Compositions and methods for treating liver disease with siRNA targeting GPAM

Abstract

Disclosed herein is a composition comprising siRNA that can downregulate the gene expression of glycerol-3-phosphate acyltransferase, mitochondrial (GPAM) or its variant.Disclosed herein is also a method of using such composition in treating liver disease or injury, such as fatty liver disease (FLD), non-alcoholic fatty liver disease (NAFLD) or non-alcoholic steatohepatitis (NASH).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 328,593, filed April 7, 2022, and U.S. Provisional Patent Application No. 63 / 491,262, filed March 20, 2023, the disclosures of which are incorporated by reference in their entireties herein for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted in xml format via EFS-Web, the entire contents of which are incorporated herein by reference. The xml copy created on April 7, 2023 is named 106546-755472.xml and is 329KB in size. [Background technology]

[0003] 1. Field The present inventive concepts relate to compositions and methods of use thereof for the treatment of liver diseases, particularly metabolic liver diseases, including but not limited to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0004] 2. Description of Related Technology NAFLD is a group of chronic liver disorders that encompasses nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH). NASH, a common cause of chronic liver disease, is defined as having at least 5% hepatic steatosis and inflammation with or without fibrosis. If left untreated, over time, NASH progresses to cirrhosis and even hepatocellular carcinoma (HCC). Furthermore, NAFLD / NASH is strongly associated with obesity and type II diabetes, which together affect more than 50% of the US population and lead to a heavy economic burden. Unfortunately, treatment options for NASH remain limited, with only modest benefits observed from vitamin E or obeticholic acid treatment. Although NASH is the leading cause of chronic liver disease and cirrhosis, there is currently no clinically approved therapy. Thus, new targets, therapeutic agents, and their combinations are needed to accelerate clinical progress in the treatment of liver disease. Summary of the Invention

[0005] The present disclosure is based in part on the novel discovery that positive selection of somatic mutations in the liver of NASH patients can increase cellular fitness and competitiveness through the reduction of lipid accumulation in NASH.By screening somatic mutations for genes that promote clonal fitness through the reversal of lipotoxicity, GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) was identified.Therefore, the present disclosure provides novel compositions for modulating the expression of GPAM, as well as methods for preventing, attenuating, and / or treating liver disease.

[0006] In some embodiments, the disclosure encompasses compositions comprising nucleic acid molecules that downregulate expression of GPAM or variants thereof. In some embodiments, the nucleic acid is an siRNA, a cluster regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a transactivating crRNA (tracrRNA), a plasmid DNA (pDNA) or a viral vector. In some embodiments, the nucleic acid is a small interfering RNA (siRNA) molecule or encodes an siRNA molecule. In some embodiments, the composition comprises a plasmid or a viral vector, and the plasmid or viral vector comprises a nucleic acid encoding an siRNA molecule disclosed herein. In some embodiments, the siRNA molecule comprises a nucleotide sequence that is 2-30 nucleotides in length and is at least 80% homologous to at least 2-30 contiguous nucleotides of the human GPAM cDNA sequence, the human GPAM cDNA sequence being SEQ ID NO:1. In some embodiments, the siRNA molecule targets the open reading frame or 5' or 3' UTR of the GPAM gene. In some embodiments, the siRNA molecule comprises at least one sense sequence, at least one antisense sequence, or at least one sense sequence and at least one antisense sequence. In some embodiments, the siRNA molecule comprises the nucleotide sequence of SEQ ID NO: 2-165 or any combination thereof. In some embodiments, at least one sense sequence comprises SEQ ID NO: 2-83. In some embodiments, at least one antisense sequence comprises SEQ ID NO: 84-165.

[0007] In some embodiments, the present disclosure also encompasses compositions comprising a nucleic acid molecule that downregulates expression of GPAM, the nucleic acid being an sgRNA or encoding an sgRNA. In some embodiments, the composition comprises a plasmid or viral vector, the plasmid or viral vector comprising a first nucleic acid encoding an sgRNA molecule disclosed herein and optionally a second nucleic acid encoding an RNA-guided nuclease. In some embodiments, the target sequence of the sgRNA is at least about 80% identical to any one of SEQ ID NOs: 177-276. In some embodiments, the RNA-guided nuclease is a Cas endonuclease.

[0008] In some embodiments, the siRNA molecule disclosed herein specifically downregulates the gene expression of at least one variant of GPAM. In some embodiments, the sgRNA molecule disclosed herein specifically downregulates the gene expression of at least one variant of GPAM. In some embodiments, at least one variant of GPAM is associated with liver disease. Non-limiting examples of liver disease include fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer, or any combination thereof. In some embodiments, at least one variant of GPAM comprises rs10787429 C>T, rs7096937 T>C, rs11446981 T>TA, rs7898213 T>C, rs2792759 C>T, rs2297991 T>C, rs1129555 A>G, rs2254537 T>A, or any combination thereof.

[0009] In some embodiments, the nucleic acid molecules disclosed herein can be conjugated to at least one targeting ligand. In some embodiments, the at least one targeting ligand comprises a liver targeting ligand. In some embodiments, the liver targeting ligand comprises at least one N-acetylgalactosamine (GalNAc) conjugate. In some embodiments, the nucleic acid molecule is conjugated to about 1 to about 3 GalNAc conjugates. In some embodiments, the nucleic acid molecule comprises at least one chemical modification. In some embodiments, the nucleic acid molecule comprises a modification in at least one ribosugar moiety of its nucleotide sequence. In some embodiments, at least one ribosugar moiety is modified with 2 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, 2'-C-allyl, or any combination thereof. In some embodiments, between about less than 10% and about 70% of the ribosugar moieties of the entire nucleotide sequence are modified.

[0010] In some embodiments, the present disclosure also encompasses a pharmaceutical composition comprising any one of the compositions disclosed herein and at least one pharma- ceutically acceptable carrier.In some embodiments, the pharmaceutical composition further comprises nanoparticles.In some embodiments, the pharmaceutical composition further comprises lipid.

[0011] In some embodiments, the disclosure also encompasses a method of treating a subject in need thereof, comprising administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein. In some embodiments, the subject in need thereof is a human subject having or suspected of having liver disease. In some embodiments, the liver disease comprises fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer, or any combination thereof. In some embodiments, the administration method comprises parenteral administration. In some embodiments, administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein increases the life expectancy of the subject compared to an untreated subject with the same disease state and predicted outcome. In some embodiments of the method, administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein increases the liver function of the subject compared to an untreated subject with the same disease state and predicted outcome. In some aspects of the method, administering a therapeutically effective amount of the composition disclosed herein or the pharmaceutical composition disclosed herein attenuates liver fibrosis in the subject compared to an untreated subject with the same disease state and predicted outcome.In some aspects, administering a therapeutically effective amount of the composition disclosed herein or the pharmaceutical composition disclosed herein prevents further liver fibrosis in the subject compared to an untreated subject with the same disease state and predicted outcome.

[0012] In some aspects, the present disclosure also encompasses kits comprising a container holding a composition disclosed herein or a pharmaceutical composition disclosed herein; a pharmaceutical administration means; and instructions for use.

[0013] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which can be better understood by reference to the drawings in combination with the detailed description of specific embodiments provided herein. [Brief description of the drawings]

[0014] [Figure 1A] Figure 1 shows the screening of 63 known NASH-related genes. [Figure 1B] FIG. 1 shows the results of a screen of 63 known NASH-associated genes. The most expanded clones are frequently associated with deletions of adipogenic genes such as GPAM. [Figure 1C] FIG. 1 provides the top gene hits of the screen, including GPAM. [Figure 1D] Figure 1 shows the pathways involved in the gene hits. This screen helped to show that genes mutated in human cirrhotic tissues may be good drug targets for NASH. GPAM is one such gene that is frequently mutated in human liver disease. [Figure 2A] Figure 1: GPAM mutant clones can outcompete wild-type clones in the liver during the development of NASH. Red blood cells were wild-type and decreased in numbers over time in the Western diet cohort. [Figure 2B] Figure 2 shows that red blood cells were GPAM mutant and increased in number over time in the Western diet cohort. Furthermore, (compare histology in Figure 2A and Figure 2B) the NASH phenotype was improved when more GPAM mutant clones were present in the liver. This experiment demonstrated that the GPAM mutant clones were more fit and reduced the level of hepatic steatosis. [Figure 3A] FIG. 1 shows the luciferase-based screen used to test the relative knockdown efficacy of various human GPAM siRNAs. [Figure 3B] FIG. 1 shows GPAM protein levels examined using Western blot analysis after siRNA knockdown. [Figure 4A] Genotyping of Gpamf / f liver tissue before and after Cre recombination. Asterisks are next to the hybrid annealing bands derived from the top and bottom PCR products. [Figure 4B]Figure 1 shows qPCR examination of Gpam mRNA levels in liver before and after Cre recombination. Primer designs are shown on the left. Two pairs of primers were used (n=3 mice for each group). [Figure 4C] Quantification of Tomato+ cells from LSL-tdTomato het liver sections (n=7, 11, 11 mice for each group). Each dot represents one image field; two fields were analyzed from each mouse liver. Statistical analysis was performed on image data averaged from individual mice. [Figure 4D] Quantification of Tomato+ cells from Gpamf / f;LSL-tdTomato het liver sections (n=7, 15, 15 mice for each group). Each dot represents one image field; two fields from each mouse liver are shown. Statistical analysis was performed on image data averaged from individual mice. [Figure 4E] FIG. 13 shows the body weight of Gpam KO mice and corresponding control mice treated with chow or WD (n=6, 16, 6, 12 mice for each group). [Figure 4F] FIG. 13 shows liver weights of whole liver Gpam KO mice and corresponding control mice treated with chow or WD (n=6, 16, 6, 12 mice for each group). [Figure 4G] Liver / body weight ratios of liver-specific Gpam WT and KO mice (n=6, 16, 6, 12 mice per group) fed a WD for 3 months. These mice were given a high dose of AAV8-TBG-Cre, resulting in liver-wide Gpam deletion in almost all hepatocytes. [Figure 4H] Representative H&E staining of Gpam WT and KO liver sections after 3 months of WD. [Figure 4I] FIG. 1 shows liver function tests by plasma ALT (n=6, 16, 6, 12 mice per group). [Figure 4J] FIG. 1 shows liver function tests by plasma AST (n=6, 16, 6, 12 mice per group). [Figure 4K] Figure 1 shows NAFLD activity scores of H&E sections. The right panel represents the total NAFLD activity score, which is the sum of the three scores on the left (n=13 and 14 mice for each group). [Figure 4L] FIG. 1 shows triglyceride measurements from liver tissue of WT and KO mice (n=6, 16, 6, 12 mice per group). [Figure 4M] FIG. 1 shows cholesterol measurements from liver tissue of WT and KO mice (n=6, 16, 6, 12 for each group). [Figure 4N] FIG. 1 shows plasma cholesterol for WT and KO mice (n=6, 16, 6, 12 for each group). [Figure 4O] FIG. 1 shows plasma triglycerides for WT and KO mice (n=6, 16, 6, 12 for each group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The drawings are not intended to limit the inventive concepts to the specific embodiments disclosed and described herein. The drawings are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of certain embodiments of the inventive concepts.

[0016] The following detailed description refers to the accompanying drawings, which illustrate various embodiments of the inventive concepts. The drawings and description are intended to describe aspects and embodiments of the inventive concepts in sufficient detail to enable those skilled in the art to practice the inventive concepts. Other components can be utilized and changes can be made without departing from the scope of the inventive concepts. Therefore, the following description should not be taken in a limiting sense. The scope of the inventive concepts is defined solely by the appended claims, along with the full scope of equivalents to which such claims are entitled.

[0017] With the rise of overnutrition and obesity, non-alcoholic fatty liver disease (NAFLD) is rapidly becoming the leading cause of liver disease worldwide. NAFLD is usually conceptualized at the organism and tissue level, but genetic heterogeneity within liver clones is rarely considered. Somatic mutations are common in most healthy individuals, and there is accumulating evidence that mutation burden increases with age and chronic tissue damage. The present disclosure is based in part on the novel discovery that positive selection of somatic mutations in the liver of NASH patients can increase cellular fitness and competitiveness through reduced lipid accumulation in NASH. In vivo genetic screening of somatically mutated genes in the liver identified additional genes, such as GPAM (glycerol-3-phosphate acyltransferase, mitochondrial), that, when mutated, promote clonal fitness through reversal of lipotoxicity. Thus, compositions and methods of use thereof are provided herein for the treatment of liver disease, particularly metabolic liver disease, such as, but not limited to, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0018] I. Terminology The terminology and nomenclature used herein are for purposes of explanation and should not be considered limiting. For example, the use of singular terms, such as "a," is not intended to limit the number of items. Also, the use of relationship terms, such as, but not limited to, "top," "bottom," "left," "right," "upper," "lower," "down," "up," and "side," are used in the description for clarity, with particular reference to the figures, and are not intended to limit the scope of the inventive concept or the appended claims.

[0019] Furthermore, since the inventive concept is susceptible to embodiment in many different forms, the present disclosure is intended to be regarded as an example of the principles of the inventive concept, and is not intended to limit the inventive concept to the specific embodiments shown and described. Any one of the features of the inventive concept may be used separately or in combination with any other feature. Reference to the term "embodiment", "embodiments" and / or the like in the description means that the feature and / or features referred to are included in at least one aspect of the description. Separate references to the term "embodiment", "embodiments" and / or the like in the description do not necessarily refer to the same embodiment, nor are they mutually exclusive, unless so expressly stated and / or readily apparent to one of ordinary skill in the art from the description. For example, a feature, structure, process, step, act or the like described in one embodiment may be included in other embodiments, but is not necessarily included. Thus, the inventive concept may include various combinations and / or integrations of the embodiments described herein. Moreover, not all aspects of the disclosure described herein are essential to its practice. Similarly, other systems, methods, features and advantages of the inventive concepts will be or become apparent to one of ordinary skill in the art upon review of the figures and description. It is intended that all such additional systems, methods, features and advantages be included within this description, be within the scope of the inventive concepts, and be encompassed by the claims.

[0020] As used herein, the term "about" can mean ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2% or ±1% of a stated value, e.g., amount, dosage, temperature, time, percentage, etc.

[0021] The terms "comprising," "including," "containing," and "having" are used interchangeably in this disclosure. The terms "comprising," "including," "containing," and "having" mean to include, but are not necessarily limited to, what is so described.

[0022] As used herein, the terms "or" and "and / or" should be construed as inclusive or meaning any one or any combination. Thus, "A, B or C" or "A, B and / or C" means any of: "A", "B" or "C"; "A and B"; "A and C"; "B and C"; "A, B and C". Exceptions to this definition occur only where combinations of elements, features, steps or acts are in some way mutually exclusive by nature.

[0023] As used herein, "biomarker" refers to any biological molecule (e.g., nucleic acid, gene, peptide, protein, lipid, hormone, metabolite, etc.) that, either alone or collectively, reflects the current state or predicts the future state of a biological system. Thus, as used herein, the presence or concentration of one or more biomarkers can be detected and correlated with a known state, e.g., a disease state. In some embodiments, the detection of the presence and / or concentration of one or more biomarkers herein can be an indicator of the risk of liver disease in a subject. In some other embodiments, the detection of the presence and / or concentration of one or more biomarkers herein can be used in treating and / or preventing chronic liver disease in a subject.

[0024] As used herein, the terms "treat," "treating," "treatment," and the like, unless otherwise indicated, can refer to reversing, alleviating, inhibiting the processes of, or preventing the disease, disorder, or condition to which such term applies, or one or more symptoms of such disease, disorder, or condition, and include administering any of the compositions, pharmaceutical compositions, or dosage forms described herein to prevent the onset of, or alleviate, a symptom or complication, or eliminate the condition or disorder.

[0025] As used herein, the term "biomolecule" refers to, but is not limited to, proteins, enzymes, antibodies, DNA, siRNA, and small molecules. As used herein, "small molecules" can refer to chemicals, compounds, drugs, and the like.

[0026] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in either single-stranded or double-stranded form. Unless otherwise specified, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses not only the sequence explicitly indicated, but also their conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0027] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that a protein or peptide sequence may contain. A polypeptide includes any peptide or protein that contains two or more amino acids linked together by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as, for example, peptides, oligopeptides and oligomers, and longer chains, commonly referred to in the art as proteins, of which there are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, mutants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0028] It is also understood that, unless expressly stated otherwise, in any method claimed herein that includes more than one step or act, the order of the method steps or acts is not necessarily limited to the order in which the method steps or acts are described.

[0029] II. Composition (a)GPAM In certain embodiments, the compositions for use in the methods disclosed herein can modulate the GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) gene. The GPAM gene encodes a mitochondrial enzyme, Gpam, which catalyzes the acylation of glycerol-3-phosphate with acyl-coenzyme A (CoA) to produce CoA and lysophosphatidic acid (LPA). This is the rate-limiting step in TAG synthesis. There are two forms of this enzyme, one present in mitochondria and one present in the endoplasmic reticulum. In addition, two alternatively spliced ​​transcript variants of the GPAM gene have been described.

[0030] As used herein, a composition that "modulates" GPAM can include any biological molecule that can reduce the expression of the GPAM gene, reduce the expression of the GPAM protein, reduce GPAM activity, or a combination thereof. In some embodiments, a biological molecule herein that can modulate GPAM can be an inhibitor of GPAM. As used herein, an inhibitor of GPAM can inhibit the direct activity of GPAM, inhibit the indirect activity of GPAM, reduce the expression of the GPAM gene, reduce the expression of the GPAM protein, or a combination thereof.

[0031] In certain embodiments, the composition for use in the methods disclosed herein can include a nucleic acid molecule. As used herein, the term "nucleic acid molecule" refers to a molecule having nucleotides. The nucleic acid can be single-stranded, double-stranded, or multi-stranded, and can include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some embodiments, the nucleic acid molecule for use herein can be double-stranded RNA. In some examples, the double-stranded RNA suitable for use herein can be small RNA, small nuclear RNA, small nucleolar RNA, small hairpin RNA, microRNA, or the like. In certain embodiments, the double-stranded RNA suitable for use herein can be small interfering RNA (siRNA).

[0032] As used herein, the term "siRNA" refers to a small inhibitory RNA duplex that induces the RNA interference (RNAi) pathway. The siRNA molecules disclosed herein may be capable of silencing, reducing and / or inhibiting expression of a target gene (e.g., GPAM). These molecules vary in length (generally about 5-50 base pairs) and may contain varying degrees of complementarity to their target mRNA in the antisense strand. Some, but not all, siRNAs have unpaired overhanging bases at the 5' or 3' end of the sense and / or antisense strand. As used herein, the term "siRNA" may include duplexes of two separate strands, as well as single strands that can form hairpin structures that include the duplex region.

[0033] In certain embodiments, the siRNA molecules disclosed herein can be any interfering RNA with a duplex length of about 2 to 60, about 5 to 50, or about 10 to 40 nucleotides in length, more typically about 2 to 30, about 5 to 25, or about 10 to 25 nucleotides in length. In some embodiments, the siRNA molecules disclosed herein can have a nucleotide sequence that is about 2 to about 30 nucleotides in length, about 5 to about 25 nucleotides in length, or about 10 to about 20 nucleotides in length. In some embodiments, the siRNA molecules disclosed herein can have a nucleotide sequence that is about 10 nucleotides in length, about 11 nucleotides in length, about 12 nucleotides in length, about 13 nucleotides in length, about 14 nucleotides in length, about 15 nucleotides in length, about 14 nucleotides in length, about 15 nucleotides in length, about 16 nucleotides in length, about 17 nucleotides in length, about 18 nucleotides in length, about 19 nucleotides in length, about 20 nucleotides in length, about 21 nucleotides in length, about 22 nucleotides in length, about 23 nucleotides in length, about 24 nucleotides in length, or about 25 nucleotides in length. Each complementary sequence of the double-stranded siRNA can be about 2 to 60, about 5 to 50, about 10 to 40, about 2 to 30, about 5 to 25, or about 10 to 25 nucleotides in length, although other non-complementary sequences may be present. For example, the siRNA duplex can include a 3' overhang of about 1 to about 4 or more nucleotides and / or a 5' phosphate terminus containing about 1 to about 4 or more nucleotides.

[0034] In certain embodiments, the siRNA molecules disclosed herein may have about 2-60, about 2-50, about 2-40, or about 2-30 contiguous nucleotides homologous to a target (e.g., GPAM) nucleotide sequence. In some aspects, the target nucleotide sequence herein may be a human GPAM nucleotide sequence or a variant thereof. In some other aspects, the target nucleotide sequence herein may be human GPAM, RefSeq:NM_001244949.2, or a variant thereof. In yet some other aspects, the target nucleotide sequence herein may be the following SEQ ID NO: 1, or a variant thereof: Human GPAM (SEQ ID NO: 1) [ka]

[0035] In certain embodiments, the siRNA molecules disclosed herein may be about 2-30 contiguous nucleotides in length and have a nucleotide sequence that is at least about 80% (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) homologous to at least about 2-30 contiguous nucleotides of the human GPAM cDNA sequence. In some embodiments, the siRNA molecules disclosed herein may be about 2-30 contiguous nucleotides in length and have a nucleotide sequence that is at least 80% (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) homologous to at least about 2-30 contiguous nucleotides of the human GPAM sequence RefSeq:NM_001244949.2 or a variant thereof. In some embodiments, the siRNA molecules disclosed herein may be about 2-30 contiguous nucleotides in length and have a nucleotide sequence that is at least 80% (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) homologous to at least about 2-30 contiguous nucleotides of SEQ ID NO: 1. In some other embodiments, the siRNA molecules disclosed herein may be about 2-30 contiguous nucleotides in length and have a nucleotide sequence that is at least ....

[0036] The siRNA molecules disclosed herein can be synthesized in any of several conformations.Those skilled in the art will recognize the type of conformation of siRNA that is used for a particular purpose.Examples of siRNA conformations include, but are not limited to, a double-stranded polynucleotide molecule that is assembled from two separate strand molecules, one strand being a sense strand and the other being a complementary antisense strand; a double-stranded polynucleotide molecule that is assembled from a single-stranded molecule that is connected by a nucleic acid-based or non-nucleic acid-based linker, a double-stranded polynucleotide molecule that has a hairpin secondary structure with complementary sense and antisense regions; or a circular single-stranded polynucleotide molecule that has a stem with two or more loop structures and self-complementary sense and antisense regions.In the case of circular polynucleotides, polynucleotides can be processed either in vivo or in vitro to generate active double-stranded siRNA molecules.

[0037] In certain embodiments, the siRNA molecule disclosed herein can be a double-stranded siRNA molecule. In some aspects, the double-stranded siRNA molecule disclosed herein can have at least one sense sequence. In some other aspects, the double-stranded siRNA molecule disclosed herein can have at least one antisense sequence. In yet some other aspects, the double-stranded siRNA molecule disclosed herein can have at least one antisense sequence and at least one sense sequence. In some embodiments, the double-stranded siRNA molecule disclosed herein can have at least one antisense sequence selected from Table 1, at least one sense sequence selected from Table 1, or both.

[0038] JPEG2025513018000003.jpg252170JPEG2025513018000004.jpg253170JPEG2025513018000005.jpg253170

[0039] In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 2-83. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one antisense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 2-83 and at least one antisense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence of SEQ ID NOs: 2-83. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one antisense sequence of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence of SEQ ID NOs: 2-83 and at least one antisense sequence of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence of SEQ ID NO: 2-63. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one antisense sequence of SEQ ID NO: 84-145. In some embodiments, the double-stranded siRNA molecules disclosed herein may have at least one sense sequence of SEQ ID NO: 2-63 and at least one antisense sequence of SEQ ID NO: 84-145. In some aspects, the double-stranded siRNA molecules disclosed herein may have a sense sequence of SEQ ID NO: 8 and an antisense sequence of SEQ ID NO: 90.In some other embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of SEQ ID NO: 9 and an antisense sequence of SEQ ID NO: 91. In yet some other embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of SEQ ID NO: 13 and an antisense sequence of SEQ ID NO: 95. In yet some other embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of SEQ ID NO: 17 and an antisense sequence of SEQ ID NO: 99.

[0040] In some embodiments, the present disclosure also encompasses nucleic acid sequences encoding the double-stranded siRNA molecules disclosed herein. In some embodiments, the nucleic acid sequences encoding the double-stranded siRNAs disclosed herein can be double-stranded DNA, single-stranded DNA, a plasmid vector, a viral vector, such as a retroviral vector, a lentiviral vector, a poxvirus vector, an adenoviral vector, or an adeno-associated viral vector. In some embodiments, the nucleic acid can encode at least one sense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 2-83. In some embodiments, the nucleic acid can encode at least one antisense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid can encode at least one sense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 2-83 and at least one antisense sequence having at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid can encode at least one sense sequence of any one of SEQ ID NOs: 2-83. In some embodiments, the nucleic acid can encode at least one antisense sequence of any one of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid can encode at least one sense sequence of SEQ ID NOs: 2-58 and at least one antisense sequence of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid can encode a sense sequence of SEQ ID NO: 9 and an antisense sequence of SEQ ID NO: 91. In some embodiments, the nucleic acid can encode the sense sequence of SEQ ID NO:13 and the antisense sequence of SEQ ID NO:95.In some embodiments, the nucleic acid molecule can encode the sense sequence of SEQ ID NO:17 and the antisense sequence of SEQ ID NO:99.

[0041] The present disclosure also provides methods for making the siRNA molecules disclosed herein. The creation of siRNA may be via chemical synthesis, or the siRNA may be encoded and transcribed into a plasmid, or vectorized by a virus engineered to express the siRNA. The siRNA may be a single-stranded molecule with a complementary sequence that self-hybridizes to a duplex with a hairpin loop. The siRNA may also be generated by cleavage of a parent dsRNA using a suitable enzyme, such as E. coli RNase III, or Dicer. The parent dsRNA may be any double-stranded RNA duplex from which siRNA can be generated, for example, a full or partial mRNA transcript. The use of cell lysates or in vitro processing may require subsequent isolation of short nucleotide siRNAs (e.g., about 2-25 nucleotides in length) from the lysate, making the process somewhat cumbersome and expensive. Chemical synthesis proceeds by creating two single-stranded RNA oligomers, followed by annealing the two single-stranded oligomers to a double-stranded RNA. Methods of chemical synthesis are diverse and can be readily adapted to synthesize a variety of siRNAs.

[0042] In certain embodiments, the siRNA molecules disclosed herein can eliminate the gene expression of GPAM.The methods known in the art for detecting and quantifying RNA expression suitable for use herein can include, but are not limited to, Northern blotting and in situ hybridization, RNAse protection assay, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative reverse transcription PCR (RT-qPCR or qPCR), sequencing-based gene expression analysis (e.g., Serial Analysis of Gene Expression (SAGE)), massively parallel signature sequencing (MPSS) gene expression analysis, etc.

[0043] The siRNA molecule of the present invention specifically downregulates the gene expression of GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) or its mutants. The term downregulate may be interchangeably expressed as reducing, inhibiting, preventing, blocking or silencing. As used herein, the phrase "downregulate gene expression" refers to any reduced level of gene expression compared to normal expression levels. For example, the reduced level of gene expression may be about 70% to 0% of normal expression levels. In other words, about 30% to 100% of gene expression is downregulated, reduced, blocked, inhibited, prevented or silenced compared to normal expression levels. In particular, the level of gene expression reduction is about 70%, 65%, 60%, 55%, 50%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 5%, 4%, 3%, 2%, 1% or 0% of the normal expression level of GPAM. The level of gene expression reduction can also be any percentage or range as above. In contrast, the term "specific" or "specifically" used in combination with downregulate refers to the downregulation of the expression of the target gene with minimal or no binding or downregulation of other nucleic acids or their expression.

[0044] In some embodiments, the siRNA molecules disclosed herein can reduce gene expression of GPAM by at least about 50%. In some aspects, the siRNA molecules disclosed herein can reduce gene expression of GPAM by about 50% to about 99%, about 55% to about 98%, or about 60% to about 95%. In some aspects, the siRNA molecules disclosed herein can reduce gene expression of GPAM by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0045] In certain embodiments, the siRNA molecules disclosed herein can eliminate the protein expression of Gpam. Methods known in the art for detecting and quantifying protein expression suitable for use herein include, but are not limited to, ELISA (enzyme-linked immunosorbent assay), immunoblot assay, flow cytometry assay, immunohistochemical assay, radioimmunoassay, Western blot assay, immunofluorescence assay, chemiluminescence assay, mass spectrometry assay, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass mapping, liquid chromatography / quadrupole time-of-flight electrospray ionization tandem mass spectrometry (LC / Q-TOF-ESI-MS / MS), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), etc.

[0046] In some embodiments, the siRNA molecules disclosed herein can reduce the protein expression of Gpam by at least about 50%. In some aspects, the siRNA molecules disclosed herein can reduce the protein expression of Gpam by about 50% to about 99%, about 55% to about 98%, or about 60% to about 95%. In some aspects, the siRNA molecules disclosed herein can reduce the protein expression of Gpam by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0047] In some embodiments, the siRNA molecules disclosed herein may have one or more chemical modifications. Non-limiting examples of chemical modifications may include terminal cap moieties, phosphate backbone modifications, and the like. Examples of classes of terminal cap moieties include, but are not limited to, inverted deoxyabasic residues, glyceryl modifications, 4',5'-methylene nucleotides, 1-(β-D-erythrofuranosyl) nucleotides, 4'-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threopentofuranosyl nucleotides, acyclic 3',4'-seconucleotides, acyclic 3,4-dihydroxybutyl nucleotides, acyclic 3,5-dihydroxypentyl nucleotides, 3'-3'-inverted nucleotide moieties, 3'-3'-inverted abasic moieties, 3'-2'-inverted nucleotide moieties, 3'-2'-inverted abasic moieties, 5'- ... These include 5'-inverted nucleotide moieties, 5'-5'-inverted abasic moieties, 3'-5'-inverted deoxy abasic moieties, 5'-amino-alkyl phosphates, 1,3-diamino-2-propyl phosphate, 3 aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidates, 5' phosphoramidates, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioates, 5'-phosphorothioates, phosphorodithioates and bridged or non-bridged methylphosphonates or 5'-mercapto moieties. Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide linkages) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal and alkylsilyl substitutions. Such chemical modifications can be present at the 5'-end and / or 3'-end of the sense strand, antisense strand or both strands of siRNA.

[0048] Chemical modification of the siRNA molecule disclosed herein can include modification of at least one ribosugar moiety of its nucleotide sequence. The ribosugar moiety can be modified with 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, 2'-C-allyl, or any combination thereof. In some embodiments, the siRNA molecule disclosed herein can have less than about 10% to about 70% (e.g., about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%) of the ribosugar moiety of the entire nucleotide sequence modified. Chemical modification of the siRNA molecule disclosed herein can include attaching a conjugate to the siRNA molecule. The type of conjugate used and the degree of conjugation to siRNA can be evaluated for improving the pharmacokinetic profile, bioavailability and / or stability of siRNA while retaining activity.Thus, those skilled in the art can use any of a variety of well-known in vitro cell culture or in vivo animal models, including the above-mentioned negatively regulated expression studies, to screen siRNA molecules with various conjugates attached to them to identify siRNA conjugates with improved properties.The conjugate can be covalently linked, for example, via a nucleic acid or non-nucleic acid linker, to the 5' and / or 3' ends of the sense and / or antisense strands of siRNA.The conjugate can be linked to siRNA via a carbamate group or other linking group (see, for example, US Patent Publication Nos. 20050074771, 20050043219 and 20050158727, the contents of each of which are incorporated herein by reference).The conjugate can be added to siRNA for any of several purposes. For example, the conjugate may be a molecular entity that facilitates delivery into a cell, or may be a molecule that includes a drug or label.Examples of conjugate molecules suitable for binding to the siRNA of the present invention include, but are not limited to, steroids, such as cholesterol, glycols, such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenes, bile acids, folates (e.g., folic acid, folate analogs and their derivatives), sugars (e.g., galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, ligands for cell receptors that can mediate cellular uptake, and combinations thereof.Other examples include lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalators, minor groove binders, cleavage agents and crosslinker conjugate molecules, as described, for example, in U.S. Patent Publication Nos. 20050119470 and 20050107325, the contents of each of which are incorporated herein by reference. Other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationic modified pyrimidines, cationic peptides, guanidinium groups, amidinium groups, cationic amino acid conjugate molecules, etc. Further examples of conjugate molecules include hydrophobic groups, membrane active compounds, cell penetrating compounds, cell targeting signals, interaction modifiers or steric hindrance stabilizers, as described in U.S. Patent Publication No. 20040167090, which is incorporated herein by reference.

[0049] In certain embodiments, the siRNA molecules disclosed herein can be conjugated to at least one targeting ligand. Targeting ligands contemplated herein include ligands suitable for targeting siRNA molecules to the liver, liver tissue and / or liver cells. Non-limiting examples of targeting ligands suitable for use herein include galactose, galactosamine, N-formyl-galactosamine, N-acetylgalactosamine, N-propionyl-galactosamine, Nn-butanoyl-galactosamine, N-iso-butanoylgalactose-amine, galactose cluster and N-acetylgalactosamine trimer, which may optionally have a pharmacokinetic modulator selected from the group consisting of a hydrophobic group having 16 or more carbon atoms, a hydrophobic group having 16 to 20 carbon atoms, palmitoyl, hexadeca-8-enoyl, oleyl, (9E,12E)-octadeca-9,12 dienoyl, dioctanoyl and C16 to C20 acyl and cholesterol. In some aspects, liver targeting ligands suitable for use herein may be N-acetylgalactosamine (GalNAc) conjugates. In some embodiments, the siRNA molecules disclosed herein may be conjugated to at least one GalNAc conjugate. In some embodiments, the siRNA molecules disclosed herein may be conjugated to about 1 to about 10 GalNAc conjugates, about 2 to about 9 GalNAc conjugates, or about 3 to about 8 GalNAc conjugates. In some embodiments, the siRNA molecules disclosed herein may be conjugated to about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 GalNAc conjugates.

[0050] Any of the siRNA molecules disclosed herein can target the open reading frame or 5' or 3' UTR of the GPAM gene or at least one variant thereof. In some embodiments, at least one variant of GPAM can include rs10787429 C>T, rs7096937 T>C, rs11446981 T>TA, rs7898213 T>C, rs2792759 C>T, rs2297991 T>C, rs1129555 A>G, rs2254537 T>A, or any combination thereof. In some embodiments, the siRNA molecules disclosed herein can specifically downregulate the gene expression of GPAM or its variants, such as rs10787429 C>T, rs7096937 T>C, rs11446981 T>TA, rs7898213 T>C, rs2792759 C>T, rs2297991 T>C, rs1129555 A>G, rs2254537 T>A, or any combination thereof. GPAM gene and / or its variants can be associated with liver disease, liver dysfunction, liver injury, and / or liver damage. Liver disease can include fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer, or any combination thereof. Liver disorders can include cirrhosis, chronic infection with hepatitis B virus (HBV), chronic infection with hepatitis C virus (HCV), nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), hereditary hemochromatosis, type 2 diabetes, obesity, smoking, alcohol abuse, long-term use of anabolic steroids, tyrosinemia, alpha-1 antitrypsin deficiency, porphyria cutanea tarda, glycogen storage disease, Wilson's disease, or any combination thereof.

[0051] In certain embodiments, the methods and compositions provided herein can include a vector comprising any one of the siRNA molecules disclosed herein. In some embodiments, a vector for use herein can be a viral vector. As used herein, the term "viral vector" can refer to a nucleic acid vector construct that contains at least one element of viral origin, has the ability to be packaged into a viral vector particle, and encodes at least one exogenous polynucleotide. In certain embodiments, the vector and / or particle can be utilized to transfer any nucleic acid into a cell, either in vitro or in vivo. Numerous viral vectors are known in the art. The term virion can refer to a single infectious viral particle. "Viral vector," "viral vector particle," and "viral particle" also refer to a complete viral particle with its DNA or RNA core and protein coat when present outside a cell. Non-limiting examples of viral vectors for use herein can include adenovirus, adeno-associated virus (AAV), herpes virus, retrovirus, lentivirus, integrase-deficient lentivirus (IDLV), and the like. In some embodiments, a viral vector disclosed herein can be a lentiviral vector. Examples of lentiviruses include, but are not limited to, human lentiviruses, such as HIV (especially HIV-1 or HIV-2), simian immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), feline immunodeficiency virus (FIV), caprine arthritis encephalitis virus (CAEV), ovine visna and progressive pneumonia virus, baboon pseudotype virus, bovine immunodeficiency virus (BIV), etc. In some embodiments, the siRNA molecules and / or vectors described herein can be prepared by conventional recombinant techniques known to those skilled in the art. In other embodiments, the siRNA molecules and / or vectors described herein can be prepared by gene editing methods known in the art (e.g., by CRISPR).In certain embodiments, the methods provided herein can include generating cells for expressing any of the siRNA molecules and / or vectors described herein. In some embodiments, the vectors, viral particles, etc. contemplated herein can be encapsulated in liposomes for delivery to a subject.

[0052] In some embodiments, the disclosure also encompasses the use of gene editing systems, such as CRISPR-based systems, to abolish or downregulate gene expression of GPAM. Thus, the disclosure also encompasses compositions comprising a nucleic acid sequence associated with the clustered regularly interspaced short palindromic repeats (CRISPR) system, such as a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a transactivating crRNA (tracrRNA), and an additional plasmid DNA (pDNA) or viral vector encoding a nucleic acid sequence associated with the clustered regularly interspaced short palindromic repeats (CRISPR) system that specifically targets GPAM or a variant thereof. In some embodiments, the gene editing system comprises at least one sgRNA that targets GPAM and an RNA-guided endonuclease, such as Cas9. In some embodiments, the target sequence of the sgRNA has at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95% or about 99%) to any one of SEQ ID NOs: 177-276. In some embodiments, the target sequence of the sgRNA is at least about 80% identical to any one of SEQ ID NOs: 177-276 provided in Table 2. In some embodiments, the sgRNA targets a sequence that is at least about 80% or about 85% or about 90% or about 95% or about 100% identical to SEQ ID NOs: 177-276. In some embodiments, the sgRNA targets 20 consecutive nucleotides of any one of SEQ ID NOs: 277-376. None of the gRNAs provided in Table 2 are expected to target the final exon or have a 10 bp off-target match.

[0053] JPEG2025513018000006.jpg255170JPEG2025513018000007.jpg255166JPEG202 5513018000008.jpg255169JPEG2025513018000009.jpg255168JPEG20255130180 00010.jpg255169JPEG2025513018000011.jpg255167JPEG2025513018000012.j pg255168JPEG2025513018000013.jpg255163JPEG2025513018000014.jpg255164

[0054] (b) Pharmaceutical Composition The siRNA molecules disclosed herein targeting GPAM for use in accordance with the methods described herein may be provided by themselves and / or as part of a pharmaceutical composition in which the modulators and / or inhibitors may be mixed with suitable carriers or excipients.

[0055] As used herein, "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical ingredients, such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of a compound to an organism. As used herein, the term "active ingredient" refers to any of the siRNA molecules disclosed herein. As used herein, the term "active ingredient" can also include any vector, medium, microorganism or cell culture in which siRNA molecules are synthesized, expressed and / or contained, such as genetically modified cells, viral vectors, plasmids, bacteria, yeast, fungi and their cultures or media.

[0056] (i) Pharmaceutically Acceptable Carriers and Excipients Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutical acceptable carrier" are used interchangeably herein to refer to a carrier or diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the compound being administered. Adjuvants are included in these phrases.

[0057] In certain embodiments, the compositions disclosed herein can further comprise one or more pharma- ceutically acceptable diluents, excipients and / or carriers.As used herein, pharma-ceutically acceptable diluents, excipients or carriers refer to materials that are suitable for administration to a subject and do not cause undesirable biological effects or interact in a harmful manner with any of the components of the composition in which they are contained.Pharmaceutically acceptable diluents, carriers and excipients can include, but are not limited to, saline, Ringer's solution, phosphate solution or buffer, buffered saline and other carriers known in the art.

[0058] In some embodiments, the pharmaceutical compositions herein may also include stabilizers, antioxidants, colorants, other medicinal or formulating agents, carriers, adjuvants, preservatives, stabilizers, wetting agents, emulsifiers, solubility enhancers, salts, solubilizers, antifoaming agents, antioxidants, dispersing agents, surfactants, or any combination thereof. As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples of excipients include, but are not limited to, calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Techniques for drug formulation and administration can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa., latest edition, which is incorporated herein by reference.

[0059] In certain embodiments, the pharmaceutical compositions described herein can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents, to facilitate the processing of genetically modified endothelial progenitor cells into preparations that can be used medicaments.In some embodiments, any of the well-known techniques, carriers and excipients can be used as appropriate and / or as understood in the art.

[0060] In certain embodiments, the pharmaceutical compositions described herein may be aqueous suspensions containing one or more polymers as suspending agents. In some embodiments, the polymers that can be included in the pharmaceutical compositions described herein include water-soluble polymers, such as cellulosic polymers, such as hydroxypropylmethylcellulose; for example, polymers containing cross-linked carboxyls; mucoadhesive polymers, such as water-insoluble polymers selected from carboxymethylcellulose, carbomer (acrylic acid polymer), poly(methyl methacrylic acid), polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate, and dextran; or combinations thereof. In some embodiments, the pharmaceutical compositions disclosed herein may contain a total amount of polymers as suspending agents of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% by weight of the total composition. In some embodiments, the pharmaceutical compositions disclosed herein may comprise a total amount of polymer as a suspending agent, by total weight of the composition, of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%.

[0061] In certain embodiments, the pharmaceutical compositions disclosed herein can include a viscous formulation. In some embodiments, the viscosity of the compositions disclosed herein can be increased by adding one or more gelling or thickening agents. In some embodiments, the compositions disclosed herein can include one or more gelling or thickening agents in an amount that provides the formulation with sufficient viscosity to remain in the tissue to be treated. In some embodiments, the pharmaceutical compositions disclosed herein can include at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% total amount of gelling or thickening agents by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can include at least about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% total amount of gelling or thickening agents by total weight of the composition. In some embodiments, suitable viscosity enhancing agents for use herein may be hydroxypropyl methylcellulose, hydroxyethyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate.In other embodiments, the viscosity enhancing agent is selected from the group consisting of acacia (gum arabic), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellan, gelatin, ghatti gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, xanthan gum, tragacanth gum, ethyl cellulose, ethyl hydroxyethyl cellulose, ethyl methyl cellulose. The polysaccharide may be cellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropylmethyl-cellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose) or any combination thereof.

[0062] In certain embodiments, the pharmaceutical compositions disclosed herein can include additional agents or additives selected from the group including surface active agents, surfactants, solvents, acidifying agents, alkaline agents, buffering agents, tonicity modifying agents, ionic additives effective to increase the ionic strength of the solution, antibacterial agents, antibiotics, antifungal agents, antioxidants, preservatives, electrolytes, antifoaming agents, oils, stabilizers, enhancers, etc. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more agents in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more agents in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% by total weight of the composition. In some embodiments, one or more of these agents can be added to improve the performance, efficacy, safety, shelf life and / or other properties of the muscarinic antagonist compositions of the present disclosure. In some embodiments, the additives can be non-harsh, non-abrasive, non-allergenic, and biocompatible.

[0063] In certain embodiments, the pharmaceutical compositions disclosed herein can include one or more acidifying agents. As used herein, "acidifying agent" refers to a compound used to provide an acidic medium. Such compounds include, by way of example and without limitation, acetic acid, amino acids, citric acid, fumaric acid, and other alpha hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid, as well as others known to those skilled in the art. In some embodiments, any pharma- ceutically acceptable organic or inorganic acid can be used. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more acidifying agents in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more acidifying agents in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% by total weight of the composition.

[0064] In certain embodiments, the pharmaceutical compositions disclosed herein can include one or more alkaline agents. As used herein, an "alkaline agent" is a compound used to provide an alkaline medium. Examples of such compounds include, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, and trolamine, as well as others known to those skilled in the art. In some embodiments, any pharma- ceutically acceptable organic or inorganic base can be used. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more alkaline agents in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more alkaline agents in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0065] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more antioxidants. As used herein, an "antioxidant" is an agent that can be used to inhibit oxidation and thereby prevent the deterioration of preparations due to the oxidative process. Examples of such compounds include, but are not limited to, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, hypophosphorous acid, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfoxylate, sodium metabisulfite, and other materials known to those skilled in the art. In some embodiments, the pharmaceutical compositions disclosed herein may include one or more antioxidants in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% by weight of the total composition. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more antioxidants in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0066] In certain embodiments, the pharmaceutical compositions disclosed herein can include a buffer system. As used herein, a "buffer system" is a composition composed of one or more buffering agents, and a "buffering agent" is a compound used to resist changes in pH upon dilution or addition of acid or alkali. Examples of buffering agents include, but are not limited to, potassium metaphosphate, potassium phosphate, monobasic sodium acetate, and sodium citrate anhydrous and dihydrate, as well as other materials known to those of skill in the art. In some embodiments, any pharma- ceutically acceptable organic or inorganic buffer can be used. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more buffering agents in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more buffering agents in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0067] In some embodiments, the amount of one or more buffering agents may be present at the desired pH level of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may have a pH of about 6 to about 9. In some embodiments, the pharmaceutical compositions disclosed herein may have a pH greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0068] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more preservatives. As used herein, "preservative" refers to an agent or combination of agents that inhibit, reduce or eliminate bacterial growth in a pharmaceutical dosage form. Non-limiting examples of preservatives include Nipagin, Nipasol, isopropyl alcohol and combinations thereof. In some embodiments, any pharma- ceutical acceptable preservative may be used. In some embodiments, the pharmaceutical compositions disclosed herein may include one or more preservatives in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include one or more preservatives in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0069] In certain embodiments, the pharmaceutical compositions disclosed herein can include one or more surface-activating agents or surfactants. In some embodiments, the surface-activating agents or surfactants can be synthetic, natural, or semi-synthetic. In some embodiments, the compositions disclosed herein can include anionic surfactants, cationic surfactants, zwitterionic surfactants, ampholytic surfactants, amphoteric surfactants, nonionic surfactants with a steroid backbone, or combinations thereof. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more surface-activating agents or surfactants in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% by weight of the total composition. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more surface-activating agents or surfactants in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% by weight of the total composition.

[0070] In certain embodiments, the pharmaceutical compositions disclosed herein can include one or more stabilizers. As used herein, "stabilizer" refers to a compound used to stabilize an active agent against physical, chemical or biochemical processes that would otherwise reduce the therapeutic activity of the agent. Examples of suitable stabilizers include, but are not limited to, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, niacinamide, sodium acetyltryptophan, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprylate and sodium saccharin, and others known to those skilled in the art. In some embodiments, the pharmaceutical compositions disclosed herein can include one or more stabilizers in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50% by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may comprise one or more stabilizers in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0071] In some embodiments, the pharmaceutical compositions disclosed herein may include one or more tonicity agents. As used herein, "tonicity agent" refers to a compound that can be used to adjust the tonicity of a liquid formulation. Suitable tonicity agents include, but are not limited to, glycerin, lactose, mannitol, dextrose, sodium chloride, sodium sulfate, sorbitol, trehalose, and others known to those skilled in the art. The osmolality of a composition may be expressed in milliosmoles per liter (mOsm / L). Osmolality may be measured using methods commonly known in the art. In some embodiments, the vapor pressure depression method is used to calculate the osmolality of a composition disclosed herein. In some embodiments, the amount of one or more tonicity agents comprising the pharmaceutical compositions disclosed herein may result in a composition osmolality of about 150 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 280 mOsm / L to about 370 mOsm / L, or about 250 mOsm / L to about 320 mOsm / L. In some embodiments, the compositions herein may have an osmolality in the range of about 100 mOsm / kg to about 1000 mOsm / kg, about 200 mOsm / kg to about 800 mOsm / kg, about 250 mOsm / kg to about 500 mOsm / kg, or about 250 mOsm / kg to about 320 mOsm / kg, or about 250 mOsm / kg to about 350 mOsm / kg, or about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, the pharmaceutical compositions described herein may have an osmolality of about 100 mOsm / L to about 1000 mOsm / L, about 200 mOsm / L to about 800 mOsm / L, about 250 mOsm / L to about 500 mOsm / L, about 250 mOsm / L to about 350 mOsm / L, about 250 mOsm / L to about 320 mOsm / L, or about 280 mOsm / L to about 320 mOsm / L.In some embodiments, the pharmaceutical compositions disclosed herein can comprise one or more tonicity modifiers in a total amount of at least about 5%, at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, by total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein can comprise one or more tonicity modifiers in a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90%, by total weight of the composition.

[0072] (ii) Dosage Formulation In certain embodiments, the present disclosure provides compositions that are formulated for one or more routes of administration.Suitable routes of administration can include, for example, oral, rectal, mucosal, nasal, intestinal, and / or parenteral delivery.In some embodiments, the compositions formulated herein can be formulated for parenteral delivery.In some embodiments, the compositions formulated herein can be formulated for intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal, and / or intranasal injection.

[0073] In certain embodiments, the compositions herein can be administered in a local or systemic manner, for example, by direct local administration of the pharmaceutical composition to a tissue region of a patient. In some embodiments, the pharmaceutical compositions disclosed herein can be administered parenterally, for example, by intravenous injection, intraventricular injection, intracisternal injection, intraparenchymal injection, or a combination thereof. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to a subject as disclosed herein. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to a human patient. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to a human patient via at least two routes of administration. In some embodiments, a combination of routes of administration is intraventricular injection and intravenous injection; intrathecal injection and intravenous injection; intracisternal injection and intravenous injection; and / or intraparenchymal injection and intravenous injection.

[0074] In certain embodiments, the pharmaceutical compositions of the present disclosure can be manufactured by processes that are well known in the art, for example, by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0075] In certain embodiments, therefore, the pharmaceutical composition used according to the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and auxiliary agents that facilitate the processing of active ingredient into preparations that can be used medicaments.Appropriate formulations depend on the route of administration selected.For injection, the active ingredient of the pharmaceutical composition herein can be formulated in aqueous solution, preferably in physiologically compatible buffer, such as Hanks' solution, Ringer's solution, physiological salt buffer or any combination thereof.

[0076] In certain embodiments, the pharmaceutical compositions described herein can be formulated in the form of nanoparticles. The nanoparticles may have a monolayer surrounding a nanoparticle core, and the siRNA molecules are disposed within the nanoparticle core. In one embodiment, the nanoparticle core comprises a solid lipid (i.e., lipid that remains solid at room temperature and body temperature) or a liquid lipid (i.e., oil that remains liquid at room temperature and body temperature, such as vegetable oil or lipid extracted from human adipose tissue). In particular, embodiments of the present disclosure include nanoparticles and compositions for controlled and / or sustained release (e.g., release at a rate predetermined to maintain a certain concentration over a certain period of time) of agents such as small interfering RNA (siRNA) from nanoparticles.

[0077] In certain embodiments, the pharmaceutical compositions described herein can be formulated for parenteral administration, for example, by bolus injection or continuous infusion.The preparations for injection herein can be provided in unit dosage form, for example, in ampoules or multi-dose containers, with optional addition of preservatives.In some embodiments, the compositions herein can be suspensions, solutions or emulsions in oily or aqueous vehicles, and / or can contain formulation agents, such as suspending agents, stabilizing agents and / or dispersing agents.

[0078] In certain embodiments, pharmaceutical compositions herein formulated for parenteral administration can include aqueous solutions of active preparations (e.g., siRNA molecules) in water-soluble form. In some embodiments, compositions herein can be prepared that include suspensions of active preparations as oil- or water-based injection suspensions. Suitable lipophilic solvents and / or vehicles for use herein can include, but are not limited to, fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. In some embodiments, compositions herein that include aqueous injection suspensions can include substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol, and / or dextran. In some embodiments, compositions herein that include suspensions can also include one or more suitable stabilizers and / or agents that increase the solubility of the active ingredient (e.g., siRNA molecules) to allow for the preparation of highly concentrated solutions.

[0079] In some embodiments, the compositions herein may contain the active ingredient in powder form for constitution with a suitable vehicle, e.g., a sterile pyrogen-free water-based solution, before use.

[0080] Pharmaceutical compositions suitable for use in the context of the present disclosure may include compositions that may contain active ingredients in an amount effective to achieve intended purpose. In some embodiments, a therapeutically effective amount refers to the amount of active ingredient (e.g., siRNA molecule) that is effective to prevent, delay, reduce or ameliorate the symptoms of the disorder (e.g., liver disease) or prolong the survival of the subject being treated.

[0081] Determination of a therapeutically effective amount is well within the capabilities of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0082] For any preparation used in the method of the present disclosure, therapeutically effective amount or dose can be estimated first from the in vitro and cell culture assays and / or screening platforms disclosed herein.For example, a dose can be formulated in animal models to achieve desired concentration or titer.Such information can be used to more accurately determine the dose that is useful in humans.

[0083] In some embodiments, the toxicity and therapeutic effect of the active ingredient disclosed herein can be determined by standard pharmaceutical procedures in vitro, in cell culture or in experimental animals. In some embodiments, the data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosages for use in human subjects. In some embodiments, the dosages for use herein can vary depending on the dosage form used and the route of administration utilized. The exact formulation, route of administration and dosage can be selected by the individual physician in consideration of the patient's condition (see, for example, Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch.1).

[0084] In certain embodiments, dosage and / or administration interval can be individually adjusted to the brain or blood level of active ingredient that is sufficient to induce or suppress biological effect (minimal effective concentration, MEC). In some embodiments, the MEC of active ingredient (e.g., siRNA molecule or composition disclosed herein) may vary with each preparation, but can be estimated from in vitro data. In some embodiments, the dosage required to achieve MEC herein may depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentration.

[0085] In certain embodiments, depending on the severity and responsiveness of the condition to be treated, dosing of the compositions herein may be a single or multiple administration, with the course of treatment lasting from several days to several weeks, or until a cure is effected or a diminution of the condition is achieved.

[0086] In certain embodiments, the amount of the compositions herein administered will depend on the subject being treated, the severity of the ailment, the manner of administration, the judgment of the prescribing physician, etc. In some embodiments, the effective amount can be extrapolated from dose-response curves derived from in vitro or in vivo test systems.

[0087] III.How to use The present disclosure provides a method for treating, attenuating and preventing liver disease in a subject in need thereof.In some embodiments, the method for treating, attenuating or preventing liver disease in a subject can comprise administering to a subject, including a human subject, an effective amount of one or more siRNA molecules that target GPAM or a nucleic acid that encodes a siRNA molecule that target GPAM disclosed herein.In some embodiments, the method for treating, attenuating or preventing liver disease in a subject can comprise administering to a subject, including a human subject, an effective amount of a suitable sgRNA or a suitable sgRNA that targets GPAM and a nucleic acid that encodes an RNA-guided endonuclease.

[0088] The methods disclosed herein can include treating a subject in need thereof by administering a therapeutically effective amount of one or more siRNA molecules or pharmaceutical compositions disclosed herein. The subject can be a human subject having, suspected of having, or at risk of having liver disease, liver disorder, liver dysfunction, liver injury. The terms "liver disease", "liver injury" or "liver dysfunction" can be used interchangeably and refer to any damage to the liver, including but not limited to liver hardening, liver scarring, reduced or abnormal biliary function, abnormal liver enzyme activity, liver cirrhosis, abnormal physiological function determined by common diagnostic methods including but not limited to ultrasound or biopsy / histopathology, liver necrosis, etc. Non-limiting examples of liver diseases treated using the methods disclosed herein can include fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer HCC, or any combination thereof. In some embodiments, the subject benefits from increased life expectancy compared to untreated subjects with the same disease state and predicted outcome.In some other embodiments, the treatment improves the liver function of the subject when compared to untreated subjects with the same disease state and predicted outcome.In still other embodiments, the treatment attenuates the liver fibrosis of the subject when compared to untreated subjects with the same disease state and predicted outcome.In some embodiments, the treatment prevents further liver fibrosis of the subject when compared to untreated subjects with the same disease state and predicted outcome.

[0089] The subject suitable for the treatment of liver disease disclosed herein as used herein can be selected based on the diagnosis of the subject. In some embodiments, the method of diagnosis can detect one or more serum markers indicative of liver disease. Non-limiting examples of serum markers indicative of liver disease (e.g., NAFLD or NASH) can include alpha fetoprotein (AFP) (e.g., AFP level of 20ng / mL or more), des-gamma-carboxy prothrombin, lentil agglutinin-reactive AFP (AFP-L3), etc. The method of diagnosis can also include the evaluation of at least one clinical symptom associated with liver disease. Non-limiting examples of clinical symptoms associated with liver disease include mild to moderate epigastric pain, weight loss, early satiety or a palpable epigastric mass, paraneoplastic syndromes, hypoglycemia, polycythemia, hypercalcemia, refractory diarrhea and associated electrolyte abnormalities (e.g., hyponatremia, hypokalemia, metabolic alkalosis), skin manifestations (e.g., dermatomyositis, pemphigus foliaceus, seborrheic keratosis, pityriasis oracia), intraperitoneal bleeding, jaundice, fever, suppurative liver abscess, and the like. Other aspects of the diagnosis may include cirrhosis of the liver at the time of diagnosis and / or determination of severity, chronic infection with Hepatitis B virus (HBV), chronic infection with Hepatitis C virus (HCV), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), hereditary hemochromatosis, type 2 diabetes mellitus, obesity, smoking, alcohol abuse, long-term use of anabolic steroids, tyrosinemia, alpha 1 antitrypsin deficiency, porphyria cutanea tarda, glycogen storage disease, Wilson's disease, or any combination thereof.

[0090] In some embodiments, a subject can be diagnosed and / or predicted to be at high or low risk for liver disease (e.g., NASH or NAFLD) by histological or imaging-based examination, for example, contrast-enhanced multiphase CT, ultrasound and / or MRI.The imaging features used for diagnosis can include liver size, dynamics, and contrast enhancement pattern, as well as growth on successive imaging, which can measure size as the maximum cross-sectional diameter of the image where lesion is most clearly seen.The histological aspects of NASH or NAFLD biopsy can include fatty degeneration, inflammation and fibrosis.

[0091] The disclosed methods and compositions are useful for treating subjects with fatty liver-related disorders, such as NAFLD and / or NASH. The subject may have normal or substantially normal biliary function. The presence of normal or abnormal biliary function can be determined in the subject using any suitable method known in the art. In general, preferred tests of biliary function in NASH patients are characterized into two groups: physiological-based tests and biochemical-based tests. Physiological-based tests can include, but are not limited to, abdominal ultrasound, abdominal CT scan, endoscopic retrograde cholangiopancreatography (ECRP), percutaneous transhepatic cholangiopancreatography (PTCA) or magnetic resonance cholangiopancreatography (MRCP). Biochemical-based tests can include, but are not limited to, GGT tests, liver function tests, bilirubin tests, alkaline phosphatase (ALP) tests, liver enzyme tests, amylase blood tests, lipase blood tests, prothrombin time and urinary bilirubin measurements. In some cases, one or more tests can be used to characterize the function of the bile duct. In some cases, a combination of tests can be used to assess bile duct function in subjects with NASH.

[0092] In some embodiments, any of the methods disclosed herein can further comprise monitoring the occurrence of one or more adverse effects in the subject.Adverse effects can include, but are not limited to, liver dysfunction, hematological toxicity, neurotoxicity, skin toxicity, gastrointestinal toxicity, or combinations thereof.If adverse effects are observed, the methods disclosed herein can further comprise reducing or increasing one or more doses of the therapeutic regimen according to one or more adverse effects of the subject.For example, if moderate to severe liver dysfunction is observed in the subject after treatment, the concentration or frequency of the composition used to treat the subject can be reduced.

[0093] In certain embodiments, treatment administered according to the methods disclosed herein can improve the patient's life expectancy compared to the life expectancy of untreated subjects with the same disease state (e.g., NAFLD or NASH) and predicted outcome. As used herein, "patient's life expectancy" is defined as the point at which 50 percent of subjects are alive and 50 percent are dead. In some embodiments, after treatment with the methods disclosed herein, the patient's life expectancy may be unclear. In other aspects, the patient's life expectancy may be increased by at least about 5% or more to at least about 100%, at least about 10% or more to at least about 95% or more, at least about 20% or more to at least about 80% or more, at least about 40% or more to at least about 60% or more compared to untreated subjects with the same disease state and predicted outcome. In some embodiments, the patient's life expectancy may be increased by at least about 5% or more, at least about 10% or more, at least about 15% or more, at least about 20% or more, at least about 25% or more, at least about 30% or more, at least about 35% or more, at least about 40% or more, at least about 45% or more, at least about 50% or more, at least about 55% or more, at least about 60% or more, at least about 65% or more, at least about 70% or more, at least about 75% or more, at least about 80% or more, at least about 85% or more, at least about 90% or more, at least about 95% or more, or at least about 100% compared to an untreated subject with the same disease state and predicted outcome.In some embodiments, the patient's life expectancy is increased by at least about 5% or more to at least about 10% or more, at least about 10% or more to at least about 15% or more, at least about 15% or more to at least about 20% or more, at least about 20% or more to at least about 25% or more, at least about 25% or more to at least about 30% or more, at least about 30% or more to at least about 35% or more, at least about 35% or more to at least about 40% or more, at least about 40% or more to at least about 45% or more, at least about 45% or more to at least or may extend by about 50% or more, from at least about 50% or more to at least about 55% or more, from at least about 55% or more to at least about 60% or more, from at least about 60% or more to at least about 65% or more, from at least about 65% or more to at least about 70% or more, from at least about 70% or more to at least about 75% or more, from at least about 75% or more to at least about 80% or more, from at least about 80% or more to at least about 85% or more, from at least about 85% or more to at least about 90% or more, from at least about 90% or more to at least about 95% or more, or from at least about 95% or more to at least about 100%.

[0094] In some embodiments, treating liver disease, e.g., NAFLD or NASH, with the methods disclosed herein can result in attenuation, shrinkage, reduction, or prevention of liver fibrosis, compared to the starting size of liver fibrosis. In some embodiments, the attenuation or shrinkage of liver fibrosis area is at least about 5% or more to at least about 10% or more, at least about 10% or more to at least about 15% or more, at least about 15% or more to at least about 20% or more, at least about 20% or more to at least about 25% or more, at least about 25% or more to at least about 30% or more, at least about 30% or more to at least about 35% or more, at least about 35% or more to at least about 40% or more, at least about 40% or more to at least about 45% or more, at least about 45% or more to at least about 50% or more, ... It can be from about 50% or more to at least about 55% or more, at least about 55% or more to at least about 60% or more, at least about 60% or more to at least about 65% or more, at least about 65% or more to at least about 70% or more, at least about 70% or more to at least about 75% or more, at least about 75% or more to at least about 80% or more, at least about 80% or more to at least about 85% or more, at least about 85% or more to at least about 90% or more, at least about 90% or more to at least about 95% or more, or at least about 95% or more to at least about 100% (meaning that liver fibrosis is completely eliminated after treatment).

[0095] In some embodiments, treating liver disease, such as NAFLD or NASH, by the methods disclosed herein can result in liver function improvement. Liver function can be examined by conventional biochemical methods. Biochemical examinations can include, but are not limited to, GGT examination, liver function examination, bilirubin examination, alkaline phosphatase (ALP) examination, liver enzyme examination, amylase blood examination, lipase blood examination, prothrombin time and urinary bilirubin measurement. In some cases, one or more examinations can be used to characterize the function of the bile duct. In some cases, a combination of examinations can be used to evaluate the function of the bile duct. The improvement of liver function can also be evaluated by the alleviation of the subject's symptoms, for example, the alleviation of the subject's fatigue, weight loss and weakness. Other alleviated symptoms can include fluid retention, muscle wasting, bleeding from the intestine, and any combination thereof.

[0096] IV. Kit The present disclosure provides a kit for using the method disclosed herein.In some embodiments, the present disclosure provides a kit for treating liver disease (such as NASH or NAFLD) and diagnosing liver disease as disclosed herein.Such a kit can include the means for holding and / or administering such siRNA composition or pharmaceutical composition.

[0097] In some embodiments, the kits disclosed herein can have medical containers that hold the compositions in a safe, stable, and durable manner. In some examples, the kits disclosed herein can also include a means for administering the composition, such as a needle or spatula.

[0098] Any of the kits may further include instructions providing guidance for using the kit for treatment. The instructions may be written for a physician or hepatologist as the intended audience.

[0099] Although several embodiments have been described, it will be recognized by those skilled in the art that various modifications, alternative configurations and equivalents can be used without departing from the spirit of the inventive concept. Moreover, some well-known processes and elements have not been described in order to avoid unnecessarily obscuring the inventive concept. Thus, this description should not be interpreted as limiting the scope of the inventive concept.

[0100] Those skilled in the art will understand that the embodiments disclosed herein are taught by way of example and not by way of limitation. Therefore, the matter contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not in a limiting sense. The following claims are intended to cover all the general and specific features described herein, as well as all descriptions of the scope of methods and assemblies that may be linguistically said to fall therebetween. EXAMPLES

[0101] The following examples are included to illustrate preferred embodiments of the present disclosure. Those skilled in the art should understand that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the implementation of the present disclosure, and therefore can be considered to constitute preferred modes for its implementation. However, those skilled in the art should recognize in light of the present disclosure that many changes can be made in the specific embodiments disclosed and still obtain the same or similar results without departing from the spirit and scope of the present disclosure.

[0102] [Example 1] To broadly understand how mutations in NASH candidate genes affect clonal competition in an unbiased manner in vivo, we developed a method to generate pools of mutant cells in tissues of Cas9-expressing mice. To generate a virus that can integrate into the genome, thereby enabling in vivo screening, we created a hybrid adeno-associated virus (AAV) carrying Sleeping Beauty transposase (SB100), Cre, and single guide (sg) RNA, flanked on either side of its payload by transposon integration sequences (SB-IR). This AAV was used to deliver sgRNA into the liver of dox-inducible Cas9-expressing mice for in vivo CRISPR screening. Briefly, we performed a pooled screen of 63 NASH genes identified by GWAS (i.e., APOB, TM6SF2, GCKR), exome sequencing (i.e., PNPLA3, MBOAT7, HSD17B13), somatic mutation sequencing efforts (i.e., PPARGC1B, FOXO1, GPAM, ACVR2A) or biochemical studies. Induction of Cas9 after AAV injection established a pool of hepatocytes carrying mosaic deletions of these NASH genes. After allowing Cas9 to work for 2 weeks, dox was discontinued and therefore no further truncations were expected to occur. These mosaic mice were then fed normal chow (NC) or a Western diet (WD: high sugar, fat, cholesterol) to understand clonal dynamics in both conditions. After 6 months, deep sequencing of sgRNAs pinpointed the genes with the greatest effect on clonal fitness, especially in the NASH setting. To exclude purely proliferative effects that may occur independent of NASH, we removed sgRNAs that were enriched or depleted in common between NC and WD conditions. Upon deletion, six genes were most associated with clonal expansion: Acvr2a, Irs1, Srebp1, Gpam, Dgat2, and Pparg (Figures 1A-1D).

[0103] Glycerol-3-phosphate acyltransferases (GPATs), including GPAT1 and GPAM (glycerol-3-phosphate acyltransferase, mitochondrial), catalyze the first committed step of glycerolipid biosynthesis and were predicted to play a central role in regulating the levels of cellular triacylglycerol and phospholipids. It is notable that GPAM mutant clones were more competitive and exerted beneficial effects in NAFLD / NASH. For example, recurrent and convergent loss-of-function mutations in genes that promote lipogenesis were identified in NASH livers (Ng et.al., Nature 2021). The abnormal accumulation of lipid droplets in hepatocytes may ultimately lead to lipotoxicity, cell death and cirrhosis. The observation of loss-of-function mutations in metabolic enzymes that generate lipids in the liver (e.g., GPAM) suggests that some somatic mutations can increase fitness by reversing the driving pathogenesis of the disease.

[0104] The top hits from the in vivo NASH mouse screen were analyzed in a mosaic liver disease model. To study GPAM mutations in a mosaic setting with liver-specific deletion or clonal mosaicism, we used a CRISPR approach to generate both whole-body GPAM knockout and GPAM floxed mice targeting exon 3 predicted to lead to a frameshift and stop codon causing premature termination. GPAM floxed mice were confirmed to achieve GPAM deletion after Cre recombination. To study how mosaic GPAM wild-type and knockout clones compete in the liver under normal and NAFLD conditions, we fed low doses of AAV-Cre to induce the deletion in 10–20% of hepatocytes, and then placed the mosaic mice on normal chow or WD conditions. This extended the survival of GPAM knockout clones after 6 months and reduced the severity of NASH (Figure 2A–2B). This data mirrored human genetic data showing that GPAM mutant clones are selected in human livers with NASH.

[0105] To further ascertain which genes are increasing clonal expansion via their effects on metabolic fitness, liver-specific GPAM knockout mice were used to assess the extent to which ideal siRNA suppression of this gene would affect hepatic and whole-body metabolism in the context of NASH. For each conditional CRISPR KO model, body weight, liver weight, histology, steatosis, fibrosis, and serum tests (liver function tests, cholesterol, TG, nonesterified free fatty acids (NEFA)) were measured. After 3 months of NASH diet, liver-specific GPAM knockout mice were protected from hepatic triglyceride accumulation.

[0106] [Example 2] To address whether mouse siRNA against GPAM can prevent and / or reverse NASH in mouse models, siRNA tool compounds used in mice are used to study the translational aspects of GPAM biology. Eight siRNAs against mouse GPAM were tested in Cos7 cells to identify the siRNA sequence with the highest knockdown efficiency based on mouse GPAM reporter assay. This siRNA was then modified and conjugated with GalNAc. Briefly, all 2' positions were modified with 2'-O-methyl to minimize nucleic acid degradation and immune response in vivo. To determine whether these stabilizing modifications affect silencing activity, siRNA activity in vitro was determined prior to GalNAc conjugation. Consistent with industry standard approaches, GalNAc conjugation can enable highly efficient delivery to hepatocytes without lipid nanoparticle packaging. The best in vivo administration regimen was determined for GalNAc-siGPAM. Three doses of siRNA (1, 5, 10, 15 mg / kg) in PBS were given subcutaneously (SC) to WT B6 mice once every 2 weeks starting at 4 weeks of age. After siRNA treatment, mice were sacrificed (n=5 mice / group) for GPAM qPCR, Western blotting and histology. The maximum tolerated dose was determined based on the absence of severe toxicity, e.g., weight loss >10%, organ (heart / kidney / liver) failure and death. In all of the following siRNA experiments, GalNAc-siLuc serves as a control.

[0107] To determine whether GalNAc-siGPAM prevents NASH, the optimal dose defined above was determined. Then, it was determined whether GalNAc-siRNA could mimic the GPAM KO model. In parallel with the genetic experiment, GalNAc-siRNA SC injections were started at 6 weeks of age, and the NASH diet was started at 8 weeks and continued for 12 weeks (total of 6 siRNA administrations). Mice were euthanized at 20 weeks of age, at which point steatosis, inflammation, and fibrosis were evaluated. Without being bound by theory, the siRNA approach mimics the genetic NASH prevention model.

[0108] To determine whether GalNAc-siGPAM can reverse NASH, siRNA dosing was initiated in mice (30 weeks old) already on a 24-week WD diet, with WD and siRNA continued for a total of 12 weeks. Livers were then evaluated for pathological features of NASH. Assessment included hepatocyte ballooning, inflammation and fibrosis, features that characterize NASH as described above. These siRNA experiments provide enormous translational value and pave the way for clinical treatment.

[0109] [Example 3] Specific siRNA sequence is designed to target human GPAM for the treatment of NASH.Based on the human somatic cell sequencing data that identifies recurrent loss-of-function GPAM mutations in NASH liver, the screening of genetic screen results by the exemplary method herein, and the biology of GPAM in mouse models of NASH and insulin resistance, GPAM represents a promising therapeutic target for human NASH.

[0110] Testing identified optimized siRNA sequences corresponding to the human sequence of human GPAM. This involved screening many candidate siRNAs for each gene target using an in vitro luciferase reporter-based assay. Briefly, full-length GPAM cDNA (without the 3'UTR) was cloned into the psicheck2 plasmid backbone containing the luciferase gene. This created a Renilla luciferase-GPAM fusion gene. The luciferase assay is a dual reporter system with firefly luciferase as a control and Renilla luciferase as a readout for transcription and translation. Thus, the ratio of Renilla to firefly signal accounts for variations in transfection efficiency and cell viability. The assay was performed in a 96-well plate format with technical replicates using a 48-hour time point for the assay readout. The reporter was co-transfected with siRNA into Cos7 monkey kidney cells using lipofectamine. After 48 hours, cells were lysed and signals were captured by a luminometer using substrates for firefly and Renilla. Effective siRNAs against target genes showed a reduction in the ratio of Renilla to firefly signals compared to non-targeting controls. A total of 39 siRNAs targeting the entire GPAM ORF were designed and tested (Figures 3A-3B). At least 21 effective siRNAs against human GPAM were identified (Table 3). Optimized siRNAs are modified by conjugation to acetylgalactosamine (GalNAC) using standard methods and tested in vivo.

[0111] JPEG2025513018000015.jpg220170

[0112] [Example 4] Some of the human siRNAs against GPAM disclosed herein are tested in human cell line models of NASH. Testing was performed using optimized siRNA and / or its conjugate with acetylgalactosamine (GalNAc) to improve liver targeting. The experimental design has three steps. First, the identified siRNA is used in human liver cancer cells, e.g., Huh7 or HepG2, to knock down the target gene of interest, i.e., GPAM. In particular, Huh7 cells are grown to 60-80% confluence and then transfected with siRNA. Opti-MEM (Thermo Fisher 31985062) containing siRNA and lipofectamine are mixed and incubated, then added to cells for transfection (400 μl / well for 6-well plates, or 2.4 ml for 100 mm plates). The transfection medium is replaced after 6 hours with medium containing 100 μM FA. The cells are then fed with lipids and labeled with lipid dyes. Finally, fluorescence-activated cell sorting (FACS) is performed to quantify lipid accumulation. FACS to purify specific cell populations based on phenotypes detected by flow cytometry. This method allows characterization of a single cell population without the influence of other cells. Cells are stained with 1 ml PBS containing 10 μg BODIPY 493 / 503 (BD) (Thermo Fisher D3922) for 15 min at room temperature and then subjected to FACS measurement. Early studies have shown that in cells stained with lipophilic fluorophores, the fluorescence intensity measured by FC reflects lipid levels (Wolins, 2018).

[0113] Aliquots of cells grown under each condition are assayed for triacylglycerol (TG). Briefly, cells are trypsinized, washed once with PBS, resuspended in PBS containing 10 mM EDTA, and then counted. Cells are split into three portions in a final volume of 200 μl in 13×100 mm glass tubes. Triolein standards (Sigma T7140) are also prepared in a final volume of 200 μl PBS / 10 mM EDTA in 13×100 mm glass tubes. TG is extracted and quantified. Briefly, 2 ml of isopropanol:hexane:water (40:10:1) is added to cells or standards, and samples are vortexed, covered, and incubated at room temperature for 30 minutes. 500 μl of a 1:1 mixture of hexane:diethyl ether is then added to the samples, followed by vortexing and incubation at room temperature for an additional 10 minutes. Next, 1 ml of water is added to the samples, the tubes are vortexed, and the layers are allowed to separate at room temperature while covered for 30-45 min. Using a Pasteur pipette, the top layer is transferred to a 12 x 75 mm glass tube and completely dried under N2. After the drying step, 400 μl of Infinity Triglyceride Reagent (Thermo Scientific TR22421) is added to each tube and vortexed. The tubes are covered and incubated at 37 °C for 90 min while shaking at 250 rpm. Finally, 300 μl of each sample is transferred to a 96-well plate and the absorbance is measured at 540 nm using a microplate reader. To determine whether fixation of the cells affected the TG measurements, TG is quantified in fixed and non-fixed cells from the same batch of Huh7 cells incubated in 2 mM FA.

[0114] [Example 5] Mutations in Gpam were further investigated since they emerged as top hits from the functional NASH screen and human somatic mutation sequencing efforts described above. GPAM catalyzes the acylation of glycerol-3-phosphate with acyl-coenzyme A (CoA) to produce CoA and lysophosphatidic acid (LPA), the rate-limiting step in triacylglycerol synthesis. As conditional KO mice required to study clonal dynamics were not available, GPAM floxed mice were generated as provided in Example 2 targeting exon 3, whose deletion is predicted to lead to a frameshift and premature stop codon. A saturating dose of AAV8-TBG-Cre led to the deletion of exon 3 and almost complete depletion of GPAM mRNA one week after injection (Figures 4A and 4B). These floxed mice were used to compare WT and KO clones under normal and WD conditions. An increased abundance of GPAM KO clones was observed in the setting of a Western diet (WD) (Figures 4C and 4D). These data demonstrate a diet- and steatosis-dependent beneficial effect of GPAM loss-of-function mutations.

[0115] To determine why the GPAM mutant clone was more fit, we induced whole livers of GPAM KO mice with AAV8-TBG-Cre. Although slight differences in body mass and liver weight were observed after 3 months of WD (Figures 4E and 4F, Table 4), the KO mice showed a trend toward reduced liver / body weight ratio (Figure 4G), reduced steatosis (Figure 4H), and improved transaminemia (Figures 4I and 4J).

[0116] JPEG2025513018000016.jpg243170JPEG2025513018000017.jpg255155JPEG2025513018000018.jpg255156 Steatosis and liver injury were quantified using the NAFLD activity scoring system adapted to rodent histology. It shows that KO livers have reduced macrodroplet steatosis, microdroplet steatosis and hepatocyte hypertrophy (Figure 4K). Consistent with the role of GPAM as a rate-limiting enzyme in triglyceride synthesis, hepatic triglyceride levels, but not cholesterol levels, were reduced in the WD-fed KO group (Figures 4L and 4M). Interestingly, plasma cholesterol, but not triglycerides, was reduced in the KO group (Figures 4N and 4O. Overall, the data indicate that in NASH, GPAM loss-of-function mutations lead to clonal expansion and that GPAM is a promising therapeutic target in NASH.

[0117] Experimental methods and subjects used in the examples Mouse strains and breeding All mice were handled in accordance with the guidelines of the Institutional Animal Care and Use Committee at UT Southwestern. All experiments were performed in an age- and sex-controlled manner unless otherwise noted. All mice used in this study were male, except those in Figure S8F-J. Mice of the C57BL / 6 strain background were used for all experiments. Mboat7 mice obtained from KOMP were tm1a(KOMP)Wtsi Gpam / H mice were originally generated by the Wellcome Trust Sanger Institute. f / fMice were generated at the CRI Mouse Genome Engineering Core at UT Southwestern. Briefly, CAS9 protein, synthetic sgRNA and single-stranded DNA containing Gpam exon 3 flanked by LoxP sites and homology arms were co-injected into C57BL / 6 mouse zygotes, which were then implanted into CD-1 mice. Genotyping and Sanger sequencing were used to confirm homologous recombination in genome-edited pups. LSL-tdTomato (strain #007914) and Rosa-rtTA;TetO-Cas9 mice (#029415) were obtained from The Jackson Laboratory. Mice homozygous for both Rosa-rtTA and TetO-Cas9 were used to ensure high Cas9 expression levels in the liver. The Western diet (WD) used for NAFLD / NASH modeling has been described in 45. This consisted of high fat solid food (ENVIGO #TD.120528) and high sugar water containing 23.1 g / L d-fructose (Sigma-Aldrich #F0127) and 18.9 g / L d-glucose (Sigma-Aldrich #G8270).

[0118] Lineage tracing in floxed mice For whole liver deletion of Mboat7 or Gpam, 5*10 10 Genomic copies of commercially available AAV8-TBG-Cre (Addgene #107787) or control AAV8-TBG-GFP (Addgene #105535) were transfected into 8-week-old Mboat7 mice. f / f or Gpam f / f For mosaic deletion of Mboat7 or Gpam and Tomato labeling of hepatocytes, 0.125*10 10 Transfect 8-week-old Mboat7 mice with 10 copies of the AAV8-TBG-Cre genome f / f ;LSL-tdTomato het, Gpam f / fAAV8-TBG-Cre was injected retro-orbitally into mice with 100% glycerol (LSL-tdTomato) or 100% control LSL-tdTomato het mice. One week after injection of low doses of AAV8-TBG-Cre, seven mice from each group were collected to determine the initial Tomato labeling percentage in the liver. The remaining mice were split into chow or WD groups and followed for an additional 4 or 6 months.

[0119] Fluorescence imaging and image processing For fluorescence imaging, liver fragments were fixed in buffered formalin (Fisherbrand #245-685) with gentle shaking for 24 hours at 4°C, then transferred to a 30% sucrose (w / v) solution with further shaking for 24 hours at 4°C. Livers were then embedded, frozen in Cryo-Gel (Leica #39475237), and sectioned at 16 μm thickness. Images were acquired using a Zeiss Axionscan Z1 system at the UTSW Whole Brain Microscopy Facility to visualize Tomato clones. For statistical analysis of the percentage of Tomato+ cells, black and white fluorescence images were obtained from the same slides using an Olympus IX83 microscope at 4× magnification. Two different fields of view were acquired from each liver. The percentage of Tomato+ cells (bright areas) was analyzed using ImageJ.

[0120] H&E, immunohistochemistry (IHC), immunofluorescence (IF), TUNEL and Sirius Red staining Liver fragments were fixed in buffered formalin (Fisherbrand #245-685) for 24 hours at 4°C with gentle shaking, then transferred to 70% EtOH and further shaken at 4°C for 24 hours. Paraffin embedding, liver sectioning (4 μm thick) and H&E staining were performed at the UT Southwestern Tissue Management Shared Resource Core. IHC was performed as previously described. Briefly, paraffin-embedded sections were dewaxed in xylene and hydrated using an ethanol gradient. Slides were then boiled for 20 minutes in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) and immersed in 3% hydrogen peroxide (in methanol) for 10 minutes. After blocking with 5% goat serum for 1 hour at room temperature, slides were incubated with primary antibodies overnight at 4°C. After washing, slides were incubated with secondary antibodies for 0.5 hours at room temperature. The secondary antibody was conjugated with HRP using the ABC-HRP Kit (Vector Laboratories, #PK-6101). The slides were developed using the DAB Kit (Vector Laboratories, #SK-4100). For IF staining, the following primary antibodies were used: RFP (Rockland #600-401-379, IF 1:500); Ki67 (Invitrogen #14-5698-82, IF 1:500); HNF4a (Abcam #ab41898, IF 1:500); and the following secondary antibodies were used: goat anti-rat IgG (H&L) Alexa Fluor Plus 488 (Invitrogen #A-48262, IF 1:500); donkey anti-rabbit IgG (H&L) Alexa Fluor 594 (Invitrogen #A-21207, IF 1:500); goat anti-mouse IgG2a Alexa Fluor 647 (Invitrogen #A-21241, IF 1:500). IF was performed on paraffin-embedded mouse liver sections using the same protocol as for IHC, except that the secondary antibody was replaced with an Alexa Fluor-conjugated antibody.TUNEL staining was performed on paraffin-embedded liver sections using the In Situ Cell Death Detection Kit, Fluorescein (Roche #C755B40) according to the manufacturer's protocol. Sirius Red staining was performed on paraffin-embedded liver sections using the Picro Sirius Red Staining Kit (Abcam #ab150681) according to the manufacturer's protocol. TUNEL staining and IHC staining of Ki67 were quantified using QuPath software. Sirius Red staining was quantified using ImageJ.

[0121] Plasma and liver metabolic assays Blood was collected from the inferior vena cava immediately after mouse sacrifice using heparinized tubes, then transferred into 1.5 ml tubes and centrifuged at 2000 g for 15 min at 4°C. The supernatant after centrifugation (plasma) was analyzed for AST, ALT, cholesterol and triglycerides (manufacturer's reference numbers 8433815, 1655281, 1669829 and 1336544, respectively) using a fully automated OCD Vitros 350 dry chemistry analyzer according to the protocol provided by the reagent kit manufacturer (Ortho Clinical Diagnostics, Raritan, NJ) at the UT Southwestern Metabolic Phenotyping Core. At the UT Southwestern Metabolic Phenotyping Core, 100-150 mg of liver per mouse was weighed and used for lipid extraction and quantification. Briefly, flash-frozen tissue samples were homogenized in a 2:1 chloroform:methanol mixture (v / v) using a multiplexed automatic tissue disruptor (TissueLYser II, Qiagen, Germantown, MD). The organic extract was transferred to a 5 ml volumetric flask, bringing the total volume to 5 ml. Total cholesterol and triacylglycerol analyses were performed in triplicate using 100 μl and 25 μl of lipid extract, respectively. Total cholesterol and triacylglycerol concentrations were determined by commercially available enzymatic colorimetric assays following the protocols described by the manufacturers (INFINITY™ Cholesterol Liquid Stable Reagent #TR13421; INFINITY™ Triglycerides Liquid Stable Reagent #TR22421; Matrix plus™ Chemistry Reference Kit #NC9592194).

[0122] Construction of MOSAICS reagents The MOSAICS plasmid uses the pX602 plasmid as the backbone. The sequence between the two AAV ITRs was removed using NsiI and NotI restriction enzymes. The following fragments were cloned between the two AAV ITRs: the first SB100-bound IR, the U6-driven sgRNA scaffold, the CAG promoter-driven SB100-P2A-Cre fusion cDNA with beta globin poly(A) signal, and the second SB100-bound IR. For library construction, mouse candidate genes for all in vivo screening were generated by using mouse homologs of human genes. A few genes were not included in the mouse gene list because of lack of homologs or because they were known tumor suppressor genes. Individual sgRNA sequences corresponding to mouse candidate genes were extracted from Brie library or obtained from GUIDES server and synthesized by CustomArray. Most genes had five different sgRNAs, four from Brie and one from GUIDES. A few genes had four targeting sgRNAs because the Brie and GUIDES sgRNA sequences overlapped. See Table 5 for sgRNA sequences for GPAM. Library construction protocols are also established in the art. Briefly, the synthesized oligonucleotide library was amplified by PCR, purified using PCR Purification Kit (Qiagen, #28104), and assembled into BsaI-digested MOSAICS vector using DNA Assembly Kit (NEB, #E5520A). 1 μl of assembled vector was then electroporated into 25 μl of competent cells (Lucigen, #60052-2). After recovery in SOC medium for 1 hour, the bacteria were spread on 245*245mm LB agar plates, which were incubated at 37°C overnight. The bacteria were then harvested and plasmids were prepared using HiSpeed ​​Plasmid Maxi Kit (Qiagen, #12663). Each sgRNA maintained >1000-fold representation during construction.For individual sgRNA cloning, forward and reverse primers were annealed and fused to BsaI-digested MOSAICS plasmid using T4 ligase. See Table 5 for primers related to sgRNAs.

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[0124] AAV generation and purification AAV8 was produced using AAV-Pro 293T cells (Takara #632273) cultured in one or more 15 cm dishes. Cells were plated at 50% confluence one day prior to transfection, which would allow the cells to reach 80-90% confluence the next day. For transfection of 15 cm dishes, 10 μg of MOSAICS vector, 10 μg of pAAV2 / 8 (Addgene #112864) and 20 μg of pAdΔF6 (Addgene #112867) plasmids were mixed with 1 ml of Opti-MEM medium in one tube. In another tube, 160 μl of PEI solution (1 mg / ml aqueous solution, pH 7.0, powder from ChemCruz #sc-360988) was mixed with 1 ml of Opti-MEM medium. The solutions in both tubes were then mixed and incubated for 10 min before adding the cell culture. 48 h after transfection, the cells were detached from the dishes and collected by centrifugation at 500g for 10 min. The supernatant was sterilized and discarded, and the cell pellet was dissolved in 1.5 ml / 15 cm dish of lysis buffer (PBS supplemented with NaCl powder to a final concentration of 200 mM and CHAPS powder to a final concentration of 0.5% (w / v)). The cell suspension was placed on ice for 10 min with intermittent vortexing, and then centrifuged at 20,000g for 10 min at 4°C. The supernatant containing AAV was collected. To set up a gravity column for AAV purification, 0.5 ml of AAV8-bound slurry beads (ThermoFisher #A30789), sufficient to purify AAV from three 15 cm dishes, was loaded into an empty column (Bio-Rad #731-1550). After the beads were tightly packed at the bottom, they were washed with 5 ml of wash buffer (PBS supplemented with NaCl powder to a final concentration of 500 mM). The supernatant containing AAV was then loaded onto the column. After all the supernatant had flowed through, the beads were washed twice with 10 ml of wash buffer. AAV was then eluted with 3 ml of elution buffer (aqueous solution of 100 mM glycine, 500 mM NaCl, pH 2.5) and the eluate was immediately neutralized with 0.12 ml of 1 M Tris-HCl (pH 7.5-8.0).AAV was concentrated using a 100k Amicon Ultra Centrifugal Filter Unit (Millipore #UFC810024) by centrifugation at 2000g for 3-5 min at 4°C. After centrifugation, the volume of AAV should be 0.5ml or less. The concentrated AAV was diluted with 4-5ml of AAV dialysis buffer (PBS supplemented with powder to a final concentration of 212mM NaCl and 5% sorbitol (w / v)) and centrifuged at 2000g for 3-5 min at 4°C. The dilution and centrifugation process was repeated three times. The final concentrated AAV was transferred into a 1.5ml tube and centrifuged at 20,000g for 5 min to remove debris. The supernatant was aliquoted, flash frozen using liquid nitrogen, and stored at -80°C.

[0125] Genomic DNA extraction, sgRNA amplification and amplicon library construction To extract genomic DNA containing the integrated sgRNA, the entire liver (excluding the small fragment used for sectioning and H&E staining) was cut into approximately 1 mm pieces using a blade. 3The livers were chopped into 100 ug pieces and weighed. Small nodules observed in some WD-fed epigenetic factor screen livers (<=3 nodules per liver in 5 of 8 livers) were excluded from samples processed for genomic DNA extraction. The chopped livers in 2 volumes (w / v) of homogenization buffer (100 mM NaCl, 25 mM EDTA, 0.5% SDS, 10 mM Tris-HCl, pH 8) were transferred into a glass Wheaton Dounce Tissue Grinder and stroked 50 times or until no bulk tissue was visible. After homogenization, 200 μl of chow-fed liver lysate or 300 pl of WD-fed liver lysate were transferred to a 15 ml tube for genomic DNA extraction using the Blood & Cell Culture DNA Midi Kit (Qiagen #13343) according to the manufacturer's protocol. The remaining lysate was frozen at -80°C as a backup sample. Briefly, 10 ml of Buffer G2 from the kit, 100 pl of Proteinase K (Roche #03115828001, or Proteinase K from the Qiagen kit) and 100 pl of RNase A (Invitrogen #12091-021) were added to the 15 ml tube containing the lysate and digested overnight in a 50°C water bath. The next day, the tube was centrifuged at 4000g for 10 min and the top lipid layer was discarded. The remaining supernatant was loaded onto the column, washed, and genomic DNA elution / precipitation was performed according to the manufacturer's protocol. The precipitated DNA was resuspended in 100 pl of 10 mM Tris (pH 8.0) and shaken on a shaker at 55°C for 2 hours to help dissolve the precipitated DNA. For amplicon library preparation, 5 pg genomic DNA, 5 pl of general forward primer mix (5 pM), 5 pl of barcode-specific reverse primer (5 pM), 1 pl of Q5 DNA polymerase, 10 pl of Q5 buffer, 10 pl of HighGC buffer, 1 pl of dNTPs and water were mixed for a 50 pl PCR reaction, and two reactions were performed for each genomic DNA sample. The PCR cycle was 95°C 3 min-(95°C 30 sec-60°C 30 sec-72°C 20 sec)*n-72°C 2 min. The number of PCR cycles was previously optimized using a smaller volume of the same PCR reaction.Cycle numbers that resulted in weak but clear bands on DNA gels were used. For the final PCR reactions, 23 cycles were used for preparation of the NASH genes, transcription factors and epigenetic factors screen, and 30 cycles were used for preparation of the guide minipool validation screen. After PCR, two tubes of reactions with the same genomic DNA template were combined (100pl total) and 70pl were separated on a DNA gel. The 250bp bands corresponding to the amplicons were excised and purified using a QIAquick Gel Extraction Kit (Qiagen #28704). DNA concentrations were determined using a Qubit kit (Invitrogen #Q32853) and high-throughput sequencing was performed using an Illumina NextSeq500 system at the CRI at UT Southwestern Sequencing Facility.

[0126] Bioinformatics analysis of MOSAICS screening results Reads derived from sequencing the above amplicon libraries were trimmed with cutadapt (version 1.9.1) to remove excess adapter sequences so that only sgRNA sequences were retained. 5' sequences were trimmed using option -O32--discard-untrimmed-g CTTTATATATCTTGTGGAAAGGACGAAACACCG. 3' sequences were trimmed using option -O12-a GTTTTAGAGCTAGAAATAGCA. Abundance of each sgRNA was calculated using the count function in MAGeCK (version 0.5.6) with default options. Trimmed fastq files were assigned to chow-fed and WD-fed groups and uploaded to a MAGeCK preloaded server along with library files containing sgRNA sequences and target gene names. Enrichment of each sgRNA was calculated using the test function in MAGeCK.

[0127] Transcriptome analysis of human NASH liver Dataset 1 (NAFLD / NASH cohort): RNA-Seq transcriptome profiles of biopsied liver tissue (GEO: GSE130970) were downloaded and analyzed from 72 NAFLD / NASH patients with various disease severity (NASH activity scores of 1-6) and associated histological features, i.e., steatosis, inflammation, fibrosis and hepatocyte ballooning. Raw sequence reads were aligned against the GENCODE human reference genome (GRCh37, p13) using STAR aligner (version 2.6.1b) and gene-level count data were generated by the feature count function of the Subread package (version 1.6.1) and GENCODE genome annotation (GRCh37, v19). Count data were normalized using the "Relative Log Expression" normalization (RLE) implemented in the DESeq2 package Dataset 2 (HCV cirrhosis cohort). We analyzed microarray gene expression profiles of formalin-fixed needle biopsy specimens from the liver of 216 patients with hepatitis C-associated early (Child-Pugh class A) cirrhosis (GEO: GSE156540). This cohort was prospectively followed for a median of 10 years in an Italian center, and relevant time-event outcomes were collected, including childbearing, death, HCC, and decay. Male and female patients were included in these studies.

[0128] Quantification and statistical analysis Data in most panels reflect multiple experiments performed on different days using mice from different litters. Variation in all panels is shown using standard deviations shown as mean ± SD. Two-tailed unpaired Student's t-test was used to test for significance between two groups. Statistical significance is indicated as ns (not significant or p>=0.05), *(p<0.05), **(p<0.01), ***(p<0.001), ****(p<0.0001) unless otherwise stated. Image analysis for quantification was blinded.

Claims

1. A composition comprising a nucleic acid molecule that downregulates the expression of GPAM or a variant thereof.

2. 2. The composition of claim 1, wherein the nucleic acid is an siRNA, a clustered regularly interspaced short palindromic repeats (CRISPR)-associated nucleic acid, a single guide RNA (sgRNA), a CRISPR-RNA (crRNA), a trans-activating crRNA (tracrRNA), a plasmid DNA (pDNA), or a viral vector.

3. The composition of claim 2 , wherein the nucleic acid is a small interfering RNA (siRNA) molecule or encodes a siRNA molecule.

4. A composition comprising a plasmid or a viral vector, the plasmid or the viral vector comprising a nucleic acid encoding the siRNA molecule of claim 3.

5. 5. The composition of any one of claims 3 or 4, wherein the siRNA molecule is 2-30 nucleotides in length and comprises a nucleotide sequence that is at least 80% homologous to at least 2-30 contiguous nucleotides of a human GPAM cDNA sequence.

6. The composition of claim 5 , wherein the human GPAM cDNA sequence is SEQ ID NO:

1.

7. The composition of claim 3 or 4, wherein the siRNA molecule targets the open reading frame or the 5' or 3' UTR of the GPAM gene.

8. The composition according to any one of claims 3 to 7, wherein the siRNA molecule comprises at least one sense sequence, at least one antisense sequence, or at least one sense sequence and at least one antisense sequence.

9. The composition of any one of claims 3 to 8, wherein the siRNA molecule comprises a nucleotide sequence of SEQ ID NO: 2 to 165, or any combination thereof.

10. The composition of claim 9, wherein the at least one sense sequence comprises SEQ ID NO: 2-83.

11. The composition of claim 9, wherein the at least one antisense sequence comprises SEQ ID NO: 84-165.

12. The composition of claim 2, wherein the nucleic acid is or encodes a sgRNA.

13. 13. A composition comprising a plasmid or viral vector, said plasmid or viral vector comprising a first nucleic acid encoding an sgRNA molecule of claim 12 and optionally a second nucleic acid encoding an RNA-guided nuclease.

14. 14. The composition of any one of claims 12 or 13, wherein the target sequence of the sgRNA is at least about 80% identical to any one of SEQ ID NOs: 177-276.

15. The composition of claim 13, wherein the RNA-guided nuclease is a Cas endonuclease.

16. The composition of any one of claims 3 to 11, wherein the siRNA molecule specifically downregulates gene expression of at least one mutant of GPAM.

17. The composition of any one of claims 12 to 14, wherein the sgRNA molecule specifically downregulates gene expression of at least one mutant of GPAM.

18. 18. The composition of any one of claims 16 or 17, wherein at least one variant of GPAM is associated with liver disease.

19. 19. The composition of claim 18, wherein the liver disease comprises fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer, or any combination thereof.

20. 20. The composition of any one of claims 16-19, wherein the at least one variant of GPAM comprises rsl0787429 C>T, rs7096937 T>C, rsl 1446981 T>TA, rs7898213 T>C, rs2792759 C>T, rs2297991 T>C, rsl 129555 A>G, rs2254537 T>A, or any combination thereof.

21. The composition of any one of claims 1 to 3, wherein the nucleic acid molecule is conjugated to at least one targeting ligand.

22. 22. The composition of claim 21, wherein the at least one targeting ligand comprises a liver targeting ligand.

23. The composition of claim 22, wherein the liver-targeting ligand comprises at least one N-acetylgalactosamine (GalNAc) conjugate.

24. The composition of claim 23, wherein the nucleic acid molecule is conjugated to about 1 to about 3 GalNAc conjugates.

25. The composition of any one of claims 1 to 3 or 12, wherein the nucleic acid molecule comprises at least one chemical modification.

26. 26. The composition of claim 25, wherein the nucleic acid molecule comprises a modification in at least one ribosugar moiety of its nucleotide sequence.

27. 27. The composition of claim 26, wherein at least one liposaccharide moiety is modified with 2 2'-O-methyl (2'OMe), 2'-deoxy-2'-fluoro (2'F), 2'-deoxy, 5-C-methyl, 2'-O-(2-methoxyethyl) (MOE), 4'-thio, 2'-amino, 2'-C-allyl, or any combination thereof.

28. 28. The composition of any one of claims 26 or 27, wherein less than about 10% to about 70% of the ribosugar moieties of all nucleotide sequences are modified.

29. A pharmaceutical composition comprising any one of the compositions according to claims 1 to 28 and at least one pharma- ceutically acceptable carrier.

30. 30. The pharmaceutical composition of claim 29, further comprising nanoparticles.

31. 31. The pharmaceutical composition of claim 29 or claim 30, further comprising a lipid.

32. A method for treating a subject in need thereof comprising administering a therapeutically effective amount of a composition according to any one of claims 1 to 28 or a pharmaceutical composition according to any one of claims 29 to 31.

33. 33. The method of claim 32, wherein the subject in need thereof is a human subject having or suspected of having liver disease.

34. 34. The method of claim 33, wherein the liver disease comprises fatty liver disease (FLD), alcoholic liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) from any etiology, liver cancer, or any combination thereof.

35. The method of any one of claims 32 to 34, wherein the method of administration comprises parenteral administration.

36. The method of any one of claims 31 to 34, wherein administration of a therapeutically effective amount of the composition of any one of claims 1 to 27 or the pharmaceutical composition of any one of claims 28 to 30 increases the life expectancy of said subject compared to an untreated subject with the same disease state and predicted outcome.

37. The method of any one of claims 32 to 36, wherein administration of a therapeutically effective amount of the composition of any one of claims 1 to 28 or the pharmaceutical composition of any one of claims 29 to 31 increases liver function in said subject compared to an untreated subject with the same disease state and predicted outcome.

38. The method according to any one of claims 32 to 37, wherein administration of a therapeutically effective amount of the composition according to any one of claims 1 to 28 or the pharmaceutical composition according to any one of claims 29 to 31 attenuates liver fibrosis in said subject compared to an untreated subject with the same disease state and predicted outcome.

39. The method according to any one of claims 32 to 38, wherein administration of a therapeutically effective amount of the composition according to any one of claims 1 to 28 or the pharmaceutical composition according to any one of claims 29 to 31 prevents further liver fibrosis in said subject compared to untreated subjects with the same disease state and predicted outcome.

40. (i) a container holding a composition according to any one of claims 1 to 28 or a pharmaceutical composition according to any one of claims 29 to 31; (ii) a pharmaceutical administration means; and (iii) Instructions Kit including:

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