Hepatocyte nuclear factor four alpha antisense RNA 1 targeting polynucleotide and method of use and treatment thereof

Abstract

The present invention comprises a pharmaceutic composition comprising one or more hepatocyte nuclear factor four alpha antisense RNA 1 (HNF4A-AS1) targeting polynucleotides, wherein the one or more HNF4A-AS1 targeting polynucleotides is capable of repressing HNF4A-AS1 in a subject resulting in upregulation of HNF4A expression and / or increasing HNF4A P1:P2 isoform ratio in the subject. The present invention also provides a method of treatment of a HNF4A-associated disease in a subject comprising the step of administering a therapeutically effective amount of the pharmaceutical composition comprising one or more HNF4A- AS1 targeting polynucleotides of the present invention. The present invention also provides a method of downregulating HNF4A P2 isoform expression in a subject comprising the step of repressing HNF4A-AS1 in the subject.

Description

[0001] HEPATOCYTE NUCLEAR FACTOR FOUR ALPHA ANTISENSE RNA 1 TARGETING POLYNUCLEOTIDE AND METHOD OF USE AND TREATMENTTHEREOF

[0002] INCORPORATION BY REFERENCE OF A SEQUENCE LISTING XML

[0003] A Sequence Listing is provided herewith as a Sequence Listing XML, “HNF4A AS1 Targeting Polynucleotide” created on January 23, 2025 and having a size of 172 KB. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.

[0004] FIELD OF THE INVENTION

[0005] The present invention provides a composition comprising one or more polynucleotide capable of targeting hepatocyte nuclear factor four alpha antisense RNA 1 (HNF4A-AS1) to regulate hepatocyte nuclear factor four alpha (HNF4A) and method of use and treatment thereof.|0006| BACKGROUND OF THE INVENTION

[0007] HNF4a is a crucial gene for the maintenance and differentiation of hepatocytes2. The HNF4a gene encodes for a DNA binding protein, highly expressed in the liver that regulates the expression of hepatic genes, a mediator of hepatocyte health. The downregulation of HNF4a has been linked to various disease pathologies, including liver fibrosis and cirrhosis. Studies investigating the effect of HNF4a in inflammatory liver disease models observed that increasing HNF4a levels can reduce disease-related cell dysfunction and even facilitate cell regeneration. This discovery has inspired the pursuit of treatments for liver diseases targeting the upregulation or restoration of HNF4a3’4.

[0008] HNF4a is expressed from one of two promoters with the expression from either promoter generating 12 varying isoforms of HNF4A which have been discovered and distinctly defined. Expression from promoter Pl generates the isoforms al-a6 whereas expression from the P2 promoter produces isoforms a7-al25. The Pl variants are highly prevalent in the adult liver as opposed to the P2 variants which are more prevalent in the pancreas, gastrointestinal system, and foetal liver. Most notably increased expression of the P2 variants has been correlated to cell proliferation, and liver cancer progression and is present in AH and NASH disease models4-5-8. Markedly, studies carried out in disease models have shown a switch between Pl and P2 isoforms of HNF4A with a significant increase in P2 isoforms and reduced Pl isoforms during liver disease. A down-regulation inthe associated IncRNA HNF4A-AS1 was also discovered in diseased liver models, indicating a possible correlation between this IncRNA and the repression of the P2 HNF4a isoforms3. Therefore, there is a need for a composition and method for targeting IncRNA using RNA interference technology to upregulate or restore HNF4A gene expression, increase or restore Pl :P2 isoform ratio and / or repressing P2 HNF 4a isoforms in a subject suffering from HNF4 A- associated diseases.

[0009] SUMMARY OF THE INVENTION

[0010] A pharmaceutical composition comprising one or more hepatocyte nuclear factor four alpha antisense RNA 1 (HNF4A-AS1) targeting polynucleotides, wherein the one or more HNF4A-AS1 targeting polynucleotides is capable of repressing HNF4A-AS1 in a subject resulting in upregulation of HNF4A expression and / or increasing HNF4A Pl :P2 isoform ratio in the subject.

[0011] A method of treatment of a HNF4A-associated disease in a subject comprising the step of administering a therapeutically effective amount of the pharmaceutical composition comprising one or more HNF4A-AS 1 targeting polynucleotides of the present invention to the subject.

[0012] A method of downregulating HNF4A P2 isoform expression in a subject comprising the step of repressing HNF4A-AS1 in the subject.

[0013] BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figures 1A and IB illustrate the schematic of the HNF4A gene locus with IncRNA (HNF4-ASla and HNF4-ASlb), mRNA (HNF4A isoforms), primers (Sets 1-9), and siRNA constructs (in red). The siRNA target sites (Figure IB, in red), and primer targets are visualized (Figure IB, in green) as detailed in Table 1 and 2, as were used to assess the effects of siRNA treatment on HNF4A expression.

[0015] Figure 2 illustrates the effect of siHNF4ASl on HNF4a mRNA and HNF4A IncRNA expression. The results of pooled HNF4AS 1 experimental delta-delta CT data represented as fractions of the control (miRN367) for all primer sets were demonstrated.Results gathered from initial technical duplicate screen, one triplicate and two quadruplicate repeated Hep G2 transfections, RT-qPCR carried out on RNA collected 72 hours posttransfection.

[0016] Figure 3 illustrates the effect of siHNF4AS2 on HNF4a mRNA and HNF4A IncRNA expression. The results of pooled HNF4AS2 experimental delta-delta CT data represented as fractions of the control (miRN367) for all primer sets were demonstrated.Results gathered from initial technical duplicate screen, one triplicate and one quadruplicate repeated Hep G2 transfections, RT-qPCR carried out on RNA collected 72 hours posttransfection.

[0017] Figure 4 illustrates the effect of siHNF4ASl&2 on HNF4a mRNA and HNF4A IncRNA expression. The results of pooled HNF4AS1&2 experimental delta-delta CT data represented as fractions of the control (miRN367) for all primer sets were demonstrated. Results gathered from initial technical duplicate screen, one triplicate and two quadruplicate repeated Hep G2, RT-qPCR carried out on RNA collected 72 hours post-transfection.

[0018] Figure 5 illustrates the effect of HNF4asl_Pro2 on HNF4a mRNA and HNF4A IncRNA expression. The results of pooled HNF4asl_Pro2 experimental delta-delta CT data represented as fractions of the control (miRN367) for all primer sets were demonstrated. Results gathered from initial technical duplicate screen, one triplicate and two quadruplicate repeated Hep G2 transfections, RT-qPCR carried out on RNA collected 72 hours post-transfection.

[0019] Figures 6A and 6B illustrate the effects of siRNA targeting of HNF4A antisense RNA on HNF4A expression. Figure 6A illustrates HepG2 cells were transfected with various HNF4A antisense RNA targeted siRNAs and the effects of this treatment on various HNF4A isoforms determined. A notable increase in HNF4A expression was observed with siRNA treatments; siHNF4asl_Pro2, siHNF4ASl&2 and siHNF4ASl. Figure 6B illustrates the effects of siHNF4ASl treatment on HNF4 differential isoform expression. HepG2 cells were transfected with various siRNAs and HNF4 antisense isoform 1 (HNF4A- ASla) expression determined by different primer sets; set 1, 3, and 5 that measure different HNF4a variants and set 4 which measures HNF4A-AS la expression. The averages of triplicate treated cultures are shown with standard errors of the mean and p values from a paired T-test.

[0020] Figures 7A,7B and 7C illustrate the effect of siHNF4ASl on HNF4a mRNA and HNF4A IncRNA expression. Figure 7A illustrates the repression of HNF4-ASla (set 2) results in repression of upstream P2 expressed HNF4A P2-Isol&2 (Set 5) and differential repression of HNF4A Pl-Iso 1 (set 6 vs set 7). Figure 7B-C illustrates the repression of HNF4-ASla by siHNF4_ASl (set 2) results in the specific repression of upstream HNF4A P2-Iso 1 (Set 9) while HNF4A P2-Iso 1 remains unchanged (set 8). Collectively these data tell us that HNF4-ASla is required for HNF4A Pl and P2-Iso-1 expression which includes the exon 10 and exon 11 3’ addition. For figures 7A-C the averages of triplicate treatedHepG2 cultures are shown with standard error of the means and p values from a single sided paired T-test with the treatments relative to siRNA control are shown.

[0021] DETAILED DESCRIPTION OF THE INVENTION

[0022] As used in this specification and in claims which follow, the singular forms “a”, “an” and “the” include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to “an ingredient” includes mixtures of ingredients, reference to “an active pharmaceutical agent” includes more than one active pharmaceutical agent, and the like.

[0023] As used herein, the term “about” as a modifier to a quantity is intended to mean + or - 5%, + or - 10%, + or - 15%, or + or - 20%, inclusive of the quantity being modified.

[0024] As used herein, the term “protein domain” or “domain” means a region of the polypeptide of a protein. In an embodiment, a protein domain is itself folded as a compact structure independent of rest polypeptide region. In an embodiment, the same or similar protein domain within different protein can share similar or identical functions. For example, ferlin proteins comprises multiple C2 domains and a transmembrane domain, and sometimes a Fer domain, DysF domain, or a combination thereof.

[0025] As used herein, the term "nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and polymers thereof in either single-, double- or multiple-stranded form, or complements thereof. The terms "polynucleotide," "oligonucleotide," "oligo" or the like refer, in the usual and customary sense, to a linear sequence of nucleotides. The term "nucleotide" refers, in the usual and customary sense, to a single unit of a polynucleotide, i.e., a monomer. Nucleotides can be ribonucleotides, deoxyribonucleotides, or modified versions thereof. Examples of nucleic acids contemplated herein include single and double stranded DNA, single and double stranded RNA, and hybrid molecules having mixtures of single and double stranded DNA and RNA. Examples of nucleic acids contemplated herein include any types of RNA (e.g., antisense RNA, mRNA, siRNA, miRNA, shRNA, guide RNA, dicer substrate RNA, dicer substrate siRNAs (dsiRNAs) (dsiRNA are cleaved by the RNase I class endoribonuclease dicer into 21-23 base duplexes having 2-base 3'-overhangs siRNA), and any type of DNA, genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. The term "duplex" in the context of nucleic acids refers, in the usual and customary sense, to double strandedness. Nucleic acids can be linear or branched. For example, nucleic acids can be a linear chain of nucleotides orthe nucleic acids can be branched, e.g., such that the nucleic acids comprise one or more arms or branches of nucleotides. Optionally, the branched nucleic acids are repetitively branched to form higher ordered structures such as dendrimers and the like. In an embodiment, the nucleotide sequence is provided using symbols ATCG (adenine (A), cytosine (C), guanine (G), and thymine (T)) for a DNA molecule, and provided with codes using symbols AUCG (adenine (A), cytosine (C), guanine (G), and uracil (U)) for a RNA molecule. In an embodiment, the symbols T and U are used interchangeably in a nucleotide sequence to illustrate the DNA and RNA molecule respectively made according to the nucleotide sequence.

[0026] As used herein, the terms "polypeptide," "peptide" and "protein" generally refer to a polymer of amino acid residues. As used herein, the term also applies to amino acid polymers in which one or more amino acids are chemical analogs or modified derivatives of corresponding naturally occurring amino acids or are unnatural amino acids. The term "protein", as generally used herein, refers to a polymer of amino acids linked to each other by peptide bonds to form a polypeptide for which the chain length is sufficient to produce tertiary and / or quaternary structure. In an embodiment, the "polypeptide," "peptide" or "protein" of the present invention is prepared from a plasmid encoding said "polypeptide," "peptide" or "protein". Therefore, the "polypeptide," "peptide" or "protein" of the present invention further comprises a nucleotide sequence encoding said "polypeptide," "peptide" or "protein" that could be converted using a genetic code such as but not limited to the standard genetic code.

[0027] As used herein, “sequence identity” and “% identity,” refers to the value determined by comparing two optimally aligned sequences over a comparison window, wherein a portion of the sequence in the comparison window may comprise additions or deletions as compared to the reference sequence for optimal alignment of the two sequences. The number of positions at which identical amino acid residues occur in both sequences is determined, yielding the number of matched positions, which is divided by the total number of positions in the window of comparison and the result multiplied by 100 to yield the percentage of sequence identity. The comparison window is the entire length of the sequence being referred to unless indicated otherwise.

[0028] As used herein, “ % similarity” is calculated as described for “% identity,” with the exception that the hydrophobic residues Ala, Vai, Phe, Pro, Leu, He, Trp, Met, and Cys are similar; the basic residues Lys, Arg, and His are similar; the acidic residues Glu and Aspare similar; and the hydrophilic, uncharged residues Gin, Asn, Ser, Thr, and Tyr are similar. The remaining natural amino acid Gly is not similar to any other amino acid in this context.

[0029] As used herein, the term “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets.

[0030] As used herein, the term “effective amount” or “a therapeutically effective amount” of a drug, compound, pharmacologically active agent or a pharmaceutical composition comprises administering an amount thereof necessary to achieve a desired result. The exact amount required will vary from subject to subject, depending on the species, age, general condition of the subject, the severity of the disease, the particular active agent, its mode of administration, the desired outcome, and the like. In certain embodiments of the present invention, a “therapeutically effective amount” of a drug, compound, pharmacologically active agent or a pharmaceutical composition is that amount effective for inhibiting progression or reversing of any disease disclosed herein in a subject or a biological sample (e.g., in cells). In certain embodiments, disease progression is inhibited by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100%. In certain embodiments, the drug, compound, pharmacologically active agent or a pharmaceutical composition inhibits disease progression by at least about 25%, at least about 50%, at least about 75%, at least about 90% or at least about 100%. In certain embodiments of the present invention, a “therapeutically effective amount” refers to an amount of a drug, compound, pharmacologically active agent or a pharmaceutical composition sufficient to cause reversal of disease. In certain embodiments, the disease is reversed by about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 100% or any numbers and number ranges falling within these values.

[0031] As used herein, the term “target” or “targeting” a polynucleotide comprises directly or indirectly regulating the expression level, biological function, or a combination thereof, of said polynucleotide. The indirect regulation of a polynucleotide comprises indirectly regulating the expression level, biological function, or a combination thereof, of a polynucleotide by regulation of one or more antisense RNA that regulates the polynucleotide being targeted. In an embodiment, in the case of direct regulation, down regulating a polynucleotide may comprise destruction or breakup of the polynucleotide being targeted or regulated. In an embodiment, in the case of indirect regulation, down regulating apolynucleotide may comprise destruction or breakup of one or more antisense RNA that regulates the polynucleotide being targeted. In an embodiment, such destruction or breakup is done using a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof targeting or regulating the polynucleotide.

[0032] HNF4a is a crucial gene for the maintenance and differentiation of hepatocytes 2. The HNF4a gene encodes for a DNA binding protein, highly expressed in the liver that regulates the expression of hepatic genes, a mediator of hepatocyte health. HNF4A is expressed from one of two promoters Pl or P2 with the expression from either promoter generating 12 varying isoforms of HNF4A which have been discovered and distinctly defined. Expression from promoter Pl generates the alpha isoforms 1-6 whereas expression from the P2 promoter produces alpha isoforms 7-12.

[0033] Example 1 in connection with figures 2-5 show that various HNF4A-AS1 targeting siRNA of the present invention repress HNF4A-AS1. At the same time, as shown in Figure 6A, various HNF4A- AS 1 targeting siRNAs of the present invention targeting HNF4A-AS1 result in upregulated expression of the HNF4A gene.

[0034] Example 1 in connection with figures 2-5 further show various HNF4A-AS 1 targeting siRNAs of the present invention differentially regulate the expression ratio between Pl promoter-expressed isoform and P2 promoter-expressed isoform. Example 1 in connection with figures 7A, 7B and 7C illustrate that repression of HNF4A-AS 1 IncRNA by siHNF4ASl, one of the more effective HNF4A-AS1 targeting polynucleotides of the present invention, results in the loss of exon 10 and 11 containing HNF4A variants. Exon 10 contains the ligand-binding domain (LBD), which plays a role in dimerization and HNF4A transcriptional activity. It is well known that HNF4A has multiple isoforms produced by alternative splicing with isoforms expressed from the Pl promoter (e.g., HNF4al-HNF4a6) typically include exon 10, while isoforms from the P2 promoter (e.g., HNF4a7-HNF4al2) exclude it (Radi, 2023 #49).

[0035] Furthermore, Example 1 in connection with figure 6B indicate that HNF4A- AS1 is a direct transcriptional enhancer / modulator involved in regulating the ratio of P2 to Pl spliced HNF4a variants and incorporation of the P2 expressed upstream exon 1. Specifically, treatment with siHNF4ASl and siHNF4asl_Pro2 significantly repressed HNF4A-AS1 expression and this suppression correlated with a loss of exon 1 incorporation into the HNF4A mRNAs (Figure 6B). Without being bound to theory, this data together suggests that HNF4A-ASla is concordantly expressed with HNF4A exon 1. This observation juxtaposedwith the relative genomic location of HNF4ASla in the HNF4A gene body as shown in figure 1 A, whereby HNF4-AS la bisects HNF4A P2 and Pl, suggests that HNF4-ASla may be active as an element that fundamentally controls HNF4A promoter usage and exon 1 incorporation into the HNF4A mRNAs which can ultimately affect the resultant HNF4A protein variant expressed, affecting P1:P2 isoform ratio. Without being bound to theory, Example 1 in connection with figures 2-5, 6A, 6B, 7A, 7B and 7C collectively demonstrate that repression of HNF4A-AS 1 by HNF4A-AS 1 targeting polynucleotides of the present invention such as siHNF4ASl increases the HNF4A Pl :P2 isoform ratio, reduces HNF4A isoforms without exon 10, results in upregulation of HNF4A gene expression or a combination thereof.

[0036] Therefore, the present invention provides one or more HNF4A-AS 1 targeting polynucleotides capable of targeting or repressing HNF4A- AS 1. In an embodiment, the one or more HNF4A-AS1 targeting polynucleotides of the present invention targeting or repressing the HNF4A-AS1 results in upregulation of HNF4A gene expression. In an embodiment, the HNF4A-AS1 comprises HNF4A-ASla, HNF4A-ASlb, any isoforms thereof, or a combination thereof. In another embodiment, the HNF4A- AS 1 comprises a HNF4A- AS 1 exon, a HNF4A-AS 1 intron, a HNF4A-AS 1 regulator element, or a combination thereof. In an embodiment, the regulatory element comprises a promoter, a transcriptional enhancer, a transcriptional repressor, or a combination thereof. In an embodiment, nucleotide sequence of the HNF4A-AS1 is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to NR_109949.1 (SEQ ID NO. 1), NR_172878.1 (SEQ ID NO. 2), or NR_172879.1 (SEQ ID NO. 3) deposited in the NCBI Nucleotide database (https: / / www.ncbi.nlm.nih.gov / nuccore / ).

[0037] In an embodiment, the HNF4A-AS1 targeting polynucleotides of the present invention is capable of increasing HNF4A the P1:P2 isoform ratio by repressing the HNF4A- AS1 IncRNA. In an embodiment, the HNF4A-AS1 targeting polynucleotide of the present invention increases the HNF4A Pl :P2 isoform ratio by upregulating expression of at least a HNF4A Pl isoform. In an embodiment, the HNF4A-AS1 targeting polynucleotides of the present invention increase the HNF4A P1:P2 isoform ratio by downregulating expression of at least a HNF4A P2 isoform. In an embodiment, the HNF4 A- AS 1 targeting polynucleotides of the present invention upregulates expression of at least a HNF4A Pl isoform or downregulates expression of at least a HNF4A P2 isoform via RNA interference. In an embodiment, the HNF4A-AS 1 targeting polynucleotides of the present invention downregulates expression of HNF4A-AS1 via RNA interference.

[0038] In an embodiment, HNF4A Pl isoform comprises HNF4A alpha isoform 1, HNF4A alpha isoform 2, HNF4A alpha isoform 3, HNF4A alpha isoform 4, HNF4A alpha isoform 5, HNF4A alpha isoform 6, any isoform that is expressed from the HNF4A Pl promoter, or a combination thereof. In an embodiment, nucleotide sequence of the HNF4A Pl isoform is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to NM_178849.3 (alpha isoform 1; SEQ ID NO. 4), NM_000457.6 (alpha isoform 2; SEQ ID NO. 5), NM_178850.3 (alpha isoform 3; SEQ ID NO. 6), or NM_001258355.2 (alpha isoform 4; SEQ ID NO. 7) deposited in the NCBI Nucleotide database (https: / / www.ncbi.nlm.nih.gov / nuccore / ). In an embodiment, HNF4A P2 isoform comprises HNF4A alpha isoform 7, HNF4A alpha isoform 8, HNF4A alpha isoform 9, HNF4A alpha isoform 10, HNF4A alpha isoform 11, HNF4A alpha isoform 12, any isoform that is expressed from the HNF4A P2 promoter, or a combination thereof. In an embodiment, nucleotide sequence of the HNF4A P2 isoform is at least about 80%, about 85%, about 90%, about 95%, or about 100% identical to NM_001030003.3 (alpha isoform 7; SEQ ID NO. 8), NM_175914.5 (alpha isoform 8; SEQ ID NO. 9), NM_001030004.3 (alpha isoform 9; SEQ ID NO. 10), NM_001287182.2 (alpha isoform 10; SEQ ID NO. 1 1), NM_001287183.2 (alpha isoform 11; SEQ ID NO. 12), or NM_001287184.2 (alpha isoform 12; SEQ ID NO. 13) deposited in the NCBI Nucleotide database (https: / / www.ncbi.nlm.nih.gov / nuccore / ).

[0039] In an embodiment, the at least a HNF4A Pl isoform upregulated by the HNF4A-AS 1 targeting polynucleotide of the present invention does not comprise a nucleotide sequence encoding at least a part of the HNF4A F domain. In an embodiment, the at least a HNF4A Pl isoform upregulated by the HNF4A-AS1 targeting polynucleotide of the present invention comprises a nucleotide sequence encoding the full length HNF4A ligand binding domain (LBD), wherein the LBD plays a role in dimerization and HNF4A transcriptional activity. In an embodiment, a full length LBD is required for the dimerization and transcriptional activity of HNF4A. In an embodiment, the HNF4A P2 isoform downregulated by the HNF4A-AS1 targeting polynucleotide of the present invention comprises a nucleotide sequence encoding the full length HNF4A ligand binding domain (LBD), wherein the LBD plays a role in dimerization and HNF4A transcriptional activity. In an embodiment, the HNF4A P2 isoform downregulated by the HNF4A- AS 1 targeting polynucleotide of the present invention does not comprise a nucleotide sequence encoding at least a part of the HNF4A F domain of the HNF4A. In an embodiment, the nucleotide sequence encoding at least a part of the HNF4A F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 14, SEQ ID NO. 15, or acombination thereof. In an embodiment, the nucleotide sequence encoding the full lengthHNF4A LBD is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 16.

[0040] SEQ ID NO. 14 (part of exon 10):GGTCCCCCAGCGATGCACCCCATGCCCACCACCCCCTGCACCCTCACCTGATGCAGGAACATATGGGAACCAACGTCATCGTTGCCAACACAATGCCCACTCACCTCAGCAACGGACAGATGT

[0041] SEQ ID NO. 15 (part of exon 11):CCACCCCTGAGACCCCACAGCCCTCACCGCCAGGTGGCTCAGGGTCTGAGCCCTATAAGCTCCTGCCGGGAGCCGTCGCCACAATCGTCAAGCCCCTCTCTGCCATCCCCCAGCCGACCATCACCAAGCAGGAAGTTATCTAGCAAGCCGCTGGGGCTTGGGGGCTCCACTGGCTCCCCCCAGCCCCCTAAGAGAGCACCTGGTGATCACGTGGTCACGGCAAAGGAAGACGTGATGCCAGGACCAGTCCCAGAGCAGGAATGGGAAGGATGAAGGGCCCGAGAACATGGCCTAAGGGCCACATCCCACTGCCACCCTTGACGCCCTGCTCTGGATAACAAGACTTTGACTTGGGGAGACCTCTACTGCCTTGGACAACTTTTCTCATGTTGAAGCCACTGCCTTCACCTTCACCTTCATCCATGTCCAACCCCCGACTTCATCCCAAAGGACAGCCGCCTGGAGATGACTTGAGGCCTTACTTAAACCCAGCTCCCTTCTTCCCTAGCCTGGTGCTTCTCCTCTCCTAGCCCCTGTCATGGTGTCCAGACAGAGCCCTGTGAGGCTGGGTCCAATTGTGGCACTTGGGGCACCTTGCTCCTCCTTCTGCTGCTGCCCCCACCTCTGCTGCCTCCCTCTGCTGTCACCTTGCTCAGCCATCCCGTCTTCTCCAACACCACCTCTCCAGAGGCCAAGGAGGCCTTGGAAACGATTCCCCCAGTCATTCTGGGAACATGTTGTAAGCACTGACTGGGACCAGGCACCAGGCAGGGTCTAGAAGGCTGTGGTGAGGGAAGACGCCTTTCTCCTCCAACCCAACCTC ATCCTCCTTCTTCAGGGACTTGGGTGGGTACTTGGGTGAGGATCCCTGAAGGCCTTCAACCCGAGAAAACAAACCCAGGTTGGCGACTGCAACAGGAACTTGGAGTGGAGAGGAAAAGCATCAGAAAGAGGCAGACCATCCACCAGGCCTTTGAGAAAGGGTAGAATTCTGGCTGGTAGAGCAGGTGAGATGGGACATTCCAAAGAACAGCCTGAGCCAAGGCCTAGTGGTAGTAAGAATCTAGCAAGAATTGAGGAAGAATGGTGTGGGAGAGGGATGATGAAGAGAGAGAGGGCCTGCTGGAGAGCATAGGGTCTGGAACACCAGGCTGAGGTCCTGATCAGCTTCAAGGAGTATGCAGGGAGCTGGGCTTCCAGAAAATGAACACAGCAGTTCTGCAGAGGACGGGAGGCTGGAAGCTGGGAGGTCAGGTGGGGTGGATGATATAATGCGGGTGAGAGTAATGAGGCTTGGGGCTGGAGAG GACAAGATGGGTAAACCCTCACATCAGAGTGACATCCAGGAGGAATAAGCTCCCAGGGCCTGTCTCAAGCTCTTCCTTACTCCCAGGCACTGTCTTAAGGCATCTGACATGCATCATCTCATTTAATCCTCCCTTCCTCCCTATTAACCTAGAGATTGTTTTTGTTTTTTATTCTCCTCCTCCCTCCCCGCCCTCACCCGCCCCACTCCCTCCTAACCTAGAGATTGTTACAGAAGCTGAAATTGCGTTCTAAGAGGTGAAGTGATTTTTTTTCTGAAACTCACACAACTAGGAAGTGGCTGAGTCAGGACTTGAACCCAGGTCTCCCTGGATCAGAACAGGAGCTCTTAACTACAGTGGCTGAATAGCTTCTCCAAAGGCTCCCTGTGTTCTCACCGTGATCAAGTTGAGGGGCTTCCGGCTCCCTTCTACAGCCTCAGAAACCAGACTCGTTCTTCTGGGAACCCTGCCCACTCCCAGGACCAAGATTGGCCTGAGGCTGCACTAAAATTCACTTAGGGTCGAGCATCCTGTTTGCTGATAAATATTAAGGAGAATTCATGACTCTTGACAGCTTTTCTCTCTTCACTCCCCAAGTCAAGGGGAGGGGTGGCAGGGGTCTGTTTCCTGGAAGTCAGGCTCATCTGGCCTGTTGGCATGGGGGTGGGACAGTGTGCACAGTGTGGGGGCAGGGGAGGGCTAAGCAGGCCTGGGTTTGAGGGCTGCTCCGGAGACCGTCACTCCAGGTGCATTCTGGAAGCATTAGACCCCAGGATGGAGCGACCAGCATGTCATCCATGTGGAATCTTGGTGGCTTTGAGGACATTCTGGAAAATGCCACTGACCAGTGTGAACAAAAGGGATGTGTTATGGGGCTGGAGGTGTGATTAGGTAGGAGGGAAACTGTTGGACCGACTCCTGCCCCCTGCTCAACACTGACCCCTCTGAGTGGTTGGAGGCAGTGCCCCAGTGCCCAGAAATCCCACCATTAGTGATTGTTTTTTATGAGAAAGAGGCGTGGAGAAGTATTGGGGCAATGTGTCAGGGAGGAATCACCACATCCCTACGGCAGTCCCAGCCAAGCCCCCAATCCCAGCGGAGACTGTGCCCTGCTCAGAGCTCCCAAGCCTTCCCCCACCACCTCACTCAAGTGCCCCTGAAATCCCTGCCAGACGGCTCAGCCTGGTCTGCGGTAAGGCAGGGAGGCTGGAACCATTTCTGGGCATTGTGGTCATTCCCACTGTGTTCCTCCACCTCCTCCCTCCAGCGTTGCTCAGACCTCTGTCTTGGGAGAAAGGTTGAGATAAGAATGTCCCATGGAGTGCCGTGGGCAACAGTGGCCCTTCATGGGAACAATCTGTTGGAGCAGGGGGTCAGTTCTCTGCTGGGAATCTACCCCTTTCTGGAGGAGAAACCCATTCCACCTTAATAACTTTATTGTAATGTGAGAAACACAAAACAAAGTTTACTTTTTTGACTCTAAGCTGACATGATATTAGAAAATCTCTCGCTCTCTTTTTTTTTTTTTTTTTTTTTTTTGGCTACTTGAGTTGTGGTCCTAAAACATAAAATCTGATGGACAAACAGAGGGTTGCTGGGGGGACAAGCGTGGGCACAATTTCCCCACCAAGACACCCTGATCTTCAGGCGGGTCTCAGGAGCTTCTAAAAATCCGCATGGCTCTCCTGAGAGTGGACAGAGGAGAGGAGAGGGTCAGAAATGAACGCTCTTCTATTTCTTGTCATTACCAAGCCAATTACTTTTGCCAAATTTTTCTGTGATCTGCCCTGATTAAGATGAATTGTGAAATTTACATCAAGCAATTATCAAAGCGGGCTGGGTCCCATCAGAACGACCCACATCTTTCTGTGGGTGTGAATGTCATTAGGTCTTGCGCTGACCCCTGAGCCCCCATCACTGCCGCCTGATGGGGCAAAGAAACAAAAAACATTTCTTACTCTTCTGTGTTTTAACAA AAGTTTATAAAACAAAATAAATGGCGCATATGTTTTCTAA

[0042] SEQ ID NO. 16 (full-length exon 9): ATGCCAAGGGGCTGAGCGATCCAGGGAAGATCAAGCGGCTGCGTTCCCAGGTGC AGGTGAGCTTGGAGGACTACATCAACGACCGCCAGTATGACTCGCGTGGCCGCT TTGGAGAGCTGCTGCTGCTGCTGCCCACCTTGCAGAGCATCACCTGGCAGATGAT CGAGCAGATCCAGTTCATCAAGCTCTTCGGCATGGCCAAGATTGACAACCTGTTG CAGGAGATGCTGCTGGGAGGTCCGTGCCAAGCCCAGGAGGGGCGGGGTTGGAGT GGGGACTCCCCAGGAGACAGGCCTCACACAGTGAGCTCACCCCTCAGCTCCTTG GCTTCCCCACTGTGCCGCTTTGGGCAAGTTGCTTAACCTGTCTGTGCCTCAGTTTC CTCACCAGAAAAATGGGAACAAGGCAATGGTCTATTTGTTCAGGCACCGAGAAC CTAGCACGTGCCAGTCACTGTTCTAAGTGCTGGCAATTCAGCAAAGAACAAGATC TTTGCCCTCGGGGAGGCTGTGTGTGTGTGAGTATGTATGGATGCGTGGATATCTG TGTATATGCCCGTATGTGCGTGCATGTGTATATAAAGCCTCACATTTTATGATTTT GAAATAAACAGGTAATATGATAGA1000431 In an embodiment, at least a HNF4A Pl isoform comprising a full lengthHNF4A LBD is not regulated by the HNF4A-AS1 targeting polynucleotide of the present invention. In an embodiment, at least a HNF4A P2 isoform comprising a full length HNF4A EBD is not regulated by the HNF4A-AS1 targeting polynucleotide of the present invention.

[0044] In an embodiment, the polynucleotide of the present invention is about 10 to about 1500 nucleotides (nt) in length such as about 10, about 20, about 30, about 40 about 50 about 60 about 70, about 80, about 90, about 100, about 150, about 200, about 250, about 300, about 350, about 400, about 450, about 500, about 550, about 600, about 650, about 700, about 750, about 800, about 850, about 900, about 950, about 1000, about 1050, about 1100, about 1150, about 1200, about 1250, about 1300, about 1350, about 1400, about 1450, or about 1500 nt in length including any lengths or length ranges falling within these values. In an embodiment, the polynucleotide of the present invention is about 15 to about 55 nt in length, about 20 to about 30 nt in length or about 21 to about 23 nt in length. In an embodiment, the polynucleotide of the present invention is double stranded or single stranded. In an embodiment, the polynucleotide of the present invention is blunt ended or comprise overhanging ends. In an embodiment, the HNF4A-AS1 targeting polynucleotides of the present invention is chemically synthesized or recombinantly produced. In an embodiment, one strand of the polynucleotide of the present invention comprises nucleotidesequence having sufficient complementarity to HNF4A-AS 1 for the polynucleotide to direct cleavage of the HNF4A- AS 1 via RNA interference.

[0045] In an embodiment, the HNF4A-AS 1 targeting polynucleotides of the present invention comprises an antisense RNA (asRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof. In an embodiment, the one or more HNF4A- AS 1 targeting polynucleotides of the present invention comprises a siRNA. In an embodiment, the HNF4A- AS 1 targeting siRNAs of the present invention comprise a 3’ deoxythymidine dinucleotide (dTdT) overhang to increase nuclease resistance. In an embodiment, the nucleotide sequence of the HNF4A-AS 1 targeting polynucleotides of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25.

[0046] In an embodiment, the one or more HNF4A-AS 1 targeting polynucleotides of the present invention comprises a shRNA. In an embodiment, the HNF4A-AS 1 targeting shRNA of the present invention comprises a first nucleotide sequence and a second nucleotide sequence wherein the second nucleotide sequence is reverse complementary to the first nucleotide sequence. In an embodiment, the first nucleotide sequence comprises a nucleotide sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, or SEQ ID NO. 25. In an embodiment, the HNF4A-AS1 targeting shRNA of the present invention further comprises a loop connecting the first nucleotide sequence and the second nucleotide sequence. In an embodiment, the loop of the shRNA of the present invention comprises nucleotide sequence of about 2-9 bp such as about 2, 3, 4, 5, 6, 7, 8, or 9 bp. In an embodiment, the loop of the HNF4 A-AS 1 targeting shRNA of the present invention comprises nucleotide sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, or SEQ ID No. 30. In an embodiment, the loop may be cleaved off in RNA interference pathway. In an embodiment, nucleotide sequence of the HNF4A- AS1 targeting shRNA of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 31 or SEQ ID NO. 32.

[0047] SEQ ID NO. 17 (siHNF4asl_Prol): UCGUGGAGGAAAGAAGCCAAG

[0048] SEQ ID NO. 18 (siHNF4asl_Pro2): UGCAGAGAAAACUGGGACUUA

[0049] SEQ ID NO. 19 (siHNF4asl_Pro3: CUUAUCAAGACAAAGAACAAA

[0050] SEQ ID NO. 20 (siHNF4as2_Prol): GUGAGCAGAAACAACCUGUG

[0051] SEQ ID NO. 21 (siHNF4as2_Pro2): CUUUAAAGGCCAGUGCUGCCC

[0052] SEQ ID NO. 22 (siHNF4ASl&2):ACUUGUUUCAGGUAACGAGAUGUGA

[0053] SEQ ID NO. 23 (siHNF4ASl): CUGACACUUGCUGAUACCAACGGCA

[0054] SEQ ID NO. 24 (siHNF4AS2): ACCAUCUUCUCCAAAAACAAAGUCA

[0055] SEQ ID NO. 25 (siHNF4AS 1_1): UGACACUUGCUGAUACCAACGG

[0056] SEQ ID NO. 26: UUGC

[0057] SEQ ID NO. 27: CCUGACCCA

[0058] SEQ ID NO. 28: AAGCACA

[0059] SEQ ID NO. 29: UGUGCUU

[0060] SEQ ID NO. 30: UUG

[0061] SEQ ID NO. 31 (shHNF4asl_Pro2):UGCAGAGAAAACUGGGACUUAUUGUAAGUCCCAGUUUUCUCUGCAAAUUUUU

[0062] SEQ ID NO. 32 (shHNF4ASl_l):UGACACUUGCUGAUACCAACGGUUGCCCGUUGGUAUCAGCAAGUGUCACUUUU U

[0063] In an embodiment, the one or more HNF4A-AS1 targeting polynucleotides of the present invention comprises an HNF4A- AS 1 targeting asRNA. In an embodiment, nucleotide sequence of the HNF4A-AS1 targeting asRNA AS1 of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or a combination thereof.

[0064] SEQ ID NO. 33 (asHNF4-l):UGCCGUUGGUAUCAGCAAGUGUCAGAUCCCAGCUCCAGGAAGAGGGCUGUCCU CUCAGAUGGGAGGCAGGGGUCCAGUGGGGUGGCUGUGGCUGCAGGGACAGCC UAGCAGAGCCUCUUGGUGGCCCAGACCCCCUGUCCCUGGACCUUGGAAGCCGC UAAAGGAAGCAGCAAGACCAGGCCAGCUGGUGGCUGCCUGGACUGGAGUUUG GUCAGUGGUUGGGGACCUAUAUUGUGAGAUAGUUAGGACUUUGUCCACAAAC UGGAAUUUGAAAUUUCAAAGGGGCAAAACUGAAGGGCCCUACCCCAGAGUAG AGAUGGCUCUUGGCUUCUUUCCUCCACGACUUUUGUUCUUUGUCUUGAUAAG UCCCAGUUUUCUCUGCACCUCUCCCGGAGUGGUGGGUGGGGAAGAAAAGAUA GGAGAUAAUGGUGUGGUGAGCGCCAAGGGGAAGUGAAGAUGCC

[0065] SEQ ID NO. 34 (asHNF4-2):UGGUGGCCCAGACCCCCUGUCCCUGGACCUUGGAAGCCGCUAAAGGAAGCAGC AAGACCAGGCCAGCUGGUGGCUGCCUGGACUGGAGUUUGGUCAGUGGUUGGG GACCUAUAUUGUGAGAUAGUUAGGACUUUGUCCACAAACUGGAAUUUGAAAU UUCAAAGGGGCAAAACUGAAGGGCCCUACCCCAGAGUAGAGAUGGCUCUUGGC UUCUUUCCUCCACGACUUUUGUUCUUUGUCUUGAUAAGUCCCAGUUUUCUCUG CAC

[0066] SEQ ID NO. 35 (asHNF4-3):GUUAGGACUUUGUCCACAAACUGGAAUUUGAAAUUUCAAAGGGGCAAAACUG AAGGGCCCUACCCCAGAGUAGAGAUGGCUCUUGGCUUCUUUCCUCCACGACUU UUGUUCUUUGUCUUGAUAAGUCCCAGUUUUCUCUGCACCUCUCCCGGAGUGGU GGGUGGGGAAGAAAAGAUAGGAGAUAAUGGUGUGGUGAGCGCCAAGGGGAAG U

[0067] In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising N-acetylgalactosamine (GalNAc) wherein the one or more HNF4A-AS1 targeting polynucleotides of the present invention is conjugated to the GalNAc. In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising GalNAc wherein the one or more siRNA of the present invention is conjugated to the GalNAc. In an embodiment, any embodiments of the pharmaceutical composition of the present invention further comprising GalNAc wherein the one or more shRNA of the present invention is conjugated to the GalNAc.

[0068] In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention upregulates the expression of at least one of the one or more HNF4A gene Pl isoform of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotide of the present invention restores the expression of at least one of the one or more HNF4A gene Pl isoform of a subject to at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the at least one of the one or more HNF4A Pl isoform of the subject when healthy. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotide of the presentinvention restores the expression of at least one of the one or more HNF4A gene Pl isoform of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the at least one of the one or more HNF4A Pl isoform of an average healthy human being. In an embodiment, any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the expression of at least one of the one or more HNF4A gene P l isoform of a human subject to at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the at least one of the one or more HNF4A Pl isoform of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc... or a combination thereof.

[0069] In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a subject to less than about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of the normal expression level of the at least one of the one or more HNF4A P2 isoform of the subject when healthy. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a human subject to less than about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of normal expression level of the at least one of the one or more HNF4A P2 isoform of an average healthy human being. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a human subject to less than about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of normal expression level of the at least one of the one or more HNF4A P2 isoform ofan average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. .. or a combination thereof.

[0070] In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention increases the P1 :P2 isoform ratio of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. Tn an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention restores the P1:P2 isoform ratio of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the one or more HNF4A Pl isoform of the subject when healthy. In an embodiment, any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the P1 :P2 isoform ratio of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A Pl isoform of an average healthy human being. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention restores the P1 :P2 isoform ratio of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A Pl isoform of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. .. or a combination thereof.

[0071] In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention upregulates the expression of the HNF4A gene of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%,about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the HNF4A gene of the subject when healthy. In an embodiment, any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the HNF4A gene of an average healthy human being. In an embodiment, any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A gene of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.

[0072] The present invention also provides a pharmaceutical composition comprising a nanoparticle wherein the nanoparticle encapsulates any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention. In an embodiment, the nanoparticle comprises chemical nanoparticles such as but not limited to lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle and biological nanoparticles such as but not limited to liposome, exosome, virus or virus-like particle.

[0073] In an embodiment, the nanoparticle comprises an exosome. In an embodiment, the exosome encapsulates one or more fusion proteins and one or more cargo RNA. In an embodiment, the cargo RNA comprises a package RNA and a packaging domain wherein the package RNA comprises any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention and wherein the packaging domain is capable of binding to the packaging protein of the fusion protein. In an embodiment, the fusion protein comprises an exosome associated transmembrane protein fused to a packaging protein. In an embodiment, the exosome associated transmembrane protein comprises CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactadherin, or PTGFRN. In an embodiment, the packaging protein comprises U1 a protein. In an embodiment, the packaging protein comprises a RNA-binding protein capable of binding tot the packaging domain such that the cargo RNA binds to the fusion protein via the RNA- binding protein and the packaging domain.

[0074] In an embodiment, the nanoparticle comprises an exosome prepared from an exosome-based packaging and delivery system of the present invention. In an embodiment, the system comprises a low immunogenic exosome-based packaging and delivery system. In an embodiment, the exosome-based RNA package and delivery system comprises an exosome producing cell, a cargo RNA encoding plasmid and one or more fusion protein encoding plasmids, wherein the cargo RNA encoding plasmid encodes a packaging RNA comprising any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention and a packaging domain capable of binding to the packaging protein of the fusion protein encoded by the one or more fusion protein encoding plasmid. In an embodiment, the fusion protein expressed by the exosome producing cell based on a fusion protein encoding plasmid comprises an exosome associated transmembrane protein fused to a packaging protein. In an embodiment, the exosome associated transmembrane protein comprises CD9, CD37, CD53, CD63, CD68, CD81, CD82, LAMP-1, LAMP-2A, LAMP-2B, LAMP-2C, lactadherin, or PTGFRN. In an embodiment, the packaging protein comprises an RNA-binding protein. In an embodiment, the packaging protein is endogenous to a subject. Various embodiments for the recombinant fusion protein comprising an exosome associated transmembrane protein and a packaging protein were disclosed in PCT application no. PCT / US2021 / 026892 filed 12 April, 2021, the contents of which are incorporated by reference herein in their entireties. In an embodiment, the packaging protein comprises Ula protein.

[0075] In an embodiment the one or more fusion proteins comprise CD63-U 1 a protein. In an embodiment, nucleotide sequence of the CD63-Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 36. In an embodiment, the amino acid sequence of the CD63-Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 37. In an embodiment the one or more fusion proteins comprise CD81-Ula protein. In an embodiment, nucleotide sequence of the CD81- Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 38. In an embodiment, the amino the acid sequence of the CD81-Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 39. In an embodiment, the one or more fusion proteins comprise PTGFRN-U1 a protein. In an embodiment, nucleotide sequence of the PTGFRN-Ula fusion protein encoding plasmid of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100%identical to SEQ ID NO. 40. In an embodiment, the amino acid sequence of the PTGFRN- Ula fusion protein is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 41.

[0076] SEQ ID NO. 36:ATGGCGGTGGAAGGAGGAATGAAATGTGTGAAGTTCTTGCTCTACGTCCTCCTGC TGGCCTTTTGCGCCTGTGCAGTGGGACTGATTGCCGTGGGTGTCGGGGCACAGCT TGTCCTGAGTCAGACCATAATCCAGGGGGCTACCCCTGGCTCTCTGTTGCCAGTG GTCATCATCGCAGTGGGTGTCTTCCTCTTCCTGGTGGCTTTTGTGGGCTGCTGCGG GGCCTGCAAGGAGAACTATTGTCTTATGATCACGTTTGCCATCTTTCTGTCTCTTA TCATGTTGGTGGAGGTGGCCGCAGCCATTGCTGGCTATGTGTTTAGAGATAAGGT GATGTCAGAGTTTAATAACAACTTCCGGCAGCAGATGGAGAATTACCCGAAAAA CAACCACACTGCTTCGATCCTGGACAGGATGCAGGCAGATTTTAAGTGCTGTGGG GCTGCTAACTACACAGATTGGGAGAAAATCCCTTCCATGTCGAAGAACCGAGTC CCCGACTCCTGCTGCATTAATGTTACTGTGGGCTGTGGGATTAATTTCAACGAGA AGGCGATCCATAAGGAGGGCTGTGTGGAGAAGATTGGGGGCTGGCTGAGGAAA AATGTGCTGGTGGTAGCTGCAGCAGCCCTTGGAATTGCTTTTGTCGAGGTTTTGG GAATTGTCTTTGCCTGCTGCCTCGTGAAGAGTATCAGAAGTGGCTACGAGGTGAT GgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTA TATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCT GTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGG AGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGC GCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTA TGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAA

[0077] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0078] SEQ ID NO. 37:MAVEGGMKCVKFLLYVLLLAFCACAVGLIAVGVGAQLVLSQTIIQGATPGSLLPVVI IAVGVFLFLVAFVGCCGACKENYCLMITFAIFLSLIMLVEVAAAIAGYVFRDKVMSEF NNNFRQQMENYPKNNHTASILDRMQADFKCCGAANYTDWEKIPSMSKNRVPDSCCI NVTVGCGINFNEKAIHKEGCVEKIGGWLRKNVLVVAAAALGIAFVEVLGIVFACCLV KSIRSGYEVMefggggsMAVPETRPNHTIYINNLNEKIKKDELKKSLYAIFSQFGQIL DILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPFYDKPMRIQYAKTDSDIIAK MK

[0079] Uppercase sequence denotes CD63, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0080] SEQ ID NO. 38 (nt CD81-Ula):ATGTCCGGACTCAGATCTCGAGCTCAAGCTTCCGGAGTGGAGGGCTGCACCAAGTGCATCAAGTACCTGCTCTTCGTCTTCAATTTCGTCTTCTGGCTGGCTGGAGGCGT GATCCTGGGTGTGGCCCTGTGGCTCCGCCATGACCCGCAGACCACCAACCTCCTG TATCTGGAGCTGGGAGACAAGCCCGCGCCCAACACCTTCTATGTAGGCATCTACA TCCTCATCGCTGTGGGCGCTGTCATGATGTTCGTTGGCTTCCTGGGCTGCTACGGG GCCATCCAGGAATCCCAGTGCCTGCTGGGGACGTTCTTCACCTGCCTGGTCATCC TGTTTGCCTGTGAGGTGGCCGCCGGCATCTGGGGCTTTGTCAACAAGGACCAGAT CGCCAAGGATGTGAAGCAGTTCTATGACCAGGCCCTACAGCAGGCCGTGGTGGA TGATGACGCCAACAACGCCAAGGCTGTGGTGAAGACCTTCCACGAGACGCTTGA CTGCTGTGGCTCCAGCACACTGACTGCTTTGACCACCTCAGTGCTCAAGAACAAT TTGTGTCCCTCGGGCAGCAACATCATCAGCAACCTCTTCAAGGAGGACTGCCACC AGAAGATCGATGACCTCTTCTCCGGGAAGCTGTACCTCATCGGCATTGCTGCCAT CGTGGTCGCTGTGATCATGATCTTCGAGATGATCCTGAGCATGGTGCTGTGCTGT GGCATCCGGAACAGCTCCGTGTACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAA GGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATC CTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTC ATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGT TTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAG ATATCATTGCCAAGATGAAATAA

[0081] Uppercase sequence denotes CD81, lower case is linker sequence, and bold sequence is the U1 small nuclear ribonucleoprotein polypeptide A (U1 snRNP A).

[0082] SEQ ID NO. 39 (aa CD81-Ula):MSGLRSRAQASGVEGCTKCIKYLLFVFNFVFWLAGGVILGVALWLRHDPQTTNLLYEELGDKPAPNTFYVGIYIEIAVGAVMMFVGFEGCYGAIQESQCEEGTFFTCEVILFACEVAAGIWGFVNKDQIAKDVKQFYDQALQQAVVDDDANNAKAVVKTFHETEDCCG SSTETAETTSVEKNNECPSGSNIISNEFKEDCHQKIDDEFSGKEYEIGIAAIVVAVIMIFEMILSMVECCGIRNSSVYEFGGGGSMAVPETRPNHTIYINNENEKIKKDELKKSEYAIFSQFGQILDILVSRSEKMRGQAFVIFKEVSSATNAERSMQGFPFYDKPMRIQYAKTDSD IIAKMK

[0083] SEQ ID NO. 40 (nt PTGFRN-Ula):ATGGGGCGCCTGGCCTCCAGGCCGCTGCTGCTGGCGCTCCTGTCGTTGGCTCTTTGCCGAGGGCGTGTGGTGAGAGTCCCCACAGCGACCCTGGTTCGAGTGGTGGGCACTGAGCTGGTCATCCCCTGCAACGTCAGTGACTATGATGGCCCCAGCGAGCAAAACTTTGACTGGAGCTTCTCATCTTTGGGGAGCAGCTTTGTGGAGCTTGCAAGCACCTGGGAGGTGGGGTTCCCAGCCCAGCTGTACCAGGAGCGGCTGCAGAGGGGCGAGATCCTGTTAAGGCGGACTGCCAACGACGCCGTGGAGCTCCACATAAAGAACGTCCAGCCTTCAGACCAAGGCCACTACAAATGTTCAACCCCCAGCACAGATGCCACTGTCCAGGGAAACTATGAGGACACAGTGCAGGTTAAAGTGCTGGCCGACTCCCTGCACGTGGGCCCCAGCGCGCGGCCCCCGCCGAGCCTGAGCCTGCGGGAGGGGGAGCCCTTCGAGCTGCGCTGCACCGCCGCCTCCGCCTCGCCGCTGCACACGCACCTGGCGCTGCTGTGGGAGGTGCACCGCGGCCCGGCCAGGCGGAGCGTCCTCGCCCTGACCCACGAGGGCAGGTTCCACCCGGGCCTGGGGTACGAGCAGCGCTACCACAGTGGGGACGTGCGCCTCGACACCGTGGGCAGCGACGCCTACCGCCTCTCAGTGTCCCGGGCTCTGTCTGCCGACCAGGGCTCCTACAGGTGTATCGTCAGCGAGTGGATCGCCGAGCAGGGCAACTGGCAGGAAATCCAAGAAAAGGCCGTGGAAGTTGCCACCGTGGTGATCCAGCCATCAGTTCTGCGAGCAGCTGTGCCCAAGAATGTGTCTGTGGCTGAAGGAAAGGAACTGGACCTGACCTGTAACATCACAACAGACCGAGCCGATGACGTCCGGCCCGAGGTGACGTGGTCCTTCAGCAGGATGCCTGACAGCACCCTACCTGGCTCCCGCGTGTTGGCGCGGCTTGACCGTGATTCCCTGGTGCACAGCTCGCCTCATGTTGCTTTGAGTCATGTGGATGCACGCTCCTACCATTTACTGGTTCGGGATGTTAGCAAAGAAAACTCTGGCTACTATTACTGCCACGTGTCCCTGTGGGCACCCGGACACAACAGGAGCTGGCACAAAGTGGCAGAGGCCGTGTCTTCCCCAGCTGGTGTGGGTGTGACCTGGCTAGAACCAGACTACCAGGTGTACCTGAATGCTTCCAAGGTCCCCGGGTTTGCGGATGACCCCACAGAGCTGGCATGCCGGGTGGTGGACACGAAGAGTGGGGAGGCGAATGTCCGATTCACGGTTTCGTGGTACTACAGGATGAACCGGCGCAGCGACAATGTGGTGACCAGCGAGCTGCTTGCAGTCATGGACGGGGACTGGACGCTAAAATATGGAGAGAGGAGCAAGCAGCGGGCCCAGGATGGAGACTTTATTTTTTCTAAGGAACATACAGACACGTTCAATTTCCGGATCCAAAGGACTACAGAGGAAGACAGAGGCAATTATTACTGTGTTGTGTCTGCCTGGACCAAACAGCGGAACAACAGCTGGGTGAAAAGCAAGGATGTCTTCTCCAAGCCTGTTAACATATTTTGGGCATTAGAAGATTCCGTGCTTGTGGTGAAGGCGAGGCAGCCAAAGCCTTTCTTTGCTGCCGGAAATACATTTGAGATGACTTGCAAAGTATCTTCCAAGAATATTAAGTCGCCACGCTACTCTGTTCTCATCATGGCTGAGAAGCCTGTCGGCGACCTCTCCAGTCCCAATGAAACGAAGTACATCATCTCTCTGGACCAGGATTCTGTGGTGAAGCTGGAGAATTGGACAGATGCATCACGGGTGGATGGCGTTGTTTTAGAAAAAGTGCAGGAGGATGAGTTCCGCTATCGAATGTACCAGACTCAGGTCTCAGACGCAGGGCTGTACCGCTGCATGGTGACAGCCTGGTCTCCTGTCAGGGGCAGCCTTTGGCGAGAAGCAGCAACCAGTCTCTCCAATCCTATTGAGATAGACTTCCAAACCTCAGGTCCTATATTTAATGCTTCTGTGCATTCAGACACACCATCAGTAATTCGGGGAGATCTGATCAAATTGTTCTGTATCATCACTGTCGAGGGAGCAGCACTGGATCCAGATGACATGGCCTTTGATGTGTCCTGGTTTGCGGTGCACTCTTTTGGCCTGGACAAGGCTCCTGTGCTCCTGTCTTCCCTGGATCGGAAGGGCATCGTGACCACCTCCCGGAGGGACTGGAAGAGCGACCTCAGCCTGGAGCGCGTGAGTGTGCTGGAATTCTTGCTGCAAGTGCATGGCTCCGAGGACCAGGACTTTGGCAACTACTACTGTTCCGTGACTCCATGGGTGAAGTCACCAACAGGTTCCTGGCAGAAGGAGGCAGAGATCCACTCCAAGCCCGTTTTTATAACTGTGAAGATGGATGTGCTGAACGCCTTCAAGTATCCCTTGCTGATCGGCGTCGGTCTGTCCACGGTCATCGGGCTCCTGTCCTGTCTCATCGGGTACTGCAGCTCCCACTGGTGTTGTAAGAAGGAGGTTCAGGAGACACGGCGCGAGCGCCGCAGGCTCATGTCGATGGAGATGGACgaattcggcggaggcgggtccATGGCAGTTCCCGAGACCCGCCCTAACCACACTATTTATATCAACAACCTCAATGAGAAGATCAAGAAGGATGAGCTAAAAAAGTCCCTGTACGCCATCTTCTCCCAGTTTGGCCAGATCCTGGATATCCTGGTATCACGGAGCCTGAAGATGAGGGGCCAGGCCTTTGTCATCTTCAAGGAGGTCAGCAGCGCCACCAACGCCCTGCGCTCCATGCAGGGTTTCCCTTTCTATGACAAACCTATGCGTATCCAGTATGCCAAGACCGACTCAGATATCATTGCCAAGATGAAATAG

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

[0085] SEQ ID NO. 41 (aa PRGFRN-Ula):MGRLASRPLLLALLSLALCRGRVVRVPTATLVRVVGTELVIPCNVSDYDGPSEQNFDWSFSSLGSSFVELASTWEVGFPAQLYQERLQRGEILLRRTANDAVELHIKNVQPSDQGHYKCSTPSTDATVQGNYEDTVQVKVLADSLHVGPSARPPPSLSLREGEPFELRCTAASASPLHTHLALLWEVHRGPARRSVLALTHEGRFHPGLGYEQRYHSGDVRLDTVGSDAYRLSVSRALSADQGSYRCIVSEWIAEQGNWQEIQEKAVEVATVVIQPSVLRAAVPKNVSVAEGKELDLTCNITTDRADDVRPEVTWSFSRMPDSTLPGSRVLARLDRDSLVHSSPHVALSHVDARSYHLLVRDVSKENSGYYYCHVSLWAPGHNRSWHKVAEAVSSPAGVGVTWLEPDYQVYLNASKVPGFADDPTELACRVVDTKSGEANVRFTVSWYYRMNRRSDNVVTSELLAVMDGDWTLKYGERSKQRAQDGDFIFSKEHTDTFNFRIQRTTEEDRGNYYCVVSAWTKQRNNSWVKSKDVFSKPVNIFWALEDSVLVVKARQPKPFF AAGNTFEMTCKVSSKNIKSPRYSVLIMAEKPVGDLSSPNETKYIISLDQDSVVKLENW TDASRVDGVVLEKVQEDEFRYRMYQTQVSDAGLYRCMVTAWSPVRGSLWREAAT SLSNPIEIDFQTSGPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFA VHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLSLERVSVLEFLLQVHGSEDQDFGN YYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKYPLLIGVGLSTVIGLLSC LIGYCSSHWCCKKEVQETRRERRRLMSMEMDEFGGGGSMAVPETRPNHTIYINNLN EKIKKDELKKSLYAIFSQFGQILDILVSRSLKMRGQAFVIFKEVSSATNALRSMQGFPF YDKPMRIQYAKTDSDIIAKMK

[0086] In an embodiment, the cargo RNA comprises a package RNA comprising any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention and a packaging domain capable of binding to the packaging protein of the fusion protein. In an embodiment, the packaging domain comprises UR domain or L2 domain. In an embodiment the nucleotide sequence of the UR domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 42 and the nucleotide sequence of the L2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 43. In an embodiment, the package RNA comprises any embodiment of the HNF4A-AS 1 targeting polynucleotides of the present invention.

[0087] In an embodiment, the packaging domain further comprises stabilizing domains. In an embodiment, the stabilizing domain comprises OH domain and / or MorrisMotif domain wherein the nucleotide sequence of the OH domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 44 and the nucleotide sequence of the MorrisMotif domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 45.

[0088] SEQ ID NO. 42 (UR): AATCCATTGCACTCCGGATT

[0089] SEQ ID NO. 43 (L2): AATCCATTGCACTCCGGATTT

[0090] SEQ ID NO. 44 (OH): GACGTCTATAGGTCGTGTCACCG

[0091] SEQ ID NO. 45 (MorrisMotif): CGCGTCGCGCGCGTCGCG

[0092] In an embodiment, the cargo RNA further comprises a SIRLOIN (SINE- derived nuclear RNA LOcalizatloN) nuclear localization sequences wherein the SIRLOIN is upstream of the UR or L2 sequence of the cargo RNA. In an embodiment, the nucleotide sequence of SIRLOIN is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 46.

[0093] SEQ ID NO. 46:CGCCTCCCGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGA

[0094] In an embodiment, the exosome-based packaging and delivery system further comprises an argonaute 2 (Ago2)-encoding plasmid. In an embodiment, the Ago2 comprises S387A mutation. Overexpression of Ago2 or the S387A mutant thereof increases the packaging efficiency of the cargo RNA into the exosomes in an exosome-producing cell.

[0095] In an embodiment, the pharmaceutical composition comprising a nanoparticle encapsulating any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention further comprises a nanoparticle payload release enhancer. In an embodiment, the nanoparticle release enhancer enhances the release of payload from the nanoparticle 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 an embodiment, the nanoparticle payload release enhancer comprises a modified myoferlin protein. In an embodiment, the modified myoferlin protein of the present invention comprises C2F, C2G, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein of the present invention consists of C2F, C2G, transmembrane domain. In an embodiment, the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO 47. In an embodiment, the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 48. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 49.

[0096] SEQ ID NO. 47 (C2F): QFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRDHYIPNTLN PVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFGSHCGIPEE YCVSGV

[0097] SEQ ID NO. 48 (C2G):PFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPGNEENKQKTDV HYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIPPRLIIQIWDND KFSLDDYLGFLELDLRH

[0098] SEQ ID NO. 49 (transmembrane domain): PDLKAMNPLKAKTASLFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEA DERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLI LLLFVAV

[0099] In an embodiment, the modified myoferlin protein comprises C2A, FerA, FerB, DysFN, transmembrane domain, or a combination thereof. In an embodiment, the modified myoferlin protein consists of C2A, FerA, FerB, DysFN, transmembrane domain. In an embodiment, the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 50. In an embodiment, the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 51. In an embodiment, the amino acid sequence of the FerB is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 52. In an embodiment, the DysFN domain comprises DysFN- 1, DysFN-2, or a combination thereof. In an embodiment, the amino acid sequence of the DysFN- 1 is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 53. In an embodiment, the amino acid sequence of the DysFN-2 is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 54. In an embodiment, the amino acid sequence of the transmembrane domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 49. [000100] SEQ ID NO. 50 (C2A):MLRVIVESASNIPKTKFGKPDPIVSVIFKDEKKKTKKVDNELNPVWNEILEFDLRGIPL DFSSSLGIIVKDFETIGQNKLIGTATVALKDLTGDQSRSLPYKLISLLNERGQDTGATID LVIGYDPPSAPHPNDLS[000101] SEQ ID NO. 51 (FerA):LQTNIEALKSGIQGKIPANQLAELWLKLIDEVIEDTRYTLPLTEGKANVTVLDTQIRK [000102] SEQ ID NO. 52 (FerB):[000103] WLDKLMQLTEEPQNSMPDIIIWMIRGEKRLAYARIPAHQVLYSTSGENASGKYCGKTQTIFLKYPQEKNNGP[000104] SEQ ID NO. 53 (DysFN-1):AVEKKFNSFAEGTFTVFAEMYENQALMFGKWGTSGLVGRHKFSDVTGKIKLKREFF LP[000105] SEQ ID NO. 54 (DysFN-2):DPERSLLTEADAGHTEFTDEVYQNESRYPGGDWKPAEDTYTDANGDKAASPSELTC P[000106] In an embodiment, any embodiment of the modified myoferlin protein of the present invention further comprises C2B, C2C, C2D, C2E domains, or a combination thereof. In an embodiment, the amino acid sequence of the C2B domain is at least about 80%, about85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 55. In an embodiment, the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 56. In an embodiment, the amino acid sequence of the C2D is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 57. In an embodiment, the amino acid sequence of the C2E is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 58.[000107] SEQ ID NO. 55 (C2B):PQDFQIRVRVIEGRQLSGNNIRPVVKVHVCGQTHRTRIKRGNNPFFDELFFYNVNMT PSELMDEIISIRVYNSHSLRADCLMGEFKIDVGFVYDEPGHAVMRKWLLLNDP [000108] SEQ ID NO. 56 (C2C): TFLLKIYRAEDIPQMDDAFSQTVKEIFGGNADKKNLVDPFVEVSFAGKKVCTNIIEKN ANPEWNQVVNLQIKFPSVCEKIKLTIYDWDRLTKNDVVGTTYLHLSKIAASGGEVED FSSSGTGAASYTVNTGETEVGFVPTFGPCYLNLYGSPREYTGFPDPYDE [000109] SEQ ID NO. 57 (C2D): TP1VSCNFDRVYIYHLRCYVYQARNLLALDKDSFSDPYAHICFLHRSKTTE1IHSTLNP TWDQTIIFDEVEIYGEPQTVLQNPPKVIMELFDNDQVGKDEFLGRSIFSPVVKLNSEM DITPKLLWHPVMNGDKA[000110] SEQ ID NO. 58 (C2E):RNMKNFQMASITSPSLVVECGGERVESVVIKNLKKTPNFPSSVLFMKVFLPKEELYM PPLVIKVIDHRQFGRKPVVG[000111] In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2A domain wherein the amino acid sequence of the C2A domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 50. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2B domain wherein the amino acid sequence of the C2B domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ DI NO. 55. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2C domain wherein the amino acid sequence of the C2C domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ DI NO. 56. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2D domain wherein the amino acid sequence of the C2D domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 57. In an embodiment, the modified myoferlin protein of the present inventiondoes not comprise the C2E domain wherein the amino acid sequence of the C2E domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 58. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2F domain wherein the amino acid sequence of the C2F domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 47. In an embodiment, the modified myoferlin protein of the present invention does not comprise the C2G domain wherein the amino acid sequence of the C2G domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 48. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerA domain wherein the amino acid sequence of the FerA domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 51. In an embodiment, the modified myoferlin protein of the present invention does not comprise the FerB domain wherein the amino acid sequence of the FerB domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 52. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-1 domain wherein the amino acid sequence of the DysFN-1 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 53. In an embodiment, the modified myoferlin protein of the present invention does not comprise the DysFN-2 domain wherein the amino acid sequence of the DysFN-2 domain is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 54.[000112] In an embodiment, amino acid sequence of the modified myoferlin protein of the present invention is at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 59.[000113] SEQ ID NO. 59 (C2F-C2G) :MVPAPPRQFRELPDSVPQECTVRIYIVRGLELQPQDNNGLCDPYIKITLGKKVIEDRD HYIPNTLNPVFGRMYELSCYLPQEKDLKISVYDYDTFTRDEKVGETIIDLENRFLSRFG SHCGIPEEYCVSGVNTWRDQLRPTQLLQNVARFKGFPQPILSEDGSRIRYGGRDYSLD EFEANKILHQHLGAPEERLALHILRTQGLVPEHVETRTLHSTFQPNISQGKLQMWVD VFPKSLGPPGPPFNITPRKAKKYYLRVIIWNTKDVILDEKSITGEEMSDIYVKGWVPG NEENKQKTDVHYRSLDGEGNFNWRFVFPFDYLPAEQLCIVAKKEHFWSIDQTEFRIP PRLIIQIWDNDKFSLDDYLGFLELDLRHTIIPAKSPEKCRLDMIPDLKAMNPLKAKTAS LFEQKSMKGWWPCYAEKDGARVMAGKVEMTLEILNEKEADERPAGKGRDEPNMNPKLDLPNRPETSFLWFTNPCKTMKFIVWRRFKWVIIGLLFLLILLLFVAVLLYSLPNYL SMKIVKPNVYPYDVPDYA[000114] The present invention also provides a modified myoferlin protein encoding polynucleotide encoding any embodiment of the modified myoferlin protein of the present invention. In an embodiment, the modified myoferlin protein encoding polynucleotide encodes a C2A domain, a C2B domain, a C2C domain, a C2D domain, a C2E domain, a C2F domain, a C2G domain, a FerA domain, a FerB domain, a DysFN-1 domain, a DysFN-2 domain, a transmembrane domain, or a combination thereof. In an embodiment, the C2A domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 60. In an embodiment, the C2B domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 61. In an embodiment, the C2C domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 62. In an embodiment, the C2D domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 63. In an embodiment, the C2E domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 64. In an embodiment, the C2F domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 65. In an embodiment, the C2G domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 66. In an embodiment, the FerA domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 67. In an embodiment, the FerB domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 68. In an embodiment, the DysFN-1 domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 69. In an embodiment, the DysFN-2 domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%,about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 70. In an embodiment, the transmembrane domain is encoded by a polynucleotide wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 71.[000115] SEQ ID NO. 60 (nt C2A):ATGCTGCGAGTGATTGTGGAATCTGCCAGCAATATCCCTAAAACGAAATTTGGCA AGCCGGATCCTATTGTTTCTGTCATTTTTAAGGATGAGAAAAAGAAAACAAAGA AAGTTGATAATGAATTGAACCCTGTCTGGAATGAGATTTTGGAGTTTGACTTGAG GGGTATACCACTGGACTTTTCATCTTCCCTTGGGATTATTGTGAAAGATTTTGAGA CAATTGGACAAAATAAATTAATTGGCACGGCGACTGTAGCCCTGAAGGACCTGA CTGGTGACCAGAGCAGATCCCTGCCGTACAAGCTGATCTCCCTGCTAAATGAAAG AGGGCAAGATACTGGGGCCACCATTGACTTGGTGATCGGCTATGATCCGCCTTCT GCTCCACATCCAAATGACCTGAGC [000116] SEQ ID NO. 61 (nt C2B):CCACAGGACTTCCAGATCCGCGTCCGAGTGATTGAGGGCCGACAGTTAAGTGGC AACAACATAAGGCCTGTGGTCAAAGTTCACGTCTGTGGCCAGACACACCGAACA AGAATCAAGAGAGGAAACAACCCTTTTTTTGATGAGTTGTTTTTCTACAATGTCA ACATGACCCCTTCTGAATTGATGGATGAGATCATCAGCATCCGGGTTTATAATTC TCACTCTCTGCGGGCAGATTGTCTGATGGGGGAATTTAAGATTGATGTTGGATTT GTTTATGATGAACCTGGCCATGCTGTCATGAGAAAGTGGCTTCTTCTCAATGACC CG[000117] SEQ ID NO. 62 (nt C2C):ACCTTCTTGCTGAAAATCTACCGAGCTGAGGACATCCCCCAGATGGATGATGCCT TCTCACAGACAGTAAAGGAAATATTTGGAGGCAATGCAGATAAGAAAAATCTCG TGGATCCTTTTGTAGAAGTTTCCTTTGCTGGAAAAAAGGTTTGTACAAACATAAT TGAGAAGAATGCAAACCCAGAGTGGAATCAGGTCGTCAATCTTCAGATCAAGTT TCCTTCAGTGTGTGAAAAAATAAAACTAACAATATATGACTGGGACCGTCTTACT AAAAATGATGTAGTTGGAACAACATATCTACACCTCTCTAAAATTGCTGCCTCTG GTGGGGAAGTGGAAGATTTCTCATCTTCGGGAACTGGGGCTGCATCATATACAGT AAACACAGGAGAAACAGAGGTAGGCTTTGTTCCAACGTTTGGACCTTGTTACCTG AATCTTTATGGAAGCCCCAGAGAGTACACGGGATTCCCAGACCCCTATGATGAG [000118] SEQ ID NO. 63 (nt C2D):ACCCCCATTGTTTCCTGCAATTTTGACAGAGTCTACATCTACCATCTGCGCTGCTA TGTCTATCAAGCCAGAAACCTCTTGGCTTTAGATAAGGATAGCTTTTCAGATCCATATGCTCATATCTGTTTCCTCCATCGGAGCAAAACCACTGAGATCATCCATTCAACCCTGAATCCCACGTGGGACCAAACAATTATATTCGATGAAGTTGAAATCTATGGGGAACCCCAAACAGTTCTACAGAATCCACCCAAAGTTATCATGGAACTTTTTGACAATGACCAAGTGGGCAAAGATGAATTTTTAGGACGAAGCATTTTCTCTCCTGTGGTGAAACTGAACTCAGAAATGGACATCACACCCAAACTTCTCTGGCACCCAGTAATGAATGGAGACAAAGCC[000119] SEQ ID NO. 64 (nt C2E):AGAAATATGAAAAACTTCCAGATGGCTTCTATCACATCCCCCAGTCTTGTTGTGGAGTGTGGAGGAGAAAGGGTGGAATCGGTGGTGATCAAAAACCTTAAGAAGACACCCAACTTTCCAAGTTCTGTTCTCTTCATGAAAGTGTTCTTGCCCAAGGAGGAATTGTACATGCCCCCACTGGTGATCAAGGTCATCGACCACAGGCAGTTTGGGCGGAAGCCTGTCGTCGGC[000120] SEQ ID NO. 65 (nt C2F):CAGTTTCGGGAATTACCTGACAGCGTCCCACAGGAATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAACAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGAAGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTACGAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATTATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAAACCGATTCCTTTCCCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGTTTCTGGAGTC[000121] SEQ ID NO. 66 (nt C2G):CCTTTCAACATCACACCCCGGAAAGCCAAGAAATACTACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCATCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATGAAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGAATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGTATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCCCACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTACTTGGGTTTCCTAGAACTTGACTTGCGTCAC[000122] SEQ ID NO. 67 (nt FerA):CTGCAAACAAATATAGAAGCTCTAAAATCAGGGATACAAGGTAAAATTCCTGCAAACCAGCTGGCTGAATTGTGGCTGAAGCTGATAGATGAAGTTATAGAAGACACGAGATACACGTTGCCTCTCACAGAAGGAAAAGCCAACGTCACAGTTCTCGATACTCAGATCCGAAAG[000123] SEQ ID NO. 68 (nt FerB):TGGCTTGATAAATTAATGCAGCTGACTGAAGAGCCACAGAACAGCATGCCTGAC ATCATCATCTGGATGATCCGGGGAGAGAAGAGACTGGCCTATGCACGAATTCCC GCACATCAGGTCTTGTACTCCACCAGTGGTGAGAATGCATCTGGAAAATACTGTG GGAAAACCCAAACCATCTTTCTGAAGTATCCACAGGAGAAAAACAACGGGCCA [000124] SEQ ID NO. 69 (nt DysFN-1):GCTGTGGAGAAGAAGTTTAACAGCTTCGCAGAAGGAACTTTCACCGTCTTTGCTG AAATGTATGAAAATCAAGCTCTCATGTTTGGAAAATGGGGTACTTCTGGATTAGT AGGACGTCATAAGTTTTCTGATGTCACAGGAAAAATAAAACTCAAGAGGGAATT TTTTCTGCCT[000125] SEQ ID NO. 70 (nt DysFN-2):GATCCTGAAAGAAGCTTGCTGACTGAGGCAGATGCAGGTCACACGGAGTTCACT GATGAAGTCTACCAGAACGAGAGCCGCTACCCCGGGGGCGACTGGAAGCCGGCC GAGGACACCTACACGGATGCGAACGGCGATAAAGCAGCATCACCCAGCGAGTTG ACTTGTCCT1000126] SEQ ID NO. 71 (nt TM):CCGGACCTCAAAGCCATGAACCCCCTTAAAGCCAAGACAGCCTCCCTCTTTGAGC AGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCG TAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCG ACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTG GACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGA CCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTT CCTGCTTATCCTGCTGCTCTTCGTGGCCGTG[000127] In an embodiment, the modified myoferlin protein of the present invention further comprises a connexin 43 protein to form a dual protein. The dual protein comprising the modified myoferlin protein and the connexin 43 protein of the present invention can be used to greatly enhance the efficacy and delivery of the RNA or nucleic acid pay loads. In an embodiment, the connexin 43 protein comprises a S368A mutation. In an embodiment, the connexin protein of the present invention comprises an amino acid sequence at least about 80%, about 85%, about 90%, about 95% or about 100% identical or similar to SEQ ID NO. 72.[000128] SEQ ID NO. 72 (connexin 43):MATTMGDWSALGKLLDKVQAYSTAGGKVWLSVLFIFRILLLGTAVESAWGDEQSA FRCNTQQPGCENVCYDKSFPISHVRFWVLQIIFVSVPTLLYLAHVFYVMRKEEKLNKKEEELKVAQTDGVNVDMHLKQIEIKKFKYGIEEHGKVKMRGGLLRTYIISILFKSIFE VAFLLIQWYIYGFSLSAVYTCKRDPCPHQVDCFLSRPTEKTIFIIFMLVVSLVSLALNII ELFYVFFKGVKDRVKGKSDPYHATSGALSPAKDCGSQKYAYFNGCSSPTAPLSPMSP PGYKLVTGDRNNSSCRNYNKQASEQNWANYSAEQNRMGQAGSTISNSHAQPFDFP DDNQNSKKLAAGHELQPLAIVDQRPSSRAASRASSRPRPDDLEI[000129] The present invention further provides a polynucleotide encoding the dual protein comprising a modified myoferlin protein of the present invention fused to a connexin 43 protein S368A mutant wherein nucleotide sequence of the polynucleotide is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to SEQ ID NO. 73.[000130] SEQ ID NO. 73:ATGGTGCCAGCCCCTCCCAGACAGTTTCGGGAATTACCTGACAGCGTCCCACAGG AATGCACGGTTAGGATTTACATTGTTCGAGGCTTAGAGCTCCAGCCCCAGGACAA CAATGGCCTGTGTGACCCTTACATAAAAATAACACTGGGCAAAAAAGTCATTGA AGACCGAGATCACTACATTCCCAACACTCTCAACCCAGTCTTTGGCAGGATGTAC GAACTGAGCTGCTACTTACCTCAAGAAAAAGACCTGAAAATTTCTGTCTATGATT ATGACACCTTTACCCGGGATGAAAAAGTAGGAGAGACAATTATTGATCTGGAAA ACCGATTCCTTTCCCGCTTTGGGTCCCACTGCGGCATACCAGAGGAGTACTGTGT TTCTGGAGTCAATACCTGGCGAGATCAACTGAGACCAACACAGCTGCTTCAAAAT GTCGCCAGATTCAAAGGCTTCCCACAACCCATCCTTTCCGAAGATGGGAGTAGAA TCAGATATGGAGGACGAGACTACAGCTTGGATGAATTTGAAGCCAACAAAATCC TGCACCAGCACCTCGGGGCCCCTGAAGAGCGGCTTGCTCTTCACATCCTCAGGAC TCAGGGGCTGGTCCCTGAGCACGTGGAAACAAGGACTTTGCACAGCACCTTCCA GCCCAACATTTCCCAGGGAAAACTTCAGATGTGGGTGGATGTTTTCCCCAAGAGT TTGGGGCCACCAGGCCCTCCTTTCAACATCACACCCCGGAAAGCCAAGAAATAC TACCTGCGTGTGATCATCTGGAACACCAAGGATGTTATCTTGGATGAGAAAAGCA TCACAGGAGAGGAAATGAGTGACATCTACGTCAAAGGCTGGGTTCCTGGCAATG AAGAAAACAAACAGAAAACAGATGTCCATTACAGATCTTTGGATGGTGAAGGGA ATTTTAACTGGCGATTTGTTTTCCCGTTTGACTACCTTCCAGCCGAACAACTCTGT ATCGTTGCGAAAAAAGAGCATTTCTGGAGTATTGACCAAACGGAATTTCGAATCC CACCCAGGCTGATCATTCAGATATGGGACAATGACAAGTTTTCTCTGGATGACTA CTTGGGTTTCCTAGAACTTGACTTGCGTCACACGATCATTCCTGCAAAATCACCA GAGAAATGCAGGTTGGACATGATTCCGGACCTCAAAGCCATGAACCCCCTTAAA GCCAAGACAGCCTCCCTCTTTGAGCAGAAGTCCATGAAAGGATGGTGGCCATGCTACGCAGAGAAAGATGGCGCCCGCGTAATGGCTGGGAAAGTGGAGATGACATTGGAAATCCTCAACGAGAAGGAGGCCGACGAGAGGCCAGCCGGGAAGGGGCGGGACGAACCCAACATGAACCCCAAGCTGGACTTACCAAATCGACCAGAAACCTCCTTCCTCTGGTTCACCAACCCATGCAAGACCATGAAGTTCATCGTGTGGCGCCGCTTTAAGTGGGTCATCATCGGCTTGCTGTTCCTGCTTATCCTGCTGCTCTTCGTGGCCGTGCTCCTCTACTCTTTGCCGAACTATTTGTCAATGAAGATTGTAAAGCCAAATGTGTACCCATACGACGTCCCAGACTACGCTTAGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGGGTGACTGGAGCGCCTTAGGCAAACTCCTTGACAAGGTTCAAGCCTACTCAACTGCTGGAGGGAAGGTGTGGCTGTCAGTACTTTTCATTTTCCGAATCCTGCTGCTGGGGACAGCGGTTGAGTCAGCCTGGGGAGATGAGCAGTCTGCCTTTCGTTGTAACACTCAGCAACCTGGTTGTGAAAATGTCTGCTATGACAAGTCTTTCCCAATCTCTCATGTGCGCTTCTGGGTCCTGCAGATCATATTTGTGTCTGTACCCACACTCTTGTACCTGGCTCATGTGTTCTATGTGATGCGAAAGGAAGAGAAACTGAACAAGAAAGAGGAAGAACTCAAGGTTGCCCAAACTGATGGTGTCAATGTGGACATGCACTTGAAGCAGATTGAGATAAAGAAGTTCAAGTACGGTATTGAAGAGCATGGTAAGGTGAAAATGCGAGGGGGGTTGCTGCGAACCTACATCATCAGTATCCTCTTCAAGTCTATCTTTGAGGTGGCCTTCTTGCTGATCCAGTGGTACATCTATGGATTCAGCTTGAGTGCTGTTTACACTTGCAAAAGAGATCCCTGCCCACATCAGGTGGACTGTTTCCTCTCTCGCCCCACGGAGAAAACCATCTTCATCATCTTCATGCTGGTGGTGTCCTTGGTGTCCCTGGCCTTGAATATCATTGAACTCTTCTATGTTTTCTTCAAGGGCGTTAAGGATCGGGTTAAGGGAAAGAGCGACCCTTACCATGCGACCAGTGGTGCGCTGAGCCCTGCCAAAGACTGTGGGTCTCAAAAATATGCTTATTTCAATGGCTGCTCCTCACCAACCGCTCCCCTCTCGCCTATGTCTCCTCCTGGGTACAAGCTGGTTACTGGCGACAGAAACAATTCTTCTTGCCGCAATTACAACAAGCAAGCAAGTGAGCAAAACTGGGCTAATTACAGTGCAGAACAAAATCGAATGGGGCAGGCGGG AAGCACCATCTCTAACTCCCATGCACAGCCTTTTGATTTCCCCGATGATAACCAG AATTCAAAAAAACTAGCTGCTGGACATGAATTACAGCCACTAGCCATTGTGGAC CAGCGACCTTCAAGCAGAGCCGCCAGTCGTGCCAGCAGCAGACCTCGGCCTGAT GACCTGGAGATCTGA[000131] In an embodiment, the various components comprising the fusion protein or any variant thereof of the present invention, the Ago2 protein or any variant thereof of the present invention, the modified myoferlin protein or any variant thereof of the present invention, or the connexin 43 protein or any variant thereof of the present invention that could be incorporated or encapsulated to the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention for the enhanced delivery and uptake of said nanoparticle in a subject can be incorporated or encapsulated to the nanoparticle in all possible combinations. In an embodiment, the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention further comprises a fusion protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A-AS1 targeting polynucleotides of the present invention further comprises an Ago2 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention further comprises a modified myoferlin protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A-AS1 targeting polynucleotides of the present invention further comprises a connexin 43 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention does not comprise a fusion protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A-AS 1 targeting polynucleotides of the present invention does not comprise an Ago2 protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention does not comprise a modified myoferlin protein or any variant thereof of the present invention. In an embodiment, the nanoparticle encapsulating the one or more HNF4A- AS 1 targeting polynucleotides of the present invention does not comprise a connexin 43 protein or any variant thereof of the present invention. In an embodiment where the nanoparticle comprises an exosome encapsulating one or more HNF4 A- AS 1 targetingpolynucleotides of the present study and a fusion protein comprising an exosome-associated transmembrane protein fused to a packaging protein of the present invention, the one or more HNF4A- AS 1 targeting polynucleotides is fused to a packaging domain or any variant thereof of the present invention. In an embodiment, the packaging domain or any variant thereof of the present invention comprises UR, L2, the OH domain, the MorrisMotif domain, the nuclear localization sequence SIRLOIN, or a combination thereof.[000132] In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A-AS1 targeting polynucleotide of the present invention upregulates the expression of at least one of the one or more HNF4A gene Pl isoform of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotide of the present invention restores the expression of at least one of the one or more HNF4A gene Pl isoform of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the at least one of the one or more HNF4A gene Pl isoform of the subject when healthy. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotide of the present invention restores the expression of at least one of the one or more HNF4A gene Pl isoform of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the at least one of the one or more HNF4A gene Pl isoform of an average healthy human being. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotide of the present invention restores the expression of at least one of the one or more HNF4A gene Pl isoform of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the at least one of the one or more HNF4A gene Pl isoform of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.[000133] In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A-AS 1 targeting polynucleotide of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotide of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a subject to less than 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of the normal expression level of the at least one of the one or more HNF4A gene P2 isoform of the subject when healthy. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotide of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a human subject to less than about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of normal expression level of the at least one of the one or more HNF4A gene P2 isoform of an average healthy human being. In an embodiment, any embodiment of the nanoparticle encapsulating the any embodiment of the HNF4A-AS 1 targeting polynucleotide of the present invention downregulates the expression of at least one of the one or more HNF4A gene P2 isoform of a human subject to less than about 500%, about 450%, about 400%, about 350%, about 300%, about 250%, about 200%, about 150%, or about 100% of normal expression level of the at least one of the one or more HNF4A gene P2 isoform of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.[000134] In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A-AS 1 targeting polynucleotides of the present invention increases the P1:P2 isoform ratio of a subject by at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention restores the P1:P2 isoformratio of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the one or more HNF4A Pl isoform of the subject when healthy. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the increases the P1:P2 isoform ratio of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A Pl isoform of an average healthy human being. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the P1:P2 isoform ratio of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A Pl isoform of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.[000135] In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention upregulates the expression of the HNF4A gene of a subject by at least about 1 %, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 150%, about 200%, about 250%, about 300%, about 350%, about 500%, about 750% or about 1000%. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A-AS1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of the normal expression level of the HNF4A gene of the subject when healthy. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the HNF4Agene of an average healthy human being. In an embodiment, any embodiment of the nanoparticle encapsulating any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention restores the expression of the HNF4A gene of a human subject to at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or about 100% of normal expression level of the one or more HNF4A gene of an average healthy human being having the same biometrics of the subject such as age, sex, height, weight etc. . . or a combination thereof.[000136] The present invention further provides a method of increasing or restoring expression of HNF4A in a subject comprising the administration of a therapeutically effective amount of any embodiment of the one or more HNF4A-AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention to the subject. In an embodiment, the method of increasing or restoring the HNF4A gene expression in a subject comprises the step of downregulating the HNF4A-AS1 expression. In an embodiment, the method of increasing or restoring HNF4A gene expression in a subject of the present invention comprises the step of suppression of HNF4A P2 isoform expression in the subject. In an embodiment, the step of suppression of HNF4A P2 isoform expression in the subject is performed by administration of any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention to the subject. In an embodiment, the method of increasing or restoration of HNF4A gene expression in a subject of the present invention comprises the step of upregulation of HNF4A Pl isoform expression in the subject. In an embodiment, the step of upregulation of HNF4A Pl isoform expression in the subject is performed by administration of any embodiment of the HNF4A- AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention to the subject. In an embodiment, the subject is diagnosed with a HNF4A- associated disease. In an embodiment, the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD), polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY). Liver failure, the result of differing causes, can also be treated.[000137] The present invention also provides a method of increasing or restoring the HNF4A P1:P2 isoform ratio in a subject comprising the administration of a therapeutically effective amount of any embodiment of the one or more HNF4A regulating polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention to the subject. In an embodiment, increasing or restoring the HNF4A P1:P2 isoform ratio in a subject comprises the step of downregulating the HNF4A-AS1 expression. In an embodiment, the method of increasing or restoring HNF4A P 1 :P2 isoform ratio of the present invention comprises the step of suppression of HNF4A P2 isoform expression. In an embodiment, the method of increasing or restoring of HNF4A P1:P2 isoform ratio of the present invention comprises the step of upregulation of HNF4A Pl isoform expression. In an embodiment, the subject is diagnosed with a HNF4A-associated disease. In an embodiment, the step of suppression of HNF4A P2 isoform expression or the step of upregulation of HNF4A Pl isoform expression is performed by administration of any embodiment of the HNF4A-A1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention to the subject. In an embodiment, the subject is diagnosed with a HNF4A-associated disease. In an embodiment, the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD), polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY). Liver failure, the result of differing causes, can also be treated.[000138] The present invention also provides a method of treatment of a HNF4A- associated disease of a subject comprising the step of altering the HNF4A Pl :P2 isoform ratio in the subject. In an embodiment, the step of altering the HNF4A Pl :P2 isoform ratio in the subject comprises increasing or restoring HNF4A Pl :P2 isoform ratio in the subject. In an embodiment, the step of increasing the HNF4A P1:P2 isoform ratio comprises the step of administration of a therapeutically effective amount of any embodiment of the pharmaceutical composition comprising one or more HNF4A regulating polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention. In an embodiment, the alteration of HNF4A P1:P2 isoform ratio comprises the suppression of HNF4A P2 isoform expression. In an embodiment, the alteration of ratio between HNF4APl -specific transcript and HNF4A P2-transcript comprises the upregulation of HNF4A Pl isoform expression.[000139] The present invention provides a method of suppressing at least one HNF4A P2 isoform expression in a subject comprising the administration of a therapeutically effective amount of any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention to the subject. In an embodiment, the at least one HNF4A P2 isoform suppressed by the method of the present invention comprises the at least a part of HNF4A F domain. In an embodiment, the at least one HNF4A P2 isoform suppressed by the method of the present invention does not comprise a full length HNF4A LBD.[000140] The present invention also provides a method of altering the HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform in a subject comprising the administration of a therapeutically effective amount of any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A-AS1 targeting polynucleotides of the present invention to the subject. In an embodiment, increasing the HNF4A P1:P2 isoform ratio of at least a pair of Pl and P2 isoform in a subject comprises the step of downregulating the HNF4A- AS 1 expression. In an embodiment, the alteration of HNF4A P1:P2 isoform ratio of at least a pair of Pl and P2 isoform comprises the suppression of at least one HNF4A P2 isoform expression. In an embodiment, the alteration of HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform comprises the upregulation of at least one HNF4A Pl isoform expression. In an embodiment, the subject is diagnosed with a HNF4 A- associated disease. In an embodiment, the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD), polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY). Liver failure, the result of differing causes, can also be treated.[000141] The present invention also provides a method of treatment of a HNF4A- associated disease of a subject comprising the step of altering the HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform in the subject. In an embodiment, the step of altering the HNF4A P1:P2 isoform ratio of at least a pair of Pl and P2 isoform in the subject comprises increasing HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform. Inan embodiment, the step of increasing the HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform comprises the step of administration of a therapeutically effective amount of any embodiment of the pharmaceutical composition comprising one or more HNF4A- AS 1 targeting polynucleotides of the present invention. In an embodiment, the alteration of HNF4A P1:P2 isoform ratio of at least a pair of Pl and P2 isoform comprises the suppression of at least one HNF4A P2 isoform expression. In an embodiment, the alteration HNF4A Pl :P2 isoform ratio of at least a pair of Pl and P2 isoform comprises the upregulation of at least one HNF4A Pl isoform expression. In an embodiment, the at least one HNF4A P2 isoform suppressed by the method of treatment of the present invention comprises at least a part of the HNF4A F domain. In an embodiment, the at least one HNF4A P2 isoform suppressed by the method of treatment of the present invention does not comprise a full length HNF4A LBD.[000142] In an embodiment, the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, nonalcoholic fatty liver disease (NAFLD), liver failure of any cause, polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY).[000143] In an embodiment, the method of treatment of a HNF4A-associated disease comprises the step of administering a therapeutic effective amount of any embodiment of the pharmaceutical composition comprising one or more HNF4A- AS 1 targeting polynucleotides of the present invention or any embodiment of the nanoparticle encapsulating any embodiment of the one or more HNF4A- AS 1 targeting polynucleotides of the present invention to a subject. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agent comprising a site-specific targeting moiety to a particular location. For example, to target liver cells, intravenous injection may be used.[000144] In an embodiment, the method of treating a HNF4A-AS1 -associated disease comprises the step of administering about 1 to about 1000 billion of any embodiment of theexosomes, LNP, or a combination thereof encapsulating the one or more HNF4A-AS1 targeting polynucleotides of the present invention such as about 1, about 10, about 50, about 100, about 250, about 500, about 750, about 1000, about 1250, about 5000, about 7500, about 10000, about 12500, about 50000, about 75000, about 100000, about 125000, about 500000, about 750000, about 1000000, about 1250000, about 5000000, about 7500000, about 10000000, about 12500000, about 50000000, about 75000000, about 100000000, about 125000000, about 500000000, about 750000000, about 1 billion, about 10 billion, about 50 billion, about 100 billion, about 300 billion, about 600 billion, or about 1000 billion including any numbers or ranges of numbers falling within these values of exosomes encapsulating the one or more HNF4A-AS 1 targeting polynucleotides of the present invention. In an embodiment, the method of treatment of a HNF4A- AS 1 -associated disease comprises the step of administering about 0.01 to about 20 mg / kg of the body weight of the subject such as about 0.01, about 0.05, about 0.1, about 0.2, about 0.4, about 0.6, about 0.8, about 1, about 1.2, about 1.4, about 1.6, about 1.8, about 2, about 4, about 6, about 8, about 10, about 12, about 14, about 16, about 18, about 20 mg / kg, or any concentration or concentration ranges falling within these values of exosomes, LNP, or a combination thereof encapsulating the one or more HNF4A-AS1 targeting polynucleotides of the present invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration. The route and site of administration may be chosen to enhance delivery or targeting of the disrupting agent comprising a site-specific targeting moiety to a particular location. For example, to target liver cells, intravenous injection may be used.[000145] The pharmaceutical compositions may be administered in the form of any embodiment of the exosomes of the present invention. As used herein the term “exosome” refers to a cell-derived small (between 20-300 nm in diameter, more preferably 40-200 nm in diameter) vesicle comprising a membrane that encloses an internal space, and which is generated from said cell by direct plasma membrane budding or by fusion of the late endosome with the plasma membrane. The any embodiment of the exosome of the present invention comprises lipid or fatty acid and polypeptide and further comprises the inhibitory nucleic acids described herein as a payload. The any embodiment of the exosome of the present invention can be derived from a producer cell, and isolated from the producer cellbased on its size, density, biochemical parameters, or a combination thereof. The any embodiment of the exosome of the present invention can be directly loaded with exogenous nucleic acids or drugs by electroporation, lipofection, sonication and contact with calcium chloride. Alternatively, purified exosomes may be loaded ex vivo by, for example, electroporation.[000146] Any embodiment of the exosome of the present invention can be produced from a cell grown in vitro or a body fluid of a subject. When exosomes are produced from in vitro cell culture, various producer cells, e.g., HEK293 cells, Chinese hamster ovary 26 cells, or mesenchymal stem cells (MSCs), can be used.[000147] The pharmaceutical compositions may also be formulated by incorporation of the inhibitory nucleic acids described herein into adenoviruses or adeno-associated viruses (AAVs), formulated with cell-penetrating peptides, lentiviral vectors, polymers, dendrimers, or prepared as siRNA bioconjugates such as the GalNAc-siRNA conjugate delivery platform. [000148] If using the exosomes or a vector as a vehicle to deliver siRNA, the candidate siRNAs are delivered as shRNAs. Both siRNAs and shRNAs can target and repress viruses and are functionally equivalent. When the candidate siRNAs are delivered as shRNAs they are derived from a cell system and packaged into exosomes or a vector (AAV or Lentiviral vector) as described above.[000149] An shRNA may be provided in an expression cassette containing a promoter contiguously linked to an siRNA as described herein. In embodiments, the promoter is a polll or a polIII promoter, such as a U6 promoter (e.g., a mouse U6 promoter) or a Hl promoter. In embodiments, the expression cassette further contains a marker gene. In embodiments, the promoter is a polll promoter. In embodiments, the promoter is a tissue-specific promoter. In embodiments, the promoter is an inducible promoter. In embodiments, the promoter is a polIII promoter. In embodiments, the promoter is U6 or Hl promoter.[000150] Also provided is a vector containing an expression cassette described herein. Examples of appropriate vectors include adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based viral vectors. In an embodiment, the vector is an adeno-associated virus (AAV) vector.[000151] An shRNA molecule comprises paired RNA sequences and a loop portion positioned between the paired RNA sequences so as to form the hairpin. The loop can vary in length. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 nucleotides in length. In certain embodiments, the loop is 18nucleotides in length. The hairpin structure can also contain 3 ' and / or 5 ' overhang portions. In some embodiments, the overhang is a 3 " and / or a 5 overhang 0, 1 , 2, 3, 4 or 5 nucleotides in length. The nucleotide sequence of the loop region may vary and could be, for example, (5’-GCAA-3’), (5’-GCGC-3’) or (5’-TTGC-3’) or other sequences as will be well understood by the skilled person.[000152] The pharmaceutical compositions described herein may be administered in dosages sufficient to inhibit the expression of the target gene or the biological activity of nontranslated target sequences (e.g. regulatory sequences) in a cell, tissue or organism under treatment. The specific dosages of the inhibitory nucleic acids described herein administered to a given subject will depend on factors such as the route of administration and physical characteristics of the subject (including health status) and so forth. For example, the appropriate dosage of a given pharmaceutical composition comprising the inhibitory nucleic acids described herein may depend on a variety of factors including, but not limited to, a subject’s physical characteristics (e.g. age, weight, sex), the progression (i.e. pathological state) of a given coronavirus infection, and other factors that will be readily recognised by one skilled in the art. Various general considerations that may be considered when determining an appropriate dosage are described, for example, in Gennaro et al. (Eds), (1990), “Remington's Pharmaceutical Sciences”, Mack Publishing Co., Easton, Pennsylvania, USA; and Gilman et al. (Eds), (1990), “Goodman And Gilman’s: The Pharmacological Bases of Therapeutics”, Pergamon Press. Non-limiting examples of suitable dosages of the inhibitory nucleic acids described herein include those in the range of 0.01 to 200 milligrams per kilogram body weight of the recipient per day such as 1 to 50 mg / kg body weight per day, 1 to 40 mg / kg body weight per day, 1 to 30 mg / kg body weight per day, 1 to 30 mg / kg body weight per day, 1 to 10 mg / kg body weight per day, 1 to 5 mg / kg body weight per day, 1 to 3 mg / kg body weight per day, 1 to 2 mg / kg body weight per day, 0.1 to 1 mg / kg body weight per day, 0. 1 to 0.9 mg / kg body weight per day, 0. 1 to 0.8 mg / kg body weight per day, 0. 1 to 0.7 mg / kg body weight per day, 0. 1 to 0.6 mg / kg body weight per day, 0. 1 to 0.5 mg / kg body weight per day, 0.1 to 0.4 mg / kg body weight per day, 0.1 to 0.3 mg / kg body weight per day, 0.1 to 0.2 mg / kg body weight per day, 0.01 to 0.1 mg / kg body weight per day, 0.01 to 0.05 mg / kg body weight per day, 0.01 to 0.02 mg / kg body weight per day, and 0.005 to 0.01 mg / kg body weight per day.[000153] Those of ordinary skill in the art will be able, by routine experimentation, to determine an effective, non-toxic amount of the pharmaceutical compositions and / orinhibitory nucleic acids described herein to include in a dosage or in a series of dosages to achieve the desired therapeutic outcome.[000154] Typically, in therapeutic applications, the treatment would be for the duration of the infection, disease state or condition. Further, it will be apparent to one of ordinary skill in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the infection, disease state or condition being treated, the form, route and site of administration, and the nature of the particular individual being treated. Such optimum conditions can also be determined using conventional techniques.[000155] In many instances, it will be desirable to have several or multiple administrations of a pharmaceutical composition described herein. For example, they may be administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The administrations may be from about one to about twelve week intervals, and in certain embodiments from about one to about four week intervals. Periodic re-administration may be desirable in the case of recurrent exposure to a particular pathogen targeted by a pharmaceutical composition described herein. [000156] It will also be apparent to one of ordinary skill in the art that the optimal course of treatment can be ascertained using conventional course of treatment determination tests.[000157] Suitable techniques for introduction of the inhibitory nucleic acids described herein into cells, tissues, and organisms include various carrier systems, vectors and reagents. Non-limiting examples include lipid nanoparticles (LNP), micelles, nucleic-acid-lipid particles, lipoplexes, liposomes, nucleic acid polymers, single chemical entity conjugates, virosomes, virus like particles (VLP), and mixtures thereof.[000158] Pharmaceutical compositions of the present invention may be administered in any suitable way, such as, for example, intravenously, buccally, parenterally, intranasally, orally, sublingually, or topically. Accordingly, the administration may be topical, pulmonary (e.g. by inhalation or insufflation of aerosols or powders including with a nebulizer), intranasal, intratracheal, epidermal, transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial (e.g. intraparenchymal, intrathecal or intraventricular) administration. In an embodiment, the pharmaceutical composition is adapted for intranasal administration. [000159] In an embodiment, a pharmaceutical composition of the present invention is formulated as a direct-acting nasal spray. In an embodiment, a nasal spray can be selfadministered at point-of-care.[000160] It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. In general, the terms used in the disclosure should not be construed to limit the technology to the specific embodiments disclosed in the specification, unless the above detailed description explicitly defines such terms. Accordingly, the actual scope of the technology encompasses the disclosed embodiments and all equivalent ways of practicing or implementing the technology.[000161] EXAMPLES[000162] Example 1: siRNAs targeted repression of long non-coding RNA antisense HNF4A-AS1 activation of HNF4a isoform selection as a therapeutic to treat and control liver diseases.[000163] Materials and methods[000164] Cell culture and transfection[000165] HEPG2 (Hepatocellular carcinoma derived cells) cells were cultured in Dulbecco’s Modified Eagle Medium supplemented with 10% Foetal Bovine Serum and 1% L-Glutamine and grown in a humidified incubator at 37 °C with 5% CO2. Cells were cultured in T25 flasks from frozen cell pellets and expanded into T75 flasks. Cell cultures were passaged regularly to maintain healthy growth after 80-90% confluency was reached.[000166] The cell transfections were conducted in 12-well plates with the seeding density of 0.1 x 106 cells per well. Once source cell culture reached 80% confluency, cells were harvested through the treatment with trypsin using 1 ml for a T75 flask (scaled down for T25 and 12-well trypsinization) and resuspension of cells. The resuspension was then counted in a hemocytometer to calculate the amount required for seeding. 12-well plates were then supplemented with 1 ml complete DMEM (as described above) per well. 12-well plates were allowed to adhere and grow for 16-24 hours before transfection once 60% confluency was reached. Each siRNA was prepared for transfection at 50nM where 0.5 1 of lOOnM siRNA was diluted in lOOpl of Opti-MEM reduced serum media. 2p I of Lipofectamine 2000 was diluted in 100pl of Opti-MEM and incubated at room temperature for 5 minutes. Diluted siRNA and lipofectamine 2000 were then combined and left to encapsulate for 20 minutes at room temperature. Transfection solution was then added dropwise to each well of the seeded 12-well plate and media was changed 5-24 hours later. Cells were left to grow for 48-72 hours before collection. Transfection efficiency was verified by transfecting the cells with aGFP plasmid using the same transfection protocol used with the siRNA transfections. Cells were transfected with 1.6pg of the GFP plasmid, 500ng of the GFP plasmid (pcDNA3.1- GFP), and no plasmid (mock) in duplicate. After 72 hours the cells were analysed using fluorescence microscopy and results were photographed.[000167] RNA isolation and Reverse Transcription[000168] The transfected cells were collected after trypsin treatment then centrifuged at 8100 x g for 10 minutes. The RNA of the cells was harvested using the Qiagen RNeasy Maxi Kit, including the optional DNase treatment step. The RNA from transfected cells was then reverse transcribed into cDNA using the Qiagen QuantiTect Reverse Transcription Kit and associate protocol.[000169] qPCR Protocol[000170] Two slightly differing protocols were used for the qPCRs run on the cDNA converted from RNA harvested from transfected cells. The Promega GoTaq qPCR Sybr green master mix protocol was used for some preliminary screening technical duplicate experiments. This used lOpl of master mix with 2ul of each forward and reverse primer, 5p 1 of H2O and 1 pl of cDNA under the cycling condition of 95 °C for 10 minutes for initial denaturation 95 °C for 30 seconds for denaturation 55 °C for 30 seconds for annealing and 72 °C for 1 minute for extension for 40 cycles. The protocol used for subsequent qPCR runs used the Kappa Biosystems Sybr fast qPCR master mix with 7pl of Sybr green master mix with 1.4pl of each primer forward and reverse (Table 1), 3.08pl of DNase free H2O and Ipl of cDNA. The PCR conditions for this mix were 95 °C for 10 minutes for initial denaturation 95 °C for 10 seconds for denaturation 55 °C for 15 seconds for annealing and 72 °C for 20 seconds for extension for 40 cycles. These were run using both the Qaigen Rotor-gene PCR cycler and the Bio Molecular Systems MIC qPCR PCR cycler. Next the data was analysed primarily using Windows Excel’s calculation, graphing and statistical analysis functions. Results from qPCR experimentation were yielded as cycle threshold (CT) values which were processed using the delta-delta CT method in Excel. The HNF4A levels were standardised against B-actin levels in transfected cells and expressed as fraction of the control values using siRN367 transfected cells as the control values. The values used to generate error bars were standard deviation values. The fraction of control values were attributed significance via paired two-tailed T-tests comparing the siRNA treated AACT values and the miRN367 (control) treated AACT, p-values of <0.2 were deemed as sufficiently significant. The figures displaying collected data were generated using Windows Excel, Adobe Illustrator and BioRender. To understand the context of significant changes the University of California,Santa Cruise Genome Browser was used to analyse and generate images of the HNF4a gene region, associated IncRNAs and primer targets.[000171] Table 1 qPCR Primer sequences targeting HNF4a mRNA and HNF4a IncRNA.000172] Isoform detection[000173] The alternative primer sets were able to distinguish changes in HNF4a specific isoform expression and expression changes in the HNF4a IncRNA including differentially between the two HNF4A-AS1 isoforms. The target regions of each individual designed primer are depicted in Figure 1. Set 5 (I+J, Figure 1) specifically registers the mRNA expression of HNF4A isoform 1 containing the upstream exon 1. Set 3 (E+F, Figure 1) detects the amount of HNF4a mRNA isoforms 1 and 2 with intron spliced variants at exons 9 and 10. Set 4 (G+H) and Set 4a (K+H) specifically target HNF4A-ASlb, IncRNA, as opposed to Set 2 (C+D) and Set 2a (K+D) which specifically target HNF4A-ASla IncRNA (Figure 1).[000174] Results[000175] Effect of siRNAs targeting HNF4a IncRNAs on HNF4a mRNA expression.[000176] To understand the relationship more clearly between HNF4A and HNF4A-AS 1 several siRNA constructs were designed to specifically target the individual isoforms of HNF4A-AS1 separately and together (Figure 1). Nine siRNA constructs (Table 2) were designed to target and silence particular regions of the targeted IncRNA constructs expressed from the HNF4A gene locus. The siRNA constructs, siHNF4ASl, siHNF4asl_Prol,siHNF4asl_Pro2, siHNF4asl_Pro3 were developed to target HNF4ASla for silencing, whereas siHNF4as2_Pro2, siHNF4as2_Pro 1 and siHNF4ASl&2 target both isoforms of HNF4AS1, and siHNF4AS2 specifically targets HNF4AS lb (Figure 2).[000177] Table 2 siRNAs to screen for targeting of HNF4a IncRNAs and effect on HNF4a mRNA expression. Top candidates for activating HNF4a mRNA expression are in bold.[000178] To determine the silencing action of the siRNA constructs, HepG2 cells were transfected and changes in HNF4A RNA expression determined 72hrs later by qRT-PCR.The siRNAs that yielded significant increases in HNF4a mRNA levels were as follows; siHNF4ASl (specifically targets HNF4AS la), siHNF4AS2 (specifically targets HNF4ASlb),and siHNF4asl_Pro2 (specifically targets HNF4ASla). These siRNAs, all targeted to HNF4AS transcripts and independently resulted in the activation of HNF4A mRNA expression (Figure 2-8), suggesting that HNF4ASla and HNF4ASlb may be involved in negatively regulating HNF4A expression. These candidate siRNAs (siHNF4AS 1 , siHNF4AS2, siHNF4ASl&2, siHNF4asl_Pro2, Table 2) were selected for evaluation and screening with various alternative primer sets that uniquely detect individual HNF4a mRNA isoforms and HNF4AS1 isoforms.[000179] To further validate the initial siRNA screen, HepG2 cells were treated with the top candidate siRNAs (siHNF4ASl, siHNF4AS2, siHNF4ASl&2, siHNF4asl_Pro2, Table 2) and characterized for changes in HNF4A expression. The siRNA siHNF4ASl repressed HNF4A-AS 1 transcript expression (Sets 2 and 4, Figure 2) which resulted in a concomitant reduction in P2 promoter usage (Set 5, Figure 2). Interestingly, siHNF4ASl not only resulted in a significant reduction in the targeted IncRNA HNF4A-ASla isoform (Set 2 & 2a, Figure 2) but also caused the reduction of the alternate HNF4A-ASlb isoform (Set 4, Figure 2), suggesting that both HNF4A-ASla and HNF4A-ASlb are linked and may, in fact, be the same transcript (Figure 2). This data also suggests that siHNF4ASl can repress HNF4A-AS1 expression and that the loss of this antisense transcript results in reduced use of the P2 promoter and retention of P2 HNF4A isoforms. Such an observation supports the notion that the HNF4A- AS 1 IncRNA is a functional enhancer element involved in controlling HNF4A promoter usage.[000180] To explore this notion, that perhaps HNF4A-AS1 is a regulator of P2 vs Pl promoter usage and ensure that the observations with siHNF4AS 1 are consistent with a theme, we treated HEPG2 cells with HNF4A-ASlb specific siRNA construct siHNF4AS2 which suppressed HNF4A IncRNA isoform expression (Set 4, Figure 3) and similarly to observations with HNF4A-ASla (Figure 2), siHNF4AS2 treatment inhibited both HNF4A- AS1 isoforms (Figure 3). A similar observation was also found in cells treated with siHNF4ASl&2 (Figure 4), or siHNF4asl_Pro2 (Figure 5), whereby the siRNAs repressed the HNF4A-AS1 IncRNA resulting in a shift from the P2 to Pl promoted HNF4A transcripts. Collectively, this data suggests that HNF4A-ASla and HNF4A-ASlb function as P2 promoter enhancers, which when lost results in the transcriptional shutdown of the P2 HNF4A promoter.[000181] To interrogate this notion further the top-candidate siRNA, (siHNF4ASl) was assessed further with the effects of this siRNA treatment on HepG2 liver cells and the various known dominant splice variants for HNF4A (Figure 1 ) determined. Specifically, we assessedthe four dominant isoforms from the Pl and P2 promoters (Figure 1). We find that siHNF4ASl repressed HNF4-ASla expression (Figure 7A, Set 2) which resulted in concomitant repression of HNF4 A P 1 isoform 1 and reduced incorporation of P2 Exon 1 (Figure 7A, Set 5), but had little effect on Pl Isoform 2 (Figure 7A, Set 7). Interesting, both HNF4A Pl-Iso- 1 and P2-Iso-1, isoform expression includes the 3’ addition of exons 10 and 11 into the final HNF4A protein product. Such an observations suggests that one possible function for HNF4-ASla is to control the incorporation of exons 10 and 11 by modulation of upstream transcriptional HNF4A specific events. To determine this supposition further siHNF4ASl and control siRNA (N367) was transfected into HepgG2 cells and found to repress HNF4-ASla expression (Figure 7B). This repression of HNF4-AS 1 a correlated with a concomitant repression of HNF4A P2-Iso-1 expression (Figure 7C). Collectively these data tell us that HNF4-ASla is required for HNF4A Pl and P2-Iso-1 expression and the inclusion of exons 10 and 11 into the resultant HNF4A mRNA.[000182] Discussion:[000183] The loss or repression of hepatic function associated with various liver pathologies commonly correlates with the loss or repression of HNF4a expression3. Various models have been proposed for the mechanism of HNF4a downregulation within hepatocytes in the diseased liver. Recently, long noncoding RNAs have also been linked to various gene pathologies, including HNF4A-AS1 having been identified in various cancer pathologies including HCC, whereby the cancerous cells of a cirrhotic liver expressed the IncRNA more than the surrounding cirrhotic tissue1’2’9,10. HNF4A-AS 1 was also found to be downregulated in Crohn’s disease (an inflammatory disease of the intestinal system) indicating a relationship between HNF4A-AS1 and inflammation14. This link between the HNF4a IncRNA and inflammation indicates a possible connection between HNF4A-AS 1 and the proposed inflammatory mechanism of HNF4a suppression2,15. The mechanisms underlying the influence of IncRNA such as HNF4A-AS1 in gene expression regulation remain largely a mystery. Several potential mechanisms have been theorised to explain the correlation between IncRNAs and nearly every step of gene regulation, including transcription and translation. However, there are few IncRNAs that have been deeply investigated to reveal their explicit functions and roles.[000184] We report here that siHNF4ASla is one of 3 top-candidates capable of repressing HNF4A-AS1 IncRNA expression in liver cells. We find that the repression of HNF4A-AS1 IncRNA by siHNF4ASl results in the loss of exon 10 and 11 containing HNF4A variants. It is well known that HNF4A has multiple isoforms produced by alternativesplicing with isoforms expressed from the Pl promoter (e.g., HNF4al-HNF4a6) typically include exon 10, while isoforms from the P2 promoter (e.g., HNF4a7-HNF4al2) exclude it (Radi, 2023 #49).[000185] Exon 10 contains the ligand-binding domain (LBD), which plays a role in dimerization and HNF4A transcriptional activity. The LBD regulates the expression of genes involved in liver homeostasis, including those for gluconeogenesis, lipid metabolism, and detoxification (Ehle, 2024 #50). The absence of exon 10 may alter the LBD’s stability and its ability to form functional homodimers, potentially reducing transcriptional efficiency. Isoforms with or without exons 10 and 11 show differential expression across tissues. For instance, Pl -driven isoforms (with exon 10) are more common in hepatocytes, while P2- driven isoforms (without exon 10) are expressed in other tissues like pancreatic P-cell (Radi, 2023 #49). Splicing alterations affecting exon 10 have been observed in alcoholic hepatitis (AH) with the increased inclusion of exon 10 found in AH livers correlating with deregulated HNF4A splicing and impaired liver synthetic function (Argemi, 2019 #31).[000186] HNF4A is an essential transcription factor involved in hepatocyte differentiation and the reacquisition of liver- specific functions during liver regeneration (LR). Isoforms without exon 10 may be preferentially expressed during the early proliferative phase, as they have been linked to dedifferentiation and stress responses and maybe predominant in AH where liver damage is chronic as is LR to repair the damaged tissues. Precedence for this interpretation is in observations with the upregulation P2 isoforms being unique to alcoholic hepatitis with marked deregulation of HNF4a splicing associated with alcoholic hepatitis disease states3. This impaired splicing impairs the stability and activity of HNF4a, ultimately inhibiting its natural ability to repair and regenerate healthy hepatocytes and likely has a major role in the hepatocytic dysfunction present in various liver diseases, especially alcoholic hepatitis.Moreover, when analysing the effect of the upregulation of the P2 isoforms, it has consistently been observed to negatively regulate the expression of HNF4A target genes, and in the absence of P2 isoforms, the Pl isoform was upregulated resulting in higher protein expression of HNF4A and HNF4A target genes3’4. Argemi et al.3justified that the dysregulation of HNF4a isoform expression was resultant of changes in the expression of the HNF4a transcription regulator TGFB1. TGFpi was discovered to mediate the HNF4a P1-P2 isoform ratio and was associated with increased expression of P2 isoforms and decreased Pl isoform expression3 17. This may indicate that TGFB 1 interacts with the antisense IncRNAs, HNF4A-ASla and HNF4A-ASlb, forming a transcriptional enhancer complex to affect P2promoter usage. While only supposition at this stage, it is reasonable to speculate that transcription factors and gene-associated IncRNAs would interact to functionally modulate gene transcriptional and epigenetic states. While the exact mechanism of how HNF4A-AS la and HNF4A-ASlb influence P2 promoter usage is not clear, the ability to repress these IncRNAs to affect HNF4A expression, slanting liver cell function to regenerative and healthy with the lack of exons 10 and 11 and the HNF4A LBD, is profound. HNF4a’s role in hepatocyte function maintenance and liver morphogenesis is crucial to the health and regeneration of damaged liver cells, hence various liver pathologies have been associated with altered HNF4a isoform ratios, localisation, and expression4 18. In this research, several siRNAs have been investigated that can affect the differential regulation of HNF4A mRNA isoforms by specifically suppressing HNF4A- AS 1 a and HNF4A-ASlb expression. Collectively this work demonstrates the power of targeting antisense IncRNAs to ultimately control downstream protein function and in the case of HNF4A may prove relevant in reversing the dysregulation of the HNF4A P1:P2 isoform ratio associated with severe inflammatory liver diseases.1000187] Example 2 : Nanoparticle development for the activation of HNF 4 Pl variant expression as a treatment for liver fibrosis and failure.[000188] Materials and methods[000189] Screen top siRNAs (Table 3) in human liver iPSCs for changes in Pl vs. P2 HNF4 expression (Table 4). Determine optimal siRNA LNP formulation for liver fibrosis / failure.[000190] Exp 1 Screen siRNAs in primary human iPSC liver cells (lOOnM, siRNA, assay by qRTPCR with primer sets 72hrs later). Select top siRNAs for LNP formulation. [000191] Table 3 HNF4 activating siRNAs for LNP or EV delivery.[000192] Exp 2 Screen shRNA (HNF4asl_Pro2, HNF4AS) vs asRNA (HNF4aslPro2, HNF4AS1) EV packaging systems and stable iPSC liver cells.[000193] Table 4 shRNA EV RNAs to screen for activation of HNF4. All constructs are under puromycin selection.000194] Screen antisense EV RNAs for activation of HNF4. All constructs expressTGS inducing antisense RNAs and are puromycin selection. Bold sequence is the transcribed RNA.[000195] >asHNF4-l (SEQ ID NO. 102)[000196] TGCCGTTGGTATCAGCAAGTGTCAGATCCCAGCTCCAGGAAGAGGGCTGTCCTCTCAGATGGGAGGCAGGGGTCCAGTGGGGTGGCTGTGGCTGCAGGGACAGCCTAGCAGAGCCTCTTGGTGGCCCAGACCCCCTGTCCCTGGACCTTGGAAGCCGCTAAAGGAAGCAGCAAGACCAGGCCAGCTGGTGGCTGCCTGGACTGGAGTTTGGTCAGTGGTTGGGGACCTATATTGTGAGATAGTTAGGACTTTGTCCACAAACTGGAATTTGAAATTTCAAAGGGGCAAAACTGAAGGGCCCTACCCCAGAGTAGAGATGGCTCTTGGCTTCTTTCCTCCACGACTTTTGTTCTTTGTCTTGATAAGTCCCAGTTTTCTCTGCACCTCTCCCGGAGTGGTGGGTGGGGAAGAAAAGATAGGAGATAATGGTGTGGTGAGCGCCAAGGGGAAGTGAAGATGCC[000197] asHNF4-l Transcript generated (5’-3’) (SEQ ID NO. 33):[000198] UGCCGUUGGUAUCAGCAAGUGUCAGAUCCCAGCUCCAGGAAGAGGGCUGUCCUCUCAGAUGGGAGGCAGGGGUCCAGUGGGGUGGCUGUGGCUGCAGGGACAGCCUAGCAGAGCCUCUUGGUGGCCCAGACCCCCUGUCCCUGGACCUUGGAAGCCGCUAAAGGAAGCAGCAAGACCAGGCCAGCUGGUGGCUGCCUGGACUGGAGUUUGGUCAGUGGUUGGGGACCUAUAUUGUGAGAUAGUUAGGACUUUGUCCACAAACUGGAAUUUGAAAUUUCAAAGGGGCAAAACUGAAGGGCCCUACCCCAGAGUAGAGAUGGCUCUUGGCUUCUUUCCUCCACGAC UUUUGUUCUUUGUCUUGAUAAGUCCCAGUUUUCUCUGCACCUCUCCCGGAGUGGUGGGUGGGGAAGAAAAGAUAGGAGAUAAUGGUGUGGUGAGCGCCAAGGGGAAGUGAAGAUGCC[000199] >asHNF4-2 (SEQ ID NO. 103)[000200] TGGTGGCCCAGACCCCCTGTCCCTGGACCTTGGAAGCCGCTAAAGGAAGCAGCAAGACCAGGCCAGCTGGTGGCTGCCTGGACTGGAGTTTGGTCAGTGGTTGGGGACCTATATTGTGAGATAGTTAGGACTTTGTCCACAAACTGGAATTTGAAATTTCAAAGGGGCAAAACTGAAGGGCCCTACCCCAGAGTAGAGATGGCTCTTGGCTTCTTTCCTCCACGACTTTTGTTCTTTGTCTTGATAAGTCCCAGTTTTCTCTGCAC[000201] asHNF4-2 Transcript generated (5’-3’) (SEQ ID NO. 104):[000202] UGCCGUUGGUAUCAGCAAGUGUCAGAUCCCAGCUCCAGGAAGAGGGCUGUCCUCUCAGAUGGGAGGCAGGGGUCCAGUGGGGUGGCUGUGGCUGCAGGGACAGCCUAGCAGAGCCUCUUGGUGGCCCAGACCCCCUGUCCCUGGACCUUGGAAGCCGCUAAAGGAAGCAGCAAGACCAGGCCAGCUGGUGGCUGCCUGGACUGGAGUUUGGUCAGUGGUUGGGGACCUAUAUUGUGAGAUAGUUAGGACUUUGUCCACAAACUGGAAUUUGAAAUUUCAAAGGGGCAAAACUGAAGGGCCCUACCCCAGAGUAGAGAUGGCUCUUGGCUUCUUUCCUCCACGACUUUUGUUCUUUGUCUUGAUAAGUCCCAGUUUUCUCUGCACCUCUCCCGGAGUGGUGGGUGGGGAAGAAAAGAUAGGAGAUAAUGGUGUGGUGAGCGCCAAGGGGAAGUGAAGAUGCC[000203] >asHNF4-3 (SEQ ID NO. 105)[000204] GTTAGGACTTTGTCCACAAACTGGAATTTGAAATTTCAAAGGGGCAAAACTGAAGGGCCCTACCCCAGAGTAGAGATGGCTCTTGGCTTCTTTCCTCCACGACTTTTGTTCTTTGTCTTGATAAGTCCCAGTTTTCTCTGCACCTCTCCCGGAGTGGTGGGTGGGGAAGAAAAGATAGGAGATAATGGTGTGGTGAGCGCCAAGGGGAAGT[000205] asHNF4-3 Transcript generated (5 ’-3’) (SEQ ID NO. 35):[000206] GUUAGGACUUUGUCCACAAACUGGAAUUUGAAAUUUCAAAGGGGCAAAACUGAAGGGCCCUACCCCAGAGUAGAGAUGGCUCUUGGCUUCUUUCCUCCACGACUUUUGUUCUUUGUCUUGAUAAGUCCCAGUUUUCUCUGCACCUCUCCCGGAGUGGUGGGUGGGGAAGAAAAGAUAGGAGAUAAUGGUGUGGUGAGCGCCAAGGGGAAGU[000207] Table 5 qPCR Primer sequences for measuring HNF4a Pl vs P2 mRNA expression.[000208] Reference1. Chen, L., Bao, Y., Jiang, S., and Zhong, X.B. (2020). The Roles of Long Noncoding RNAs HNFlalpha-ASl and HNF4alpha-AS 1 in Drug Metabolism and Human Diseases. Noncoding RNA 6. 10.3390 / ncrna6020024.2. Dubois, V., Staels, B., Lefebvre, P., Verzi, M.P., and Eeckhoute, J. (2020). Control of Cell Identity by the Nuclear Receptor HNF4 in Organ Pathophysiology. Cells 9. 10.3390 / cells9102185.3. Argemi, J., Latasa, M.U., Atkinson, S.R., Blokhin, I.O., Massey, V., Gue, J.P., Cabezas, J., Lozano, J.J., Van Booven, D., Bell, A., Cao, S., et al. (2019). DefectiveHNF4alpha-dependent gene expression as a driver of hepatocellular failure in alcoholic hepatitis. Nat Commun 10, 3126. 10. 1038 / s41467-019-l 1004-3.4. Diaz-Aragon, R., Coard, M.C., Amimeni, S., Faccioli, L., Haep, N., Malizio, M.R., Motomura, T., Kocas-Kilicarslan, Z.N., Ostrowska, A., Florentino, R.M., and Frau, C. (2021). Therapeutic Potential of HNF4alpha in End-stage Liver Disease.Organogenesis 17, 126-135. 10.1080 / 15476278.2021.1994273.5. Lambert, E., Babeu, J.P., Simoneau, J., Raisch, J., Lavergne, L., Levesque, D., Jolibois, E., Avino, M., Scott, M.S., Boudreau, F., and Boisvert, F.M. (2020). HumanHepatocyte Nuclear Factor 4-alpha Encodes Isoforms with Distinct Transcriptional Functions. Mol Cell Proteomics 19, 808-827. 10.1074 / mcp. RAI 19.001909.6. Aguilo, F., Zhou, M.M., and Walsh, M.J. (201 1). Long noncoding RNA, polycomb, and the ghosts haunting INK4b-ARF-INK4a expression. Cancer Res 71, 5365-5369. 10.1158 / 0008-5472.CAN-10-4379.7. Kotake, Y., Nakagawa, T., Kitagawa, K., Suzuki, S., Liu, N., Kitagawa, M., and Xiong, Y. (2011). Long non-coding RNA ANRIL is required for the PRC2 recruitment to and silencing of pl5(INK4B) tumor suppressor gene. Oncogene 30, 1956-1962. 10.1038 / onc.2010.568.8. Florentino, R.M., Fraunhoffer, N.A., Morita, K., Takeishi, K., Ostrowska, A., Achreja, A., Animasahun, O., Haep, N., Arazov, S., Agarwal, N., Collin de 1'Hortet, A., et al. (2020). Cellular Location of HNF4alpha is Linked With Terminal Liver Failure in Humans. Hepatol Commun 4, 859-875. 10.1002 / hep4.1505.9. Heo, M.J., Yun, J., and Kim, S.G. (2019). Role of non-coding RNAs in liver disease progression to hepatocellular carcinoma. Arch Pharm Res 42, 48-62. 10.1007 / s 12272- 018-01104-x.10. Wang, P„ Chen, S., Wang, Y„ Wang, X., Yan, L„ Yang, K„ Zhong, X.B., Han, S„ and Zhang, L. (2021). The Long Noncoding RNA Hepatocyte Nuclear Factor 4alpha Antisense RNA 1 Negatively Regulates Cytochrome P450 Enzymes in Huh7 Cells via Histone Modifications. Drug Metab Dispos 49, 361-368. 10.1124 / dmd.l20.000316.11. Guo, S., and Lu, H. (2019). Novel mechanisms of regulation of the expression and transcriptional activity of hepatocyte nuclear factor 4alpha. J Cell Biochem 120, 519- 532. 10.1002 / jcb.27407.12. Allo, M., and Komblihtt, A.R. (2010). Gene silencing: small RNAs control RNA polymerase II elongation. Curr Biol 20, R704-707. 10.1016 / j.cub.2010.07.013.13. Weinberg, M.S., and Morris, K.V. (2016). Transcriptional gene silencing in humans. Nucleic Acids Res 44, 6505-6517. 10.1093 / nar / gkwl39.14. Marcil, V., Sinnett, D., Seidman, E., Boudreau, F., Gendron, F.P., Beaulieu, J.F., Menard, D., Lambert, M., Bitton, A., Sanchez, R., Amre, D., et al. (2012). Association between genetic variants in the HNF4A gene and childhood-onset Crohn's disease. Genes Immun 13, 556-565. 10.1038 / gene.2012.37.15. Dubois, V., Gheeraert, C., Vankrunkelsven, W., Dubois -Chevalier, J., Dehondt, H., Bobowski-Gerard, M., Vinod, M., Zummo, F.P., Guiza, E, Ploton, M., Dorchies, E.,et al. (2020). Endoplasmic reticulum stress actively suppresses hepatic molecular identity in damaged liver. Mol Syst Biol 16, e9156. 10.15252 / msb.20199156.16. Marcil, V., Amre, D., Seidman, E.G., Boudreau, F., Gendron, F.P., Menard, D., Beaulieu, J.F., Sinnett, D., Lambert, M., and Levy, E. (2015). Hepatocyte nuclear factor 4 alpha polymorphisms and the metabolic syndrome in French-Canadian youth.PLoS One 10, e0117238. 10.1371 / journal.pone.0117238.17. Babeu, J.P., and Boudreau, F. (2014). Hepatocyte nuclear factor 4-alpha involvement in liver and intestinal inflammatory networks. World J Gastroenterol 20, 22-30. 10.3748 / wjg. v20.il.22. 18. Liu, L., Yannam, G.R., Nishikawa, T., Yamamoto, T., Basma, H., Ito, R., Nagaya,M., Dutta-Moscato, J., Stolz, D.B., Duan, F., Kaestner, K.H., et al. (2012). The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis. Hepatology 55, 1529-1539. 10.1002 / hep.24815.[000209]

Claims

CLAIMS1. A pharmaceutical composition comprising one or more hepatocyte nuclear factor four alpha antisense RNA 1 (HNF4A-AS1) targeting polynucleotides, wherein the one or more HNF4A-AS1 targeting polynucleotides is capable of repressing HNF4A-AS1 in a subject resulting in upregulation of HNF4A expression and / or increasing HNF4A Pl :P2 isoform ratio in the subject.

2. The pharmaceutical composition of claim 1 , wherein the nucleotide sequence of each of the one or more HNF4A-AS1 targeting polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, or a combination thereof.

3. The pharmaceutical composition of claim 1, wherein the one or more HNF4A-AS1 targeting polynucleotides comprise a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof.

4. The pharmaceutical composition of claim 1 , wherein the nucleotide sequence of the one or more HNF4A-AS1 targeting polynucleotides is at least about 80%, about 85%, about 90%, about 95% or about 100% identical to the nucleotide sequence of SEQ ID NO. 31 or SEQ ID NO. 32.

5. The pharmaceutical composition of claim 4, wherein the HNF4A-AS1 comprises HNF4A-ASla, HNF4A-ASlb, or a combination thereof.

6. The pharmaceutical composition of claim 1 further comprising a nanoparticle wherein the nanoparticle encapsulates the one or more HNF4A-AS 1 targeting polynucleotides.

7. The pharmaceutical composition of claim 6, wherein the nanoparticle comprises lipid nanoparticle, polymer nanoparticle, lipid-polymer hybrid nanoparticle, liposome, exosome, virus or virus-like particle.

8. The pharmaceutical composition of claim 7, further comprising a fusion protein and a packaging domain wherein the nanoparticle comprises exosome, wherein the one or more HNF4 A- AS 1 targeting polynucleotides is fused to the packaging domain, wherein the fusion protein comprises an exosome-associated transmembrane protein fused to a packaging protein, wherein the packaging domain comprises an UR domain, a L2 domain, or a combination thereof and wherein the packaging domain is capable ofbinding with the packaging protein.

9. The pharmaceutical composition of claim 6, wherein the one or more HNF4A-AS1 targeting polynucleotides is further fused to a nuclear localization sequence SIRLOIN.

10. The pharmaceutical composition of claim 8, wherein the fusion protein comprises CD63-Ula, CD81-Ula, PTGFRN-Ula, or a combination thereof.

11. The pharmaceutical composition of claim 7, wherein the exosome further comprises an Ago2 protein or a S387 A mutant thereof.

12. The pharmaceutical composition of claim 7, wherein the exosome further comprises a modified myoferlin protein.

13. The pharmaceutical composition of claim 12, wherein the modified myoferlin protein comprises C2F, C2G, transmembrane domain of the myoferlin protein, or a combination thereof.

14. The pharmaceutical composition of claim 12, wherein the modified myoferlin protein further comprises a connexin 43 protein or a S368 A mutant thereof.

15. The pharmaceutical composition of claim 8, wherein the exosome is prepared using an exosome-based packaging and delivery system comprising one or more cargo RNA encoding plasmid encoding the one or more HNF4A- AS 1 targeting polynucleotides of claim 1 fused to the packaging domain, and one or more fusion protein encoding plasmids encoding the fusion protein comprising an exosome-associated transmembrane protein fused to a packaging protein.

16. The pharmaceutical composition of claim 15, wherein the exosome-based delivery and packaging system further comprises a plasmid encoding a modified myoferlin protein, a connexin 43 protein or a S368A mutant thereof, or a combination thereof.

17. A method of treatment of a HNF4A-associated disease in a subject comprising the step of administering a therapeutically effective amount of the pharmaceutical composition of claims 1 to the subject.

18. The method of claim 17, wherein the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, and nonalcoholic fatty liver disease (NAFLD), liver failure of any cause, polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY).

19. A method of downregulating HNF4A P2 isoform expression in a subject comprising the step of repressing HNF4A-AS1 in the subject.

20. The method of claim 19, wherein the method increases the HNF4A P1:P2 isoform expression ratio in a subject.

21. The method of claim 19, wherein the method increases the HNF4A P1:P2 isoform expression ratio of at least a pair of Pl and P2 isoform in a subject.

22. The method of claim 19, wherein the subject is diagnosed with a HNF4 A- associated disease.

23. The method of claim 22, wherein the HNF4A-associated disease comprises hepatocellular cancer, fatty liver (steatosis), nonalcoholic steatohepatitis (NASH), cirrhosis of the liver, accumulation of fat in the liver, inflammation of the liver, hepatocellular necrosis, liver fibrosis, nonalcoholic fatty liver disease (NAFLD), liver failure of any cause, polycystic kidney disease, inflammatory bowel disease (IBD), and maturity onset diabetes of the young (MODY).

24. The method of claim 19, wherein the step of repressing HNF4A-AS1 is performed by administration of HNF4A-AS1 targeting polynucleotides capable of repressing HNF4A- AS 1 via RNA interference wherein the HNF4A- AS 1 targeting polynucleotides comprises a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), a ribozyme, a deoxyribozyme, an aptamer, or a combination thereof.

25. The method of claim 19, the step of repressing HNF4A-AS1 in the subject is performed by administration of the pharmaceutical composition of claim 1 to the subject.

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