., an exosome) that encapsulates the one or more polynucleotides. The present disclosure also provides a method of using the one or more polynucleotides of any embodiment of the present disclosure to treat an A1AT deficiency-related disease or disorder.

Description

[0001] Incorporation of sequence list XML references

[0002] The accompanying sequence listing XML is provided under the name "A1AT Upregulating Polynucleotide SequenceListing.xml", created on January 15, 2025, and is 72 KB in size. The contents of this sequence listing XML are incorporated herein by reference in full. Technical Field

[0003] The present invention provides a composition comprising one or more polynucleotides capable of upregulating the expression of alpha-1 anti-trypsin (A1AT) and a method thereof. Background Technology

[0004] A1AT deficiency is an autosomal dominant genetic disorder that can lead to chronic liver disease, cirrhosis, hepatocellular carcinoma in children, and an increased risk of emphysema in adults. 1 Chronic obstructive pulmonary disease (COPD) is a major progressive lung disease characterized by chronic bronchitis and / or emphysema, leading to severe shortness of breath, and has been found to be associated with A1AT expression defects. COPD is the third leading cause of chronic disease incidence and death worldwide, costing more than US$2 trillion annually globally and US$9 billion annually in Australia. Current treatments for COPD primarily target symptoms, aiming to reduce acute exacerbations, but cannot stop disease progression or reverse the disease.

[0005] The main mechanism of COPD pathogenesis lies in the imbalance of protease-antiprotease function. A1AT is a 52-kDa glycoprotein, a serum trypsin inhibitor, produced only by hepatocytes and subsequently migrated to the lungs. Its function is to inhibit neutrophil elastase and various other proteases. These proteases in the lungs function to clear debris and digest damaged tissue. However, when not properly regulated, their activity can lead to tissue damage, resulting in abnormal deposition of extracellular matrix (ECM) proteins. The lung ECM is a dynamic structure composed of various proteins, glycoproteins, and proteoglycans, providing structural support and facilitating functional processes. Elastin and various forms of collagen are two major types of ECM proteins, and uncontrolled degradation of these ECM proteins by proteases can lead to lung damage and subsequently emphysema. 2Excessive fibrosis (airway remodeling) and tissue damage, along with emphysema, impair lung function and gas exchange, leading to severe respiratory distress. A1AT blocks the activity of proteases and prevents the overexpression of these elastases and lung damage. Patients with A1AT gene defects develop severe early-onset emphysema and COPD. Patients in this group do not express or have significantly reduced A1AT levels, or express nonfunctional A1AT (which is generated in the liver and released into the bloodstream), resulting in premature loss of elastase regulation, which in turn damages lung tissue and leads to early-onset emphysema and COPD. A1AT enhancement therapy (intravenous infusion of recombinant A1AT protein) is a lifelong treatment with poor efficacy, and because the injected A1AT degrades rapidly, it only slows lung damage. 3 In essence, A1AT deficiency is incurable. Therefore, the primary treatment goal is to permanently restore functional A1AT gene expression in these patients.

[0006] RNA interference 4 This is an inherent mechanism in mammalian cells that specifically and potently shuts down protein production in the cell. It operates by introducing siRNAs (15-30 bp double-stranded RNAs), which specifically target Argonaute-2 (AGO2) mRNA through sequence complementarity, leading to its subsequent degradation. 5 Currently, five RNAi drugs have been approved by the FDA. RNAi can temporarily suppress gene expression through post-transcriptional gene silencing (PTGS) or long-term gene expression through transcriptional gene silencing (TGS). The difference between PTGS and TGS lies in the target of the siRNA; when targeting the mRNA matrix, the result is PTGS; while when targeting the promoter in the genome that serves as the mRNA promoter, the result is transcriptional gene silencing (TGS).

[0007] Long non-coding RNAs (lncRNAs) are a form of RNA that plays an important role in regulating gene transcription and participating in a variety of other cellular processes. 18 We and other researchers have learned that inhibiting these lncRNAs, which act as repressive regulators of protein-coding genes, via RNAi can activate gene expression. This siRNA-regulated gene activation is a result of "repressing repressors." 20, 21Several genes have been reported to be regulated by lncRNAs, and we have discovered a lncRNA (ENSG00000289541). Figure 1 This is an antisense sequence of the A1AT promoter, which can inhibit A1AT expression. Therefore, there is an urgent need for an effective composition and method that utilizes RNA interference to treat A1AT deficiency-related diseases by regulating lncRNAs that inhibit A1AT expression. Summary of the Invention

[0008] A pharmaceutical composition comprising one or more α-1 antitrypsin (A1AT) upregulated polynucleotides, said polynucleotides being capable of upregulating the expression of A1AT protein encoded by the A1AT gene, wherein said one or more A1AT upregulated polynucleotides target at least a portion of the promoter and / or 5' UTR region of the antisense RNA of said A1AT gene.

[0009] A method for upregulating A1AT protein expression in an individual, comprising the step of using RNA interference in the individual to target at least a portion of the promoter and / or 5' UTR region of the antisense RNA of the A1AT gene.

[0010] A method for upregulating A1AT protein expression in an individual, comprising the step of using RNA interference in the individual to target at least a portion of the promoter and / or 5' UTR region of the antisense RNA of the A1AT gene. Attached Figure Description

[0011] Figure 1 This diagram illustrates the A1AT genomic locus in humans. The figure shows A1AT, along with the antisense A1AT lncRNA (ENSG00000289541) and the siRNAs found to effectively target the asA1AT promoter. It also shows the target susceptibility region within the asA1AT promoter.

[0012] Figure 2 This study illustrates siRNA-regulated A1AT activation in Huh7 cells. A double-repeat selection of siRNAs targeting and inhibiting ENSG00000289541 was performed in Huh7 cells. The figure shows the mean values ​​of transfected cells (100 nM siRNA and Lipofectamine 2000 (L2K)) 48 hours post-transfection, along with the standard error of the mean and the p-value for the paired T test.

[0013] Figure 3This study illustrates A1AT activation regulated by siRNA in HepG2 cells. Quadruple screening with siRNAs was performed in HepG2 cells. The figure shows the mean A1AT expression in HepG2 cells 48 hours post-transfection (100 nM, L2K transfection), along with the standard error of the mean and the p-value for the paired T test.

[0014] Figure 4 This study illustrates the effect of TSA treatment on siRNA-activated A1AT. HEPG2 cells transfected with siRNA (100 nM, L2K) were treated with TSA (day 1, 0.05 µM), and A1AT expression was assessed after 48 hours. The figures show the mean values ​​of cells treated in three replicates, along with the standard error of the mean.

[0015] Figure 5A -C illustrates the vector map of various plasmids used for the delivery of shRNA exosomes targeting asA1AT in this invention. Figure 5A The vector map of plasmid pLV_shGFP_B220606-1427pzm is shown, and its performance is compared with shGFP, Ago-2 and Cnx43 to achieve exosome delivery of shRNA. Figure 5B This section describes the vector map of plasmid pLV_shA1AT_P1_VB221027-1180pww, which shows shP1, Ago-2, and Cnx43 to achieve exosome delivery of shRNA. Figure 5C This section describes the vector map of plasmid pLV_asA1At_shP4_VB221027-1182fwc, showing its representation of shP4, Ago-2, and Cnx43 to achieve exosome delivery of shRNA.

[0016] Figure 6 This study demonstrates that shRNA targeting asA1AT is transfected into hepatocytes via extracellular vesicles (EVs). HEK producer cells were transfected with plasmids overexpressing Ago-2, connexin 43, and shRNA, and co-cultured with HepG2 hepatocytes for 48 hours. The changes in A1AT expression relative to β-actin in cultures (including producer HEK293 and recipient HepG2 cells) were then assessed. The figures show the mean values ​​of triplet treatment cultures, along with standard deviations and p-values ​​for paired T-tests.

[0017] Figure 7This study aimed to investigate the effect of extracellular vesicle (EV) transfection of asA1AT-targeting shRNA to hepatocytes on antisense A1AT expression. HEK producer cells were transfected with Ago-2, connexin 43 overexpression, and shRNA expression plasmids and co-cultured with HepG2 hepatocytes for 48 hours. The changes in asA1AT expression relative to β-actin in cultures (including producer HEK293 and recipient HepG2 cells) were then assessed.

[0018] Figure 8 The vector map of the dual shRNA 7 bp hairpin loop vector is shown. The vector pLV[Exp]-Puro-Rev(hU6>asA1At-P1_7bp Loop)-H1>asA1ATP4_7bp was generated to simultaneously express the previously discovered shRNAs asA1At-P1 and asA1ATP4, which can induce A1AT expression activation.

[0019] Figure 9 This study demonstrates the activation of A1AT by 7 bp dual shRNA. HEK293 cells were transfected with a plasmid expressing shRNA (100 ng shRNA plasmid, L3K), and A1AT expression was assessed after 72 hours. The figures show the mean, standard deviation, and p-values ​​for paired T tests. Detailed Implementation

[0020] As used in this specification and the appended claims, the singular forms “a,” “an,” and “described” include plural references unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes a mixture of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and so on.

[0021] As used in this article, the term “about” as a quantity modifier means including the modified quantity within the range of ±5%, ±10%, ±15%, or ±20%.

[0022] As used herein, the term "nucleic acid" refers to a nucleotide (e.g., deoxyribonucleotide or ribonucleotide) and its polymers, or complementary forms, in single-stranded, double-stranded, or multi-stranded form. The terms "polynucleotide," "oligonucleotide," "oligomery," or similar terms, in their usual and conventional sense, refer to a linear sequence of nucleotides. The term "nucleotide," in its usual and conventional sense, refers to a single unit, or monomer, of a polynucleotide. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified forms thereof. Examples of nucleic acids covered herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules comprising mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids covered in this article include any type of RNA (e.g., antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, Dicer-receptor RNAs, Dicer-receptor siRNAs (dsiRNAs) (dsiRNAs are cleaved by the type I RNase ribonuclease Dicer into 21-23 base double-stranded siRNAs with 2-base 3' overhangs)), and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragment thereof. The term "double-stranded" in the context of nucleic acids, in its usual and conventional sense, refers to a double-stranded state. Nucleic acids can be linear or branched. For example, nucleic acids can be linear chains of nucleotides, or they can be branched, such that they contain one or more nucleotide arms or branches. Where desired, the branched nucleic acids are repeating branches to form higher-order structures, such as dendritic polymers. In one specific embodiment, the nucleotide sequence uses the symbols ATCG (adenine (A), cytosine (C), guanine (G), and thymine (T)) to code DNA molecules and the symbols AUCG (adenine (A), cytosine (C), guanine (G), and uracil (U)) to code RNA molecules. In one specific embodiment, the symbols T and U are used interchangeably in the nucleotide sequence to describe DNA and RNA molecules made according to the nucleotide sequence, respectively.

[0023] As used herein, the terms “polypeptide,” “peptide,” and “protein” generally refer to polymers of amino acid residues. As used herein, these terms also apply to polymers of amino acids in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids, or are non-natural amino acid polymers. As generally used herein, the term “protein” refers to a polymer of amino acids linked together by peptide bonds to form a polypeptide, the chain length of which is sufficient to produce tertiary and / or quaternary structures. In one specific embodiment, the “polypeptide,” “peptide,” or “protein” of the present invention is prepared from a plasmid encoding the “polypeptide,” “peptide,” or “protein.” Therefore, the “polypeptide,” “peptide,” or “protein” of the present invention further comprises a nucleotide sequence encoding the “polypeptide,” “peptide,” or “protein,” the sequence of which can be converted using the genetic code (e.g., but not limited to, the standard genetic code).

[0024] As used herein, "sequence identity" and "% identity" refer to numerical values ​​determined by comparing two best-aligned sequences within a comparison window, where the sequence portion may include additions or deletions compared to a reference sequence to achieve optimal alignment. The number of positions where identical amino acid residues appear in both sequences is determined, generating a match number. This number is divided by the total number of positions in the comparison window, and the result is multiplied by 100 to obtain the sequence identity percentage. Unless otherwise indicated, the comparison window is the full length of the reference sequence.

[0025] As used herein, “% similarity” is calculated as described in “% identity”, with the exception of hydrophobic residues Ala, Val, Phe, Pro, Leu, Ile, Trp, Met, and Cys, which are considered similar; basic residues Lys, Arg, and His, which are considered similar; acidic residues Glu and Asp, which are considered similar; and hydrophilic, uncharged residues Gln, Asn, Ser, Thr, and Tyr, which are considered similar. The remaining native amino acid Gly is not similar to any other amino acid in this context.

[0026] As used herein, the terms “subject,” “individual,” or “patient” are used interchangeably and refer to vertebrates, preferably mammals, and more preferably humans. Mammals include, but are not limited to, rodents, apes, humans, farm animals, sporting animals, and pets.

[0027] As used herein, an “effective amount” or “therapeutic effective amount” of a drug, compound, pharmacologically active agent, or pharmaceutical composition includes the amount necessary to achieve the desired outcome. The exact amount required will vary from person to person, depending on species, age, individual general condition, disease severity, specific active agent, mode of administration, desired outcome, etc. In certain embodiments of the invention, a “therapeutic effective amount” of a drug, compound, pharmacologically active agent, or pharmaceutical composition is the amount that effectively inhibits or reverses any disease progression disclosed herein in an individual or biological sample (e.g., 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 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, "therapeuticly effective amount" refers to an amount of drug, compound, pharmacologically active agent, or pharmaceutical composition sufficient to cause disease reversal. In certain embodiments, disease reversal is approximately 1%, approximately 5%, approximately 10%, approximately 20%, approximately 30%, approximately 40%, approximately 50%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 100%, or any value and range falling within these values.

[0028] As used herein, the term "target" or "targeting" a polynucleotide encompasses the direct or indirect regulation of the expression level, biological function, or a combination thereof of the polynucleotide. Indirect regulation of a polynucleotide includes indirectly regulating the expression level, biological function, or a combination thereof of the polynucleotide by regulating one or more antisense RNAs that regulate the targeted polynucleotide. In one specific embodiment, in the case of direct regulation, downregulating a polynucleotide may include disrupting or breaking down the targeted or regulated polynucleotide. In one specific embodiment, in the case of indirect regulation, downregulating a polynucleotide may include disrupting or breaking down one or more antisense RNAs that regulate the targeted polynucleotide. In one specific embodiment, the disruption or breakdown is performed using small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozyme, deoxyribozyme, aptamer, or a combination thereof that targets or regulates the polynucleotide.

[0029] This invention provides one or more α-1 antitrypsin (A1AT) upregulating polynucleotides and pharmaceutical compositions thereof capable of upregulating α-1 antitrypsin (A1AT) expression. In one specific embodiment, the one or more A1AT upregulating polynucleotides of this invention increase or de-inhibit the expression level of A1AT by inhibiting the expression level of one or more A1AT antisense RNA regulators. In one specific embodiment, the A1AT antisense RNA regulator is antisense to the A1AT promoter. In one specific embodiment, the one or more A1AT upregulating polynucleotides of this invention target a portion of the promoter, 5' UTR, or a combination thereof of the one or more A1AT antisense RNA regulators to inhibit the expression level of the one or more A1AT antisense RNA regulators. In one specific embodiment, one or more A1ATs of the present invention upregulate a portion of the promoter, 5' UTR, or combination thereof of the one or more A1AT antisense RNA regulators of a length of about 10 to about 40 nucleotides (nt), 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 length or length range falling within these values), to inhibit the expression of the one or more A1AT antisense RNA regulators. In one specific embodiment, the nucleotide sequence of the promoter, 5' UTR, or combination thereof of the one or more A1AT antisense RNA regulators targeted by the polynucleotide upregulated by the A1AT of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 1.

[0030] SEQ ID NO. 1 (ENST00000693506.1 promoter and 5' UTR): ACCCAGGAAGTAGACTTCGGGTGGAGGCAGTAGGCTGGGGAGGGGCGGGGAGCTTGGACAGGAAGGAGCCTTGCTCATTGCCCGGCAGACACAAGACTGGGCCCTCATAAACTCAGACAGCCCGGCATGTCACCTGTTGTACCTGCCCTTTCAGCTCTGTGACCCGGGACAAGTCACCCTCTCCCTTTGAGTTGCCGCAAGA

[0031] In one specific embodiment, the one or more A1AT antisense RNA regulatory factors are transcribed from a genomic region located on approximately chromosome 14: nucleotides 94,390,771 to 94,420,211 (GRCh38 / hg38). In one specific embodiment, the one or more A1AT antisense RNA regulatory factors are transcribed from a genomic region containing a nucleotide sequence having at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of ENSG00000289541 registered in the Ensembl database (https: / / asia.ensembl.org / index.html). In one specific embodiment, the nucleotide sequence of the one or more A1AT antisense RNA regulators has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2) registered in the Ensembl database (https: / / asia.ensembl.org / index.html).

[0032] SEQ ID NO. 2 (ENST00000693506.1):

[0033] GCCCGGCAGACACAAGACTGGGCCCTCATAAACTCAGACAGCCCGGCATGTCACCTGTTGTACCTGCCCTTTCAGCTCTGTGACCCGGGACAAGTCACCCTCTCCCTTTGAGTTGCCGCAAGAGGCTCATAGGTGGAAGGGACTTGCC TTGTCTCAGATGAAACATTGGACTTGGACTTTTGGATTAATGCTGGAATGAGTTAAGACTTTGGGGGACTGTTGGAAGAGCATGATCGTGTTTTGAAATGTGACGACGTGAAATTTGGAAGGGGCCAGGGGCAGAAATATATGGTTTG GCCCTGTGTCTCCACCCAAATCTCACTTTGAATTGTAATAATCCCCACATGTCCAGGGCAGGGTCAGGCAGAGTTAATGGAATCATGAGGGTGGTTTCCCCCATGCTGTTCTTGTGGTAGTGAATAAGTCTCATGAGAGCTGATGGTT TTATAAATTGGAGTCCCCTTGCACAAGCTCTCTTGCTTGCCACCATGTAAGATGTGACTTTGCTCCTCATTCTCCATCCACCATGATTGGGAGGCCTCCCCAGCCTTGTGGAACTGTGAGTCCATTAAACCTCTTTCCTGTATAAATTA

[0034] In one specific embodiment, the length of one or more AlAT upregulated polynucleotides of the present invention is from about 10 to about 100 nucleotides (nt), for example, about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, or about 100 nt, including any length or length range falling within these values. In one specific embodiment, the length of the AlAT upregulated polynucleotides of the present invention is from about 15 to about 55 nt, from about 20 to about 30 nt, or from about 21 to about 23 nt. In one specific embodiment, the AlAT upregulated polynucleotides of the present invention are double-stranded or single-stranded. In one specific embodiment, the AlAT upregulated polynucleotides of the present invention are blunt-ended or include overhangs. In one specific embodiment, the AlAT upregulated polynucleotides of the present invention are chemically synthesized or recombinant produced. In one specific embodiment, one strand of the A1AT upregulated polynucleotide of the present invention comprises a nucleotide sequence that is sufficiently complementary to the one or more A1AT antisense RNA regulators, such that the polynucleotide guides the cleavage of the A1AT antisense RNA regulators via RNA interference.

[0035] In one specific embodiment, one or more AlAT-upregulated polynucleotides of the present invention comprise small interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), ribozymes, deoxyribozymes, aptamers, or combinations thereof. In one specific embodiment, one or more AlAT-upregulated polynucleotides of the present invention comprise siRNA. In one specific embodiment, the siRNA of the present invention comprises a nucleotide sequence having at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. In one specific embodiment, the siRNA of the present invention comprises a 3' deoxythymidine dinucleotide (dTdT) overhang to increase resistance to nuclease degradation.

[0036] In one specific embodiment, one or more A1AT upregulated polynucleotides of the present invention comprise one or more first siRNAs and one or more second siRNAs, wherein the nucleotide sequence of the one or more first siRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with 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 second siRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with 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 second siRNAs differs from the nucleotide sequence of the one or more first siRNAs, or wherein the difference between the nucleotide sequence of the one or more second siRNAs and the nucleotide sequence of the one or more first siRNAs is greater than 60%.

[0037] In one specific embodiment, one or more A1AT upregulated polynucleotides of the present invention comprise one or more first siRNAs and one or more second siRNAs, wherein the nucleotide sequence of the one or more first siRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of SEQ ID NO. 3, and the nucleotide sequence of the one or more second siRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of SEQ ID NO. 6.

[0038] SEQ ID NO. 3 (asA1At_P1): GUAGGCUGGGGAGGGGCGGG

[0039] SEQ ID NO. 4 (asA1At_P2): UUGGACAGGAAGGAGCCUUG

[0040] SEQ ID NO. 5 (asA1At_P3): ACCCAGGAAGUAGACUUCGGG

[0041] SEQ ID NO. 6 (asA1At_P4): CGGGGAGCUUGGACAGGAAGG

[0042] In one specific embodiment, one or more A1AT upregulated polynucleotides of the present invention comprise one or more shRNAs. In one specific embodiment, each of the one or more shRNAs comprises a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence is anticomplementary to the first nucleotide sequence. In one specific embodiment, the nucleotide sequence of the first nucleotide sequence of each of the one or more shRNAs of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6. In one specific embodiment, the shRNA of the present invention further comprises a loop connecting the first nucleotide sequence and the second nucleotide sequence. In one specific embodiment, the loop of the shRNA of the present invention comprises a nucleotide sequence of about 2-9 bp in length, for example, about 2, 3, 4, 5, 6, 7, 8, or 9 bp. In one specific embodiment, the nucleotide sequence of the loop of the shRNA of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequences of SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, or SEQ ID NO. 11. In one specific embodiment, the loop can be cleaved and removed in an RNA interference pathway.

[0043] SEQ ID NO. 7: UUGC

[0044] SEQ ID NO. 8: CCUGACCCA

[0045] SEQ ID NO. 9: AAGCACA

[0046] SEQ ID NO. 10: UGUGCUU

[0047] SEQ ID NO. 11: UUG

[0048] In one specific embodiment, one or more A1AT upregulated polynucleotides of the present invention comprise one or more first shRNAs and one or more second shRNAs, wherein each of the one or more first or one or more second shRNAs comprises a first nucleotide sequence and a second nucleotide sequence, wherein the second nucleotide sequence is anticomplementary to the first nucleotide sequence, and wherein the first nucleotide sequence of the one or more first shRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 3, and the first nucleotide sequence of the one or more second shRNAs has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 6.

[0049] In one specific embodiment, any embodiment of the A1AT upregulated polynucleotide of the present invention further comprises N-acetylgalactosamine (GalNAc), wherein one or more A1AT upregulated polynucleotides of the present invention are conjugated to said GalNAc. In one specific embodiment, any embodiment of the siRNA of the present invention further comprises GalNAc, wherein one or more siRNAs of the present invention are conjugated to said GalNAc. In one specific embodiment, any embodiment of the shRNA of the present invention further comprises GalNAc, wherein one or more shRNAs of the present invention are conjugated to said GalNAc.

[0050] The present invention also provides an A1AT upregulating pharmaceutical composition comprising a therapeutically effective amount of one or more A1AT upregulating polynucleotides according to any specific embodiment of the present invention. In one specific embodiment, the A1AT upregulating pharmaceutical composition of the present invention upregulates A1AT gene expression in an individual 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 one specific embodiment, the A1AT upregulating pharmaceutical composition of the present invention restores an individual's A1AT gene expression 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 A1AT expression level in a healthy individual. In one specific embodiment, the A1AT upregulating pharmaceutical composition of the present invention restores the individual's A1AT gene expression to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 6%, about 7%, 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 A1AT expression level in an average healthy human. In one specific embodiment, the A1AT upregulating pharmaceutical composition of the present invention restores an individual's A1AT gene expression 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 average normal A1AT expression level of a healthy human with the same biometrics (e.g., age, sex, height, weight, etc.) or combinations thereof as the individual.

[0051] The present invention also provides an A1AT upregulating pharmaceutical composition comprising nanoparticles encapsulating a therapeutically effective amount of one or more A1AT upregulating polynucleotides according to any specific embodiment of the present invention. In one specific embodiment, the nanoparticles of the present invention comprise chemical nanoparticles, such as, but not limited to, lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, and biological nanoparticles, such as, but not limited to, liposomes, exosomes, viruses, and virus-like particles.

[0052] In one embodiment, the nanoparticles of the present invention comprise exosomes. In one embodiment, the exosomes encapsulate one or more fusion proteins and one or more RNA cargoes. In one embodiment, the RNA cargo comprises packaging RNA and a packaging domain, wherein the packaging RNA comprises one or more telomerase-upregulated polynucleotides according to any embodiment of the present invention. In one embodiment, the fusion protein comprises an exosome-associated transmembrane protein fused to the packaging protein. In one 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 one embodiment, the packaging protein comprises a U1a protein. In one embodiment, the packaging protein comprises an RNA-binding protein capable of binding to the packaging domain, such that the RNA cargo binds to the fusion protein via the RNA-binding protein and the packaging domain.

[0053] In one specific embodiment, the nanoparticles of the present invention comprise exosomes prepared by an exosome-based RNA packaging and delivery system. In one specific embodiment, the system comprises a low-immunogenic exosome-based RNA packaging and delivery system. In one specific embodiment, the exosome-based RNA packaging and delivery system of the present invention comprises exosome-producing cells, RNA cargo particles, and one or more fusion protein particles, wherein the RNA cargo particles encode packaging RNA, and the packaging RNA comprises one or more ALAT-upregulated polynucleotides according to any specific embodiment of the present invention and a packaging domain capable of binding to a packaging protein of the fusion protein encoded by the one or more fusion protein particles. In one specific embodiment, the fusion protein expressed by the exosome-producing cells based on the fusion protein particles comprises an exosome-associated transmembrane protein fused with the packaging protein. In one specific 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 one specific embodiment, the packaging protein comprises an RNA-binding protein capable of binding to the packaging domain. In one specific embodiment, the packaging protein is endogenous to an individual. Various specific embodiments of recombinant fusion proteins comprising exosome-associated transmembrane proteins and packaging proteins have been disclosed in PCT Application No. PCT / US2021 / 026892, filed April 12, 2021, the contents of which are incorporated herein by reference in their entirety. In one specific embodiment, the packaging protein comprises the U1a protein.

[0054] In one specific embodiment, the one or more fusion proteins comprise the CD63-U1a protein. In one specific embodiment, the nucleotide sequence of the plasmid encoding the CD63-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 12. In one specific embodiment, the amino acid sequence of the CD63-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity with SEQ ID NO. 13. In one specific embodiment, the one or more fusion proteins comprise the CD81-U1a protein. In one specific embodiment, the nucleotide sequence of the plasmid encoding the CD81-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 14. In one specific embodiment, the amino acid sequence of the CD81-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 15. In one specific embodiment, the one or more fusion proteins comprise the PTGFRN-U1a protein. In one specific embodiment, the nucleotide sequence of the plasmid encoding the PTGFRN-U1a fusion protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity to SEQ ID NO. 16. In one specific embodiment, the amino acid sequence of the PTGFRN-U1a fusion protein has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 17.

[0055]

[0056] The uppercase sequence represents CD63, the lowercase letters are connector sequences, and the bold sequence is the U1 small nucleus ribonucleoprotein A polypeptide (U1 snRNP A).

[0057] SEQ ID NO. 13 (aa CD63-U1a): MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVIIAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEFNNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCI NVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLVKSIRSGYEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0058] The uppercase sequence represents CD63, the lowercase letters are connector sequences, and the bold sequence is the U1 small nucleus ribonucleoprotein A polypeptide (U1 snRNP A).

[0059] SEQ ID NO. 14 (nt CD81-U1a):

[0060]

[0061] The uppercase sequence represents CD81, the lowercase letters are connector sequences, and the bold sequence is the U1 small nucleus ribonucleoprotein A polypeptide (U1 snRNP A).

[0062] SEQ ID NO. 15 (aa CD81-U1a):

[0063] MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYLELGDKPAPNTFYVGIYILIAVGAVMMFVGFLGCYGAIQESQCLLGTFFTCLVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETLDCCGSSTLTAL TTSVLKNNLCPSGSNIISNLFKEDCHQKIDDLFSGKLYLIGIAAIVVAVIMIFEMILSMVLCCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0064] SEQ ID NO. 16 (nt PTGFRN-U1a):

[0065]

[0066] The uppercase sequence represents PTGFRN, the lowercase letters are connector sequences, and the bold sequence is the U1 small nucleus ribonucleoprotein A polypeptide (U1 snRNP A).

[0067] SEQ ID NO. 17 (aa PRGFRN-U1a):

[0068] MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFFAAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENWTDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAATSLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGGGGSMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAKMK

[0069] In one specific embodiment, the RNA cargo comprises packaging RNA, which includes one or more A1AT upregulated polynucleotides according to any specific embodiment of the present invention and a packaging domain capable of binding to the packaging protein of the fusion protein. In one specific embodiment, the packaging domain comprises a UR domain or an L2 domain. In one specific embodiment, the nucleotide sequence of the UR domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 18, and the nucleotide sequence of the L2 domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 19.

[0070] In one specific embodiment, the packaging domain further comprises a stabilizing domain. In another specific embodiment, the stabilizing domain comprises an OH domain and / or a Morris motif domain, wherein the nucleotide sequence of the OH domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 20, and the nucleotide sequence of the Morris motif domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 21.

[0071] SEQ ID NO. 18 (UR): AATCATTGCACTCCGGATT

[0072] SEQ ID NO. 19 (L2): AATCATTGCACTCCGGATTT

[0073] SEQ ID NO. 20 (OH): CTGCAGATATCCAGCACAGTGGC

[0074] SEQ ID NO. 21 (MorrisMotif): GCGCAGCGCGCGCAGCGC

[0075] In one specific embodiment, the RNA cargo further comprises a SIRLOIN (SINE-derived nuclear RNA localization) nuclear localization sequence, wherein the SIRLOIN is located upstream of the UR or L2 sequence of the RNA cargo. In one specific embodiment, the nucleotide sequence of the SIRLOIN has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 22.

[0076] SEQ ID NO. 22 (SIRLOIN): CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA

[0077] In one embodiment, the exosome-based packaging and delivery system further comprises an Argonaute 2 (Ago2) encoding plasmid. In one embodiment, the Ago2 contains an S387A mutation. Overexpression of Ago2 or its S387A mutant can increase the efficiency of packaging RNA cargo into exosomes in exosome producer cells.

[0078] In one specific embodiment, the pharmaceutical composition comprising nanoparticles encapsulating one or more ALAT upregulated polynucleotides according to any specific embodiment of the present invention further comprises a nanoparticle-loaded release enhancer. In one specific embodiment, the nanoparticle-loaded release enhancer enhances the release of the load from the nanoparticles by at least about 2-fold, at least about 5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, or at least about 25-fold. In one specific embodiment, the nanoparticle-loaded release enhancer comprises modified myoferlin. In one specific embodiment, the modified myoferlin comprises C2F, C2G, a transmembrane domain, or a combination thereof. In one specific embodiment, the modified myoferlin is composed of C2F, C2G, a transmembrane domain, or a combination thereof. In one specific embodiment, the amino acid sequence of the C2F domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 23. In one specific embodiment, the amino acid sequence of the C2G domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 24. In one specific embodiment, the amino acid sequence of the transmembrane domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 25.

[0079] SEQ ID NO. 23 (C2F): QFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRDHYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEEYCVSGV

[0080] SEQ ID NO. 24 (C2G): PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDNDKFSLDDYLGFLELDLRH

[0081] SEQ ID NO. 25 (transmembrane domain): PDLKAMNPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLLILLLFVAV

[0082] In one specific embodiment, the modified myotensin comprises C2A, FerA, FerB, DysFN, a transmembrane domain, or a combination thereof. In one specific embodiment, the modified myotensin is composed of C2A, FerA, FerB, DysFN, and a transmembrane domain. In one specific embodiment, the amino acid sequence of the C2A domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 26. In one specific embodiment, the amino acid sequence of the FerA domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 27. In one specific embodiment, the amino acid sequence of the FerB domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 28. In one specific embodiment, the DysFN domain comprises DysFN-1, DysFN-2, or a combination thereof. In one specific embodiment, the amino acid sequence of DysFN-1 has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 29. In one specific embodiment, the amino acid sequence of DysFN-2 has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 30. In one specific embodiment, the amino acid sequence of the transmembrane domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 25.

[0083] SEQ ID NO. 26 (C2A): MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRGIPLDFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATIDLVIGYDPPSAPHPNDLS

[0084] SEQ ID NO. 27 (FerA): LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIRK

[0085] SEQ ID NO. 28 (FerB):

[0086] WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCGKTQTIFLKYPQEKNNGP

[0087] SEQ ID NO. 29 (DysFN-1): AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKREFFLP

[0088] SEQ ID NO. 30 (DysFN-2): DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSELTCP

[0089] In one specific embodiment, any specific embodiment of the modified myotropic protein of the present invention further comprises C2B, C2C, C2D, C2E domains or combinations thereof. In one specific embodiment, the amino acid sequence of the C2B domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 31. In one specific embodiment, the amino acid sequence of the C2C domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 32. In one specific embodiment, the amino acid sequence of the C2D domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 33. In one specific embodiment, the amino acid sequence of C2E has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 34.

[0090] SEQ ID NO. 31 (C2B): PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVNMTPSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP

[0091] SEQ ID NO. 32 (C2C): TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIEKNANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVEDFSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE

[0092] SEQ ID NO. 33 (C2D): TPIVSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTEIIHSTLNPTWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEMDITPKLLWHPVMNGDKA

[0093] SEQ ID NO. 34 (C2E): RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEELYMPPLVIKVIDHRQFGRKPVVG

[0094] In one specific embodiment, the modified myotensin of the present invention does not contain a C2A domain, wherein the amino acid sequence of the C2A domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 26. In one specific embodiment, the modified myotensin of the present invention does not contain a C2B domain, wherein the amino acid sequence of the C2B domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 31. In one specific embodiment, the modified myotensin of the present invention does not contain a C2C domain, wherein the amino acid sequence of the C2C domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 32. In one specific embodiment, the modified myotensin of the present invention does not contain a C2D domain, wherein the amino acid sequence of the C2D domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 33. In one specific embodiment, the modified myotensin of the present invention does not contain a C2E domain, wherein the amino acid sequence of the C2E domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 34. In one specific embodiment, the modified myotensin of the present invention does not contain a C2F domain, wherein the amino acid sequence of the C2F domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 23. In one specific embodiment, the modified myotensin of the present invention does not contain a C2G domain, wherein the amino acid sequence of the C2G domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 24. In one specific embodiment, the modified myotensin of the present invention does not contain a FerA domain, wherein the amino acid sequence of the FerA domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 27. In one specific embodiment, the modified myotensin of the present invention does not contain a FerB domain, wherein the amino acid sequence of the FerB domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 28. In one specific embodiment, the modified myotrophin of the present invention does not contain a DysFN-1 domain, wherein the amino acid sequence of the DysFN-1 domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 29.In one specific embodiment, the modified myotensin of the present invention does not contain a DysFN-2 domain, wherein the amino acid sequence of the DysFN-2 domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 30. In another specific embodiment, the modified myotensin of the present invention does not contain a transmembrane domain, wherein the amino acid sequence of the transmembrane domain has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 25.

[0095] In one specific embodiment, the amino acid sequence of the modified myotrophin of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 35.

[0096] SEQ ID NO. 35 (C2F-C2G): MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNGLCDPYIKITLGKKVIEDRDHYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEEYCVSGVNTW RDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLDEFEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVDVFPKSLGPPGPPFNITPRKAKKYYLRVIIWNTKDVI LDEKSITGEEMSDIYVKGWVPGNEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDNDKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKA KTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLLILLLFVAVLLYSLPNYLSMKIVKPNVYPYDVPDYA

[0097] This invention also provides a polynucleotide encoding a modified myotensin, wherein the polynucleotide encodes the modified myotensin of any specific embodiment of the invention. In one specific embodiment, the polynucleotide encoding the modified myotensin 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 one specific embodiment, the C2A domain is encoded by a polynucleotide, wherein the nucleotide sequence of the polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 36. In one embodiment, the C2B domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 37. In one embodiment, the C2C domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 38. In one embodiment, the C2D domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 39. In one embodiment, the C2E domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 40. In one embodiment, the C2F domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 41. In one embodiment, the C2G domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 42. In one embodiment, the FerA domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 43. In one embodiment, the FerB domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 44.In one embodiment, the DysFN-1 domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 45. In one embodiment, the DysFN-2 domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 46. In one embodiment, the transmembrane domain is encoded by a polynucleotide, wherein the nucleotide sequence of said polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 47.

[0098] SEQ ID NO. 36 (nt C2A): Atgctgcgagtgattgtggaatctgccagcaatatccctaaaacgaaatttggcaagccggatcctattgtttctgtcatttttaaggatgagaaaaagaaa acaaagaaagttgataatgaattgaaccctgtctggaatgagattttggagtttgacttgaggggtataccactggacttttcatcttcccttgggattatt gtgaaagattttgagacaattggacaaaataaattaattggcacggcgactgtagccctgaaggacctgactggtgaccagagcagatcctgccgtacaagctgatctccctgctaaatgaaagagggcaagatactggggccaccattgacttggtgatcggctatgatccgccttctgctccacatccaaatgacctgagc

[0099] SEQ ID NO. 37 (nt C2B): Ccacaggacttccagatccgcgtccgagtgattgagggccgacagttaagtggcaacaacataaggcctgtggtcaaagttcacgtctgtggccagacacaccgaacaaaatcaagagaggaaacaaccctttttttgatgagttgtttttctacaatgtcaac atgacccctctgaattgatggatgagatcatcagcatccgggtttaataattctcactctctgcgggcagattgtctgatgggggaatttaagattgatgttggatttgtttatgatgaacctggccatgctgtcatgagaaagtggcttcttctcaatgacccg

[0100] SEQ ID NO. 38 (nt C2C): ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGCCTTCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCGTGGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAAAAAGGTTTGTACAAACATAATTGAGAAGAATGCAAACCCAGAGTGGAATCAGGTCGTCAATCTTCAGATCAAGTTTCCTTCAGTGTGTGAAAAAATAAAACTAACAATATATGACTGGGACCGTCTTACTAAAAATGATGTAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTGGTGGGGAAGTGGAAGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGTAAACACAGGAGAAACAGAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTGAATCTTTATGGAAGCCCCAGAGAGTACACGGGATTCCCAGACCCCTATGATGAG

[0101] SEQ ID NO. 39 (nt C2D): Acccccattgtttcctgcaattttgacagagtctacatctaccatctgcgctgctatgtctatcaagccagaaacctcttggctttagataaggatagcttttcagatccatatgctcatatctgtttcctccatcggagcaaaaccactgagatcatccattcaaccctgaatcccacgtgggaccaaacaattatattcgatgaagttgaaatctatggggaaccccaaacagttctacagaatccacccaaagttatcatggaactttttgacaatgaccaagtgggcaaagatgaatttttaggacgaagcattttctctcctgtggtgaaactgaactcagaaatggacatcacacccaaacttctctggcacccagtaatgaatggagacaaagcc

[0102] SEQ ID NO. 40 (nt C2E): AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTGGAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACACCCAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTACATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAAGCCTGTCGTCGGC

[0103] SEQ ID NO. 41 (nt C2F): Cagtttcgggaattacctgacagcgtcccacaggaatgcacggttaggatttacattgttcgaggcttagagctccagccccaggacaacaatggcctgtgtgacccttacataaaaataacactgggcaaaaaagtcattgaagaccgagatcactacattcccaacactctcaacccagtctttggcaggatgtacgaactgagctgctacttacctcaagaaaaagacctgaaaatttctgtctatgattatgacacctttacccgggatgaaaaagtaggagagacaattattgatctggaaaaccgattcctttcccgctttgggtcccactgcggcataccagaggagtactgtgtttctggagtc

[0104] SEQ ID NO. 42 (nt C2G): cctttcaacatcacaccccggaaagccaagaaatactacctgcgtgtgatcatctggaacaccaaggatgttatcttggatgagaaaagcatcacaggagaggaaatgagtgacatctacgtcaaaggctgggttcctggcaatgaagaaaacaaacagaaaacagatgtccattacagatctttggatggtgaagggaattttaactggcgatttgttttcccgtttgactaccttccagccgaacaactctgtatcgttgcgaaaaaagagcatttctggagtattgaccaaacggaatttcgaatcccacccaggctgatcattcagatatgggacaatgacaagttttctctggatgactacttgggtttcctagaacttgacttgcgtcac

[0105] SEQ ID NO. 43 (nt FerA): Ctgcaaacaaatatagaagctctaaaatcagggatacaaggtaaaattcctgcaaaccagctggctgaattgtggctgaagctgatagatgaagttatagaagacacgagatacacgttgcctctcacagaaggaaaagccaacgtcacagttctcgatactcagatccgaaag

[0106] SEQ ID NO. 44 (nt FerB): Tggcttgataaattaatgcagctgactgaagagccacagaacagcatgcctgacatcatcatctggatgatccggggagagaagagactggcctatgcacgaattcccgcacatcaggtcttgtactccaccagtggtgagaatgcatctggaaaatactgtgggaaaacccaaaccatctttctgaagtatccacaggagaaaaacaacgggcca

[0107] SEQ ID NO. 45 (nt DysFN-1): Gctgtggagaagaagtttaacagcttcgcagaaggaactttcaccgtctttgctgaaatgtatgaaaatcaagctctcatgtttggaaaatggggtacttctggattagtaggacgtcataagttttctgatgtcacaggaaaaataaaactcaagagggaattttttctgcct

[0108] SEQ ID NO. 46 (nt DysFN-2): gatcctgaaagaagcttgctgactgaggcagatgcaggtcacacggagttcactgatgaagtctaccagaacgagagccgctaccccgggggcgactggaagccggccgaggacacctacacggatgcgaacggcgataaagcagcatcacccagcgagttgacttgtcct

[0109] SEQ ID NO. 47 (nt TM): Ccggacctcaaagccatgaacccccttaaagccaagacagcctccctctttgagcagaagtccatgaaaggatggtggccatgctacgcagagaaagatggcgcccgcgtaatggctgggaaagtggagatgacattggaaatcctcaacgagaaggaggccgacgagaggccagccg ggaaggggcgggacgaacccaacatgaaccccaagctggacttaccaaatcgaccagaaacctccttcctctggttcaccaacccatgcaagaccatgaagttcatcgtgtggcgccgctttaagtgggtcatcatcggcttgctgttcctgcttatcctgctgctcttcgtggccgtg

[0110] In one specific embodiment, the modified myotropic protein of the present invention further comprises connexin 43 protein, and the bifunctional protein comprising the modified myotropic protein and the connexin 43 protein can be used to significantly enhance the efficacy and delivery of RNA or nucleic acid loads. In one specific embodiment, the connexin 43 protein comprises the S368A mutation. In one specific embodiment, the amino acid sequence of the connexin 43 protein of the present invention has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity or similarity to SEQ ID NO. 48.

[0111] SEQ ID NO. 48 (connector protein 43): MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSAFRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLY LAHVFYVMRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFEVAFLLIQWYIYGFSLSAVYTCKR DPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNIIELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSPPGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFPDDNQNSKKLAAGHELQPLAIVDQRPSSRAASRASSRPRPDDLEI

[0112] The present invention further provides a bifunctional protein polynucleotide encoding a bifunctional protein comprising a modified myotrophin of the present invention fused with a S368A mutant of connectin 43 protein, wherein the nucleotide sequence of the bifunctional protein polynucleotide has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 49.

[0113]

[0114] In one specific embodiment, various components of the fusion protein or any variant thereof, the Ago2 protein or any variant thereof, the modified myokinin or any variant thereof, or the linker protein 43 or any variant thereof, may be integrated or encapsulated in all possible combinations into nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention to enhance the delivery and absorption of the nanoparticles in an organism. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention further comprise the fusion protein or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention further comprise the Ago2 protein or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention further comprise the modified myokinin or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention further comprise the linker protein 43 or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention do not contain the fusion protein of the present invention or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention do not contain the Ago2 protein of the present invention or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention do not contain the modified myotrophin of the present invention or any variant thereof. In one specific embodiment, the nanoparticles encapsulating one or more A1AT upregulated polynucleotides of the present invention do not contain the linker protein 43 protein of the present invention or any variant thereof. In specific embodiments where the nanoparticles contain a fusion protein encapsulating one or more A1AT upregulated polynucleotides of the present invention and a fusion protein containing an exosome-related transmembrane protein fused with the packaging protein of the present invention, the one or more A1AT-targeting upregulated polynucleotides are fused with the packaging domain of the present invention or any variant thereof. In one specific embodiment, the packaging domain of the present invention or any variant thereof contains UR, L2, OH domains, MorrisMotif domains, nuclear localization sequences SIRLOIN, or combinations thereof.

[0115] In one specific embodiment, any embodiment of encapsulating one or more A1AT upregulated polynucleotide nanoparticles of the present invention upregulated A1AT gene expression in an individual 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 one specific embodiment, any embodiment of encapsulating one or more A1AT upregulated polynucleotides of the present invention restores an individual's A1AT gene expression 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 A1AT expression level in a healthy individual. In one specific embodiment, any embodiment of encapsulating one or more A1AT upregulated polynucleotides of the present invention restores an individual's A1AT gene expression to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 7%, about 7%, about 80%, about 90%, or about 100% of the normal A1AT expression level in an average healthy human. In one specific embodiment, any embodiment of encapsulating one or more A1AT upregulated polynucleotides of the present invention restores an individual's A1AT gene expression 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 A1AT expression level of an individual with the same biometrics (e.g., age, sex, height, weight, etc.) or combinations thereof as the individual.

[0116] This invention provides a method for upregulating A1AT expression in an individual, comprising administering to the individual a therapeutically effective amount of one or more A1AT-upregulated polynucleotides according to any specific embodiment of the invention. In one specific embodiment, the individual is diagnosed with an A1AT-related disease. In one specific embodiment, the A1AT-related disease includes A1AT deficiency-related lung disease or A1AT deficiency-related liver disease. In one specific embodiment, the A1AT-related disease includes chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, or panniculitis.

[0117] The present invention also provides a method for treating an individual's A1AT-related disease, comprising the step of altering the individual's A1AT expression. In one specific embodiment, the step of altering A1AT expression includes administering to the individual a therapeutically effective amount of one or more A1AT-upregulated polynucleotides of any specific embodiment of the present invention. In one specific embodiment, the step of altering A1AT expression includes administering to the individual any specific embodiment of nanoparticles encapsulating one or more A1AT-upregulated polynucleotides of the present invention.

[0118] The present invention also provides a method for treating an individual with A1AT-related diseases, comprising the step of targeting the promoter portion, 5' UTR portion, or a combination thereof of the antisense regulator of the A1AT gene. In one specific embodiment, the step of targeting the promoter portion, 5' UTR portion, or a combination thereof of the antisense regulator of the A1AT gene is performed by administering to the individual any specific embodiment of the A1AT upregulated polynucleotide of the present invention or any specific embodiment of nanoparticles containing the A1AT upregulated polynucleotide.

[0119] In one specific embodiment, the A1AT-related diseases include A1AT deficiency-related lung diseases or A1AT deficiency-related liver diseases. In another specific embodiment, the A1AT-related diseases include chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, lipoprotein lipitis, or liver failure.

[0120] In one specific embodiment, the method of treating A1AT-related diseases includes the step of administering to an individual a therapeutically effective amount of any specific embodiment of a pharmaceutical composition comprising one or more A1AT-upregulating polynucleotides of the present invention. In one specific embodiment, the method of treating A1AT-related diseases includes the step of administering to the individual a therapeutically effective amount of any specific embodiment of a pharmaceutical composition comprising nanoparticles encapsulating one or more A1AT-upregulating polynucleotides of the present invention. In one specific embodiment, the method of treating A1AT-related diseases includes administering about 1 to about 100 billion exosomes, LNPs, or combinations thereof encapsulating one or more A1AT-upregulating polynucleotides of the present invention, for example, about 1, about 10, about 50, about 100, about 250, about 500, about 750, about 1000, about 1250, about 5000, about 75000, about 100000, about 12500, about 50000, about 75000, about 100000, about 125000, Approximately 500,000, approximately 750,000, approximately 1,000,000, approximately 1,250,000, approximately 5,000,000, approximately 7,500,000, approximately 1,000,000, approximately 1,250,000, approximately 5,000,000, approximately 7,500,000, approximately 1,000,000, approximately 1,250,000,000, approximately 5,000,000, approximately 7,500,000, approximately 1,000,000,000, or any number or range of exosomes, LNPs, or combinations thereof falling within these values. In one specific embodiment, the method of treating A1AT-related diseases includes administering exosomes, LNPs, or combinations thereof encapsulated with one or more A1AT-upregulating polynucleotides of the present invention at a concentration of about 0.01 to about 20 mg / kg of individual body weight, for example, 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 range falling within these values. The pharmaceutical compositions of the present invention can be administered in various ways depending on the desired local or systemic treatment and the area to be treated. Administration can be local (including ocular, vaginal, rectal, intranasal, percutaneous), oral, or non-enteric. Non-enteric administration includes intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration. The delivery of interferons containing site-specific targeting portions can be enhanced by selecting the route and site of administration, or by targeting a specific location. For example, intravenous injection can be used to target hepatocytes.

[0121] The pharmaceutical composition may be administered in the form of any specific embodiment of the exosomes of the present invention. As used herein, the term "exosome" refers to a cell-derived small vesicle (20-300 nm in diameter, more preferably 40-200 nm) comprising a membrane surrounding its internal space and generated by said cell via direct plasma membrane budding or via late endosome fusion with the plasma membrane. Any specific embodiment of the exosomes of the present invention comprises lipids or fatty acids and polypeptides, and further comprises the inhibitory nucleic acids described herein as a loading medium. Any specific embodiment of the exosomes of the present invention may be derived from producer cells and isolated from said producer cells based on their size, density, biochemical parameters, or combinations thereof. Any specific embodiment of the exosomes of the present invention may be directly loaded with exogenous nucleic acids or drugs by electroporation, lipid transfection, ultrasonic treatment, and contact with calcium chloride. Alternatively, purified exosomes may be loaded in vitro, for example, by electroporation.

[0122] Any specific embodiment of the exosomes of the present invention can be produced from cells cultured in vitro or from the body fluids of an individual. When exosomes are produced from in vitro cell culture, various producer cells can be used, such as HEK293 cells, Chinese hamster ovary 26 cells, or mesenchymal stem cells (MSCs).

[0123] The pharmaceutical composition can also be formulated by incorporating the inhibitory nucleic acid described herein into adenovirus or adeno-associated virus (AAV), formulated with cell-penetrating peptides, lentiviral vectors, polymers, dendritic polymers, or prepared as siRNA bioconjugates (such as GalNAc–siRNA conjugation delivery platforms).

[0124] If exosomes or vectors are used as the delivery vehicle for siRNA, the candidate siRNA is delivered in the form of shRNA. Both siRNA and shRNA can target and inhibit viruses and are functionally equivalent. When the candidate siRNA is delivered in the form of shRNA, it is derived from a cellular system and packaged into an exosome or vector (AAV or lentiviral vector) as described above.

[0125] shRNA can be provided in an expression cassette containing a promoter sequentially linked to the siRNA described herein. In a specific embodiment, the promoter is a polII or polIII promoter, such as the U6 promoter (e.g., the mouse U6 promoter) or the H1 promoter. In a specific embodiment, the expression cassette further contains a marker gene. In a specific embodiment, the promoter is a polII promoter. In a specific embodiment, the activator is a tissue-specific activator. In a specific embodiment, the promoter is an inducible promoter. In a specific embodiment, the promoter is a polIII promoter. In a specific embodiment, the promoter is a U6 or H1 promoter.

[0126] The present invention also provides vectors containing the expression cassettes described herein. Examples of suitable vectors include adenovirus, lentivirus, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV), or mouse Maloney-based viral vectors. In one specific embodiment, the vector is an adeno-associated viral (AAV) vector.

[0127] The shRNA molecule comprises paired RNA sequences and a loop portion located between the paired RNA sequences to form a hairpin structure. The length of the loop can vary. In some embodiments, the loop length 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 some embodiments, the loop length is 18 nucleotides. The hairpin structure may also contain 3' and / or 5' overhangs. In some embodiments, the overhangs are 3' and / or 5' overhangs of length 0, 1, 2, 3, 4, or 5 nucleotides. The nucleotide sequence of the loop region can vary and may be, for example, (5'-GCAA-3'), (5'-GCGC-3'), or (5'-TTGC-3'), or other sequences that will be well understood by those skilled in the art to which this invention pertains.

[0128] The pharmaceutical compositions described herein can be administered to the treated cells, tissues, or individuals in doses sufficient to inhibit the expression of target genes or the biological activity of non-translated target sequences (e.g., regulatory sequences). The specific dose of the inhibitory nucleic acid described herein administered to a particular individual will depend on factors such as, for example, the route of administration and the individual's physiological characteristics (including health status). For example, the appropriate dose of a particular pharmaceutical composition containing the inhibitory nucleic acid described herein may depend on a variety of factors, including, but not limited to, the individual's physiological characteristics (e.g., age, weight, sex), the progression of a particular coronavirus infection (i.e., pathological state), and other factors that will be readily recognized by one of ordinary skill in the art to which this invention pertains. Various general considerations that may be taken into account in determining an appropriate dose are described, for example, in Gennaro et al. (eds.), (1990), "Remington's Pharmaceutical Sciences," Mack Publishing, Easton, Pennsylvania, USA; and Gilman et al. (eds.), (1990), "Goodman And Gilman's: The Pharmacological Bases of Therapeutics," Pergamon Publishing. Non-limiting examples of appropriate doses of the inhibitory nucleic acids described herein include doses ranging from 0.01 to 200 mg per kilogram of body weight per day, such as 1 to 50 mg per kilogram of body weight per day, 1 to 40 mg per kilogram of body weight per day, 1 to 30 mg per kilogram of body weight per day, 1 to 10 mg per kilogram of body weight per day, 1 to 5 mg per kilogram of body weight per day, 1 to 3 mg per kilogram of body weight per day, 1 to 2 mg per kilogram of body weight per day, 0.1 to 1 mg per kilogram of body weight per day, 0.1 to 0.9 mg per kilogram of body weight per day, etc. 0.1 to 0.8 mg per kilogram of body weight, 0.1 to 0.7 mg per kilogram of body weight per day, 0.1 to 0.6 mg per kilogram of body weight per day, 0.1 to 0.5 mg per kilogram of body weight per day, 0.1 to 0.4 mg per kilogram of body weight per day, 0.1 to 0.3 mg per kilogram of body weight per day, 0.1 to 0.2 mg per kilogram of body weight per day, 0.01 to 0.1 mg per kilogram of body weight per day, 0.01 to 0.05 mg per kilogram of body weight per day, 0.01 to 0.02 mg per kilogram of body weight per day, and 0.005 to 0.01 mg per kilogram of body weight per day.

[0129] Those skilled in the art to which this invention pertains will be able to determine, through conventional experiments, the effective non-toxic amount of the pharmaceutical composition and / or inhibitory nucleic acid described herein, in order to achieve the desired therapeutic outcome in a single dose or a series of doses.

[0130] Typically, in therapeutic applications, treatment continues until the infection, disease state, or symptom ends. Furthermore, those skilled in the art will understand that the optimal quantity and interval of individual doses will be determined by the nature and extent of the infection, disease state, or symptom being treated, the form, route, and site of application, and the nature of the specific individual being treated. Such optimal conditions can also be determined using conventional techniques.

[0131] In many cases, multiple applications of the pharmaceutical composition described herein are required. For example, it may be applied 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The application interval may be from about one week to about twelve weeks, and in some specific embodiments, from about one week to about four weeks. In cases of repeated exposure to the specific pathogen targeted by the pharmaceutical composition described herein, periodic reapplication may be necessary.

[0132] Those skilled in the art to which this invention pertains will also understand that the optimal treatment regimen can be determined using conventional treatment regimen testing.

[0133] Suitable techniques for introducing the inhibitory nucleic acids described herein into cells, tissues, and individuals include various vector systems, vehicles, and reagents. Non-limiting examples include lipid nanoparticles (LNPs), microcells, nucleic acid-lipid particles, lipoplexes, microliposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus-like particles (VLPs), and mixtures thereof.

[0134] The pharmaceutical compositions of the present invention can be administered by any suitable manner, such as intravenous, buccal, non-enteral, intranasal, oral, sublingual, or topical application. Thus, administration can be local, pulmonary (e.g., by inhalation or blowing in an aerosol or powder, including the use of a nebulizer), intranasal, intratracheal, epidermal, percutaneous, oral, or non-enteral. Non-enteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial (e.g., intraparenchymal, intrathecal, or intraventricular) administration. In one specific embodiment, the pharmaceutical composition is adapted for intranasal administration.

[0135] In one specific embodiment, the pharmaceutical composition of the present invention is formulated as a direct-acting nasal spray. In one specific embodiment, the nasal spray can be self-administered by the patient at a point of care.

[0136] It should be understood that the foregoing overview and detailed description are illustrative and explanatory only, and are not intended to limit the invention as claimed in the claims. Generally, the terminology used in this disclosure should not be construed as limiting the technology to the specific embodiments disclosed in the specification, unless these terms are explicitly defined in the foregoing detailed description. Therefore, the actual scope of this technology encompasses the disclosed specific embodiments and all equivalent ways of implementing or carrying out the technology. Specific Implementation

[0138] Materials and Methods

[0139] Small interfering RNAs (siRNAs) were purchased from SynGenis (Perth, Australia) and screened in human HEK293, HepG2, and Hep3G cells. A final concentration of 100 nM siRNA was transfected into cells in 12-well plates using Lipofectamine 2000 (L2K). Cellular RNA was collected 48 or 72 hours post-transfection using a Qiagen RNAeasy manifold (containing DNase treatment). The resulting RNA was converted to cDNA using a Thermofisher reverse transcription manifold. Subsequently, differential gene expression of cDNA relative to β-actin was assessed by quantitative PCR using primers listed in Table 1.

[0140] Table 1 – PCT primers for RNA detection

[0141] result

[0142] Example 1 – A1AT activating siRNA

[0143] Long non-coding RNAs (lncRNAs) are a form of RNA that plays an important role in regulating gene transcription and participating in a variety of other cellular processes. 6 We and other researchers have learned that inhibiting lncRNAs that suppress protein-coding genes via RNAi can activate gene expression. This siRNA-regulated gene activation is a result of "repressing repressor factors." 7,8 Several genes have been reported to be regulated by lncRNAs, and we have discovered a lncRNA (ENSG00000289541). Figure 1 This is the antisense sequence of the A1AT promoter, which can inhibit A1AT expression. When we target the promoter of this lncRNA with siRNA (Table 2), we find that Huh7 ( Figure 2 ) and HepG2 ( Figure 3A1AT expression in hepatocytes increased significantly by 2 to 4 times. Both siA1AT-P1 and siA1AT-P4 are siRNAs that target and induce TGS at the ENSG00000289541 promoter. Figure 1 To confirm that the A1AT activation observed in cells treated with siA1AT-P1 and siA1AT-P4 siRNAs is a result of epigenetic regulatory patterns, we evaluated the ability of siRNAs to activate A1AT expression in the presence of the known siRNA-guided TGS inhibitor Trichostatin A (TSA). We found that TSA inhibited the observed A1AT activation (…). Figure 4 The results show that both siA1AT-P1 and siA1AT-P4 siRNAs operate via TGS, utilizing an epigenetic process that leads to long-term stable activation of A1AT. In summary, we describe two siRNAs that can epigenetically activate A1AT (Serpina 1) expression in human hepatocytes by targeting the promoter of the A1AT regulatory lncRNA ENSG00000289541. We also define susceptibility sites in the A1AT promoter that target TGS and regulate lncRNA ENSG00000289541 without affecting A1AT expression. Figure 1 ).

[0144] Table 2. Screened siRNAs targeting lncRNA ENSG00000289541 or its promoter

[0145] siRNA target sites in the genome

[0146] asA1AT_P1: GTAGGCTGGGGAGGGGCGGG

[0147] asA1AT_P2: TTGGACAGGAAAGGAGCCTTG

[0148] asA1AT_P3: ACCCAGGAAGTAGACTTCGGG

[0149] asA1AT_P4: CGGGGAGCTTGGACAGGAAGG

[0150] Example 2 – Exosome Delivery of A1AT Activated siRNA

[0151] Exosomes are extracellular vesicles (EVs) shed from all cells. Exosomes derived from stem cells or induced pluripotent stem cell (iPSC) systems can be endowed with endogenous anti-inflammatory properties and have been found to exhibit significant immune inertia in vivo. 9 Extracellular vesicles are nanoscale (50-150 nm) and can continuously detach and be absorbed by neighboring cells. An emerging understanding in this field is that extracellular vesicles are functional entities that can be used to deliver RNA and protein therapeutics to regulate cellular function. 10 Even controlling infectious diseases 11 Although extracellular vesicles naturally contain proteins and RNA secreted by the mother cell, they can be modified to load specific RNA. 10,11 or protein 12 Therefore, it can be used as a therapeutic commodity. Thus, it can serve as a natural nanoparticle for in vivo delivery. 11 Therefore, extracellular vesicles are ideal delivery vehicles for gene therapy agents such as messenger RNA and non-coding RNA (ncRNA), recombinant proteins, and even CRISPR. 13 Extracellular vesicles are relatively inert, non-immunogenic, and possess anti-inflammatory properties. 14,15 It is biodegradable, biocompatible, and safe. 16 Unlike other types of nanoparticles, extracellular vesicles derived from stem cells (MSCs) possess inherent anti-inflammatory properties. 17 While the possibility of using iPSC-derived extracellular vesicles has only recently begun to be explored, it represents a feasible treatment approach. 18 Based on previous work and our research team's earlier findings... 11,12,19-21 We have developed a modular platform technology (RNex) that can engineer cells to package targeted therapeutic RNAs. We have also developed a system called shRex that can package shRNA into exosomes. These methods can engineer cells to produce large quantities of exosomes packaged with specific mRNAs, lncRNAs, circular RNAs, antisense RNAs (for RNex), or shRNAs (for shRex).

[0152] We have successfully packaged other gene regulatory proteins (zinc finger proteins) into exosomes. 11,12 They also discovered that this method can deliver these gene regulatory proteins to the brain. 11 We propose constructing stable exosome-producing cells containing shRNAs capable of selectively activating A1AT expression. To date, we have identified shRNAs that specifically target and activate CFTR. 22And the recently discovered A1AT si / shRNA ( Figure 2 We discovered two candidate siRNAs that target and inhibit the antisense lncRNA (asA1AT) that negatively regulates A1AT (ENSG00000289541), thereby leading to the activation (de-inhibition) of A1AT. Figure 2 To date, several research teams have published methods for packaging shRNA into exosomes, including direct transfection and embedding specific microRNA (miRNA) scaffolds into shRNA. 23 We found that direct transfection of siRNA into exosomes severely disrupted the particle structure, and the miRNA scaffold method could not be replicated in our experiments. Therefore, we instead employed a method utilizing Ago-dependent shRNA and Ago2 overexpression (Ago2 is naturally enriched in exosomes) to enhance shRNA packaging into exosomes. 24 We named this system shRex.

[0153] To determine whether the activating effect of si4 on A1AT could be transferred, we performed a Transwell analysis. In this analysis, we transfected Argonaute 2, connexin 43, and a plasmid expressing shRNA (Figure 5) into HEK293 producer cells. These transfected cells were then co-cultured with recipient HepG2 hepatocytes in Transwell plates, as A1AT is expressed in hepatocytes rather than HEK293 fibroblasts. Producer and recipient cells were harvested after 48 hours, and A1AT expression was assessed by qRT-PCR. Using this analysis, we validated the ability of the shRex system to transfer sh-asA1At_P4 to activate A1AT expression. Figure 6 Notably, sh-asA1At_P4 induces suppression of asA1AT expression in receptor cells ( ). Figure 7 The results show that sh-asA1At_P4 is an effective inhibitor of asA1AT, and this inhibition is related to the activation of A1AT.

[0154] Finally, the published results 25 The results showed that using a 7 bp hairpin loop could enhance the gene knockdown properties of short hairpin RNA (shRNA). To determine whether it was feasible to use shRNAs that target COPD activation (e.g., asA1AT_P1 and asA1AT_P4 (see Table 2)), we established new strains containing both asA1AT_P1 and asA1AT_P4 shRNAs. Figure 8 This was used to test the ability of a dual shRNA expressing a 7bp hairpin loop to activate A1AT. This vector can be used to engineer any cell type to stably produce shRNA extracellular vesicles. We found that this novel vector ( Figure 8 It can also significantly induce A1AT expression. Figure 9 The vector will be used to establish a stable A1AT-activated extracellular vesicle producer cell system. These data support the objectives of this disclosure, namely, to develop these two candidate A1AT-activated shRNAs and package them into extracellular vesicles along with other top candidate lung disease-related shRNAs (Table 2).

[0155] In summary, we report that si / shRNA targeting lncRNA ENSG00000289541 can induce activation of A1AT expression, and this activation can be long-lasting, as it depends on siRNA-induced TGS. These data suggest that sh-asA1At_P4 is a promising candidate for nanoparticle (LNP or exosome) delivery with sustained activation of A1AT expression to improve diseases such as COPD, emphysema, and idiopathic pulmonary fibrosis.

[0156] Those skilled in the art will understand that modifications can be made to the specific embodiments described above without departing from the broad inventive concept. Therefore, it should be understood that the present invention is not limited to the specific embodiments disclosed, but is intended to cover modifications within the spirit and scope of the invention as defined in the appended claims.

[0157] References (each cited in full and included in this paper)

[0158] 1.Nelson, DR, Teckman, J., Di Bisceglie, AM, and Brenner, DA(2012). Diagnosis and management of patients with alpha1-antitrypsin (A1AT)deficiency. Clin Gastroenterol Hepatol 10, 575-580. 10.1016 / j.cgh.2011.12.028.

[0159] 2.Liu, G., Cooley, M.A., Jarnicki, A.G., Borghui, T., Nair, P.M.,Tjin, G., Hsu, A.C., Haw, T.J., Fricker, M., Harrison, C.L., Jones, B., etal. (2019). Fibulin-1c regulates transforming growth factor-β activation inpulmonary tissue fibrosis JCI Insight.

[0160] 3.Barjaktarevic, I., and Campos, M. (2021). Management of lungdisease in alpha-1 antitrypsin deficiency: what we do and what we do notknow. Ther Adv Chronic Dis 12_suppl, 20406223211010172. 10.1177 / 20406223211010172.

[0161] 4.Casucci, M., Falcone, L., Camisa, B., Norelli, M., Porcellini, S.,Stornaiuolo, A., Ciceri, F., Traversari, C., Bordignon, C., Bonini, C., andBondanza, A. (2018). Extracellular NGFR Spacers Allow Efficient Tracking andEnrichment of Fully Functional CAR-T Cells Co-Expressing a Suicide Gene.Front Immunol 9, 507. 10.3389 / fimmu.2018.00507.

[0162] 5.Chi, X., Gatti, P., and Papoian, T. (2017). Safety of antisenseoligonucleotide and siRNA-based therapeutics. Drug Discov Today 22, 823-833.10.1016 / j.drudis.2017.01.013.

[0163] 6.Morris, K.V., and Mattick, J.S. (2014). The rise of regulatory RNA.Nat Rev Genet 15, 423-437. 10.1038 / nrg3722.

[0164] 7.Johnsson, P., Ackley, A., Vidarsdottir, L., Lui, W.O., Corcoran,M., Grander, D., and Morris, K.V. (2013). A pseudogene long-noncoding-RNAnetwork regulates PTEN transcription and translation in human cells. NatStruct Mol Biol. 10.1038 / nsmb.2516.

[0165] 8.Morris, K.V., Santoso, S., Turner, A.M., Pastori, C., and Hawkins,P.G. (2008). Bidirectional transcription directs both transcriptional geneactivation and suppression in human cells. PLoS Genet 4, e1000258. 10.1371 / journal.pgen.1000258.

[0166] 9.Kim, S.U. (2011). Neural stem cell-based gene therapy for braintumors. Stem Cell Rev Rep 7, 130-140. 10.1007 / s12015-010-9154-1.

[0167] 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). Designerexosomes produced by implanted cells intracerebrally deliver therapeuticcargo for Parkinson's disease treatment. Nat Commun 9, 1305. 10.1038 / s41467-018-03733-8.

[0168] 11.Shrivastava, S., Ray, R.M., Holguin, L., Echavarria, L., Grepo,N., Scott, T.A., Burnett, J., and Morris, K.V. (2021). Exosome-mediatedstable epigenetic repression of HIV-1. Nat Commun 12, 5541. 10.1038 / s41467-021-25839-2.

[0169] 12.Villamizar, O., Waters, S.A., Scott, T., Grepo, N., Jaffe, A., andMorris, K.V. (2021). Mesenchymal Stem Cell exosome delivered Zinc FingerProtein activation of cystic fibrosis transmembrane conductance regulator. JExtracell Vesicles 10, e12053. 10.1002 / jev2.12053.

[0170] 13.Cecchin, R., Troyer, Z., Witwer, K., and Morris, K.V. (2023).Extracellular vesicles: The next generation in gene therapy delivery. MolTher 31, 1225-1230. 10.1016 / j.ymthe.2023.01.021.

[0171] 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., etal. (2017). Neural Stem Cell-Based Anticancer Gene Therapy: A First-in-HumanStudy in Recurrent High-Grade Glioma Patients. Clin Cancer Res 23, 2951-2960.10.1158 / 1078-0432.CCR-16-1518.

[0172] 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 MolMed 40, 84-91. 10.3858 / emm.2008.40.1.84.

[0173] 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 humanumbilical cord mesenchymal stromal cell. Cytotherapy 18, 413-422. 10.1016 / j.jcyt.2015.11.018.

[0174] 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 A1-exosomes ease inflammation, and prevent abnormal neurogenesis and memorydysfunction after status epilepticus. Proc Natl Acad Sci U S A 114, E3536-E3545. 10.1073 / pnas.1703920114.

[0175] 18.Tu, Y.K., and Hsueh, Y.H. (2020). Extracellular vesicles isolatedfrom human olfactory ensheathing cells enhance the viability of neuralprogenitor cells. Neurol Res 42, 959-967. 10.1080 / 01616412.2020.1794371.

[0176] 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., etal. (2021). A SARS-CoV-2 targeted siRNA-nanoparticle therapy for COVID-19.Mol Ther. 10.1016 / j.ymthe.2021.05.004.

[0177] 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 directedto the spike protein inhibit SARS-CoV-2. Mol Ther Methods Clin Dev 24, 355-366. 10.1016 / j.omtm.2022.01.015.

[0178] 21.Davis, A., Morris, K.V., and Shevchenko, G. (2022). Hypoxia-directed tumor targeting of CRISPR-Cas9 and HSV-TK suicide gene therapy usinglipid nanoparticles. Mol Ther Methods Clin Dev 25, 158-169. 10.1016 / j.omtm.2022.03.008.

[0179] 22.Villamizar, O., Waters, S.A., Scott, T., Saayman, S., Grepo, N.,Urak, R., Davis, A., Jaffe, A., and Morris, K.V. (2019). Targeted Activationof Cystic Fibrosis Transmembrane Conductance Regulator. Mol Ther 27, 1737-1748. 10.1016 / j.ymthe.2019.07.002.

[0180] 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., andGibbings, D. (2020). Reduction of the therapeutic dose of silencing RNA bypackaging it in extracellular vesicles via a pre-microRNA backbone. NatBiomed Eng 4, 52-68. 10.1038 / s41551-019-0502-4.

[0181] 24.Li, Y., Xiang, G.M., Liu, L.L., Liu, C., Liu, F., Jiang, D.N., andPu, X.Y. (2015). Assessment of endogenous reference gene suitability forserum exosomal microRNA expression analysis in liver carcinoma resectionstudies. Mol Med Rep 12, 4683-4691. 10.3892 / mmr.2015.3919.

[0182] 25.Jensen, SM, Schmitz, A., Pedersen, FS, Kjems, J., and Bramsen,JB (2012). Functional selection of shRNA loops from randomized retroviral libraries. PLoS One 7 , e43095 . doi: 10.1371 / journal.pone.0043095.

[0183] 26.Argemi , J. , Latasa , MU , Atkinson , SR , Blokhin , IO , Massey ,V. , Gue , JP , Cabezas , J. , Lozano , JJ , Van Booven , D. , Bell , A. , Cao , S.,et al. (2019). Defective HNF4alpha-dependent gene expression as a driver ofhepatocellular 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 (A1AT) upregulated polynucleotides, the polynucleotides being capable of upregulating the expression of A1AT protein encoded by the A1AT gene, wherein the one or more A1AT upregulated polynucleotides target at least a portion of the promoter and / or 5' UTR region of the antisense RNA of the A1AT gene.

2. The pharmaceutical composition according to claim 1, wherein the nucleotide sequence of the antisense RNA of the A1AT gene targeted by the one or more A1AT upregulated polynucleotides has at least about 80%, about 85%, about 90%, about 95% or about 100% identity with the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2).

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

1.

4. The pharmaceutical composition according to claim 1, wherein the nucleotide sequence of each of the one or more AlAT upregulated polynucleotides has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequences of SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6 or combinations thereof.

5. The pharmaceutical composition of claim 1, wherein the one or more A1AT upregulating polynucleotides upregulate A1AT expression by downregulating the expression of antisense RNA, wherein the antisense RNA downregulates A1AT expression, and wherein the nucleotide sequence of the antisense RNA has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of ENST00000693506.1 (SEQ ID NO. 2).

6. The pharmaceutical composition according to claim 1, wherein the one or more A1AT upregulated polynucleotides each 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 AlAT upregulated polynucleotides comprise one or more first AlAT upregulated polynucleotides and one or more second AlAT upregulated polynucleotides, wherein the nucleotide sequence of the one or more first AlAT upregulated polynucleotides has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with SEQ ID NO. 3, and the nucleotide sequence of the one or more second AlAT upregulated polynucleotides has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of SEQ ID NO.

6.

8. The pharmaceutical composition of claim 1, further comprising nanoparticles, wherein the nanoparticles encapsulate the one or more ALAT upregulated polynucleotides.

9. The pharmaceutical composition of claim 8, wherein the nanoparticles comprise lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, liposomes, exosomes, viruses, or virus-like particles.

10. The pharmaceutical composition of claim 9, further comprising a fusion protein and a packaging domain, wherein the nanoparticles comprise exosomes, wherein one or more A1AT upregulated polynucleotides are fused to the packaging domain, wherein the fusion protein comprises an exosome-associated transmembrane protein fused to the packaging protein, wherein the packaging domain comprises an UR domain, an L2 domain, or a combination thereof, and wherein the packaging domain is capable of binding to the packaging protein.

11. The pharmaceutical composition of claim 10, wherein the one or more A1AT upregulated polynucleotides are further fused with the nuclear localization sequence SIRLOIN.

12. The pharmaceutical composition of claim 10, wherein the fusion protein comprises CD63-U1a, CD81-U1a, PTGFRN-U1a, or a combination thereof.

13. The pharmaceutical composition of claim 10, wherein the exosomes further comprise Ago2 protein or its S387A mutant.

14. The pharmaceutical composition of claim 10, wherein the exosomes further comprise modified myotrophin.

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

16. The pharmaceutical composition of claim 14, wherein the modified myotrophin further comprises connectin 43 protein or its S368A mutant.

17. The pharmaceutical composition of claim 10, wherein the exosomes are prepared using an exosome-based packaging and delivery system comprising one or more RNA cargo-encoding plasmids encoding one or more polynucleotides targeting AlAT as described in claim 1 and fused to the packaging domain, and one or more fusion protein-encoding plasmids encoding the fusion protein, the fusion protein comprising an exosome-associated transmembrane protein fused to the packaging protein.

18. The pharmaceutical composition of claim 17, wherein the exosome-based packaging and delivery system further comprises a plasmid encoding a modified myoglobin, a connexin 43 protein, or an S368A mutant thereof, or a combination thereof.

19. A method of treating an individual with A1AT-related disease, comprising administering a therapeutically effective amount of the pharmaceutical composition according to claim 1 to the individual.

20. The method of claim 19, wherein the A1AT-related disease comprises A1AT-related lung disease or A1AT-related liver disease.

21. The method of claim 19, wherein the A1AT-related disease includes chronic obstructive pulmonary disease (COPD), emphysema, cirrhosis, chronic liver disease, hepatocellular carcinoma, neonatal jaundice, lipoproteinemia, or liver failure.

22. A method for upregulating A1AT protein expression in an individual, comprising the step of using RNA interference in the individual to target at least a portion of the promoter and / or 5' UTR region of the antisense RNA of the A1AT gene.

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

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

25. The method of claim 22, wherein the nucleotide sequence of at least a portion of the promoter and / or 5' UTR region of the targeted A1AT gene antisense RNA has at least about 80%, about 85%, about 90%, about 95%, or about 100% identity with the nucleotide sequence of SEQ ID NO. 1.

Images