Compositions and methods for treating liver disease with GPAM-targeting siRNA

Abstract

Disclosed herein are compositions comprising a siRNA capable of down-regulating expression of a mitochondrial glycerol-3-phosphate acyltransferase (GPAM) gene or a variant thereof. Also disclosed herein are methods of treating liver diseases, such as fatty liver (FLD), non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH), or liver injury using such compositions.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 328,593, filed on April 7, 2022, and U.S. Provisional Application No. 63 / 491,262, filed on March 20, 2023, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing, which is submitted in xml format via EFS-Web, the entire text of which is incorporated herein by reference. The xml copy was created on April 7, 2023, named 106546-755472.xml, and is 329KB in size. Background Art

[0005] 1. Field

[0006] The present inventive concept relates to compositions and methods of use 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).

[0007] 2. Related technical discussions

[0008] NAFLD is a spectrum of chronic liver disorders that includes nonalcoholic fatty liver disease (NAFL) and nonalcoholic steatohepatitis (NASH). NASH is a common cause of chronic liver disease and is defined by steatosis and inflammation of at least 5% of the liver, with or without fibrosis. Over time, if left untreated, NASH may progress to cirrhosis and even hepatocellular carcinoma (HCC). In addition, NAFLD / NASH is closely associated with obesity and type II diabetes, which together affect more than 50% of the US population, resulting in a heavy economic burden. Unfortunately, treatment options for NASH remain limited, with only minor benefits from treatment with vitamin E or obeticholic acid. Although NASH is a major cause of chronic liver disease and cirrhosis, there are currently no clinically approved therapies. Therefore, new targets, therapies, and combinations thereof are needed to accelerate clinical progress in liver disease treatments. Summary of the invention

[0009] The present disclosure is based in part on a new discovery that positive selection of somatic mutations in the liver of NASH patients can improve cell fitness and competitiveness by reducing lipid accumulation in NASH. The genes screened for somatic mutations can improve clonal fitness by reversing lipotoxicity, and GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) was identified. Therefore, the present disclosure provides novel compositions for regulating GPAM expression and methods for preventing, alleviating and / or treating liver disease.

[0010] In some aspects, the present disclosure relates to a composition comprising a nucleic acid molecule that downregulates the expression of GPAM or its variants. In some aspects, the nucleic acid is siRNA, clustered regularly spaced short palindromic repeats (CRISPR)-related nucleic acids, single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), plasmid DNA (pDNA) or viral vector. In some aspects, the nucleic acid is a small interfering RNA (siRNA) molecule or the nucleic acid encodes an siRNA molecule. In some aspects, the composition includes a plasmid or a viral vector, wherein the plasmid or viral vector includes a nucleic acid encoding an siRNA molecule disclosed herein. In some aspects, the siRNA molecule includes a nucleotide sequence of 2 to 30 nucleotides in length and at least 80% homologous to at least 2 to 30 consecutive nucleotides in a human GPAM cDNA sequence, wherein the human GPAM cDNA sequence is SEQ ID NO: 1. In some aspects, the siRNA molecule targets an open reading frame or 5' or 3'UTR of the GPAM gene. In some aspects, 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 aspects, the siRNA molecule comprises a nucleotide sequence of SEQ ID NO: 2-165 or any combination thereof. In some aspects, at least one sense sequence comprises a nucleotide sequence of SEQ ID NO: 2-83. In some aspects, at least one antisense sequence comprises SEQ ID NO: 84-165.

[0011] In some aspects, the present disclosure also relates to a composition comprising a nucleic acid molecule that downregulates GPAM expression, wherein the nucleic acid is an sgRNA or the nucleic acid encodes an sgRNA. In some aspects, the composition comprises a plasmid or viral vector, wherein the plasmid or viral vector comprises a first nucleic acid encoding an sgRNA molecule disclosed herein, and optionally comprises a second nucleic acid encoding an RNA-guided nuclease. In some aspects, the target sequence of the sgRNA is at least about 80% identical to any one of SEQ ID NOs: 177-276. In some aspects, the RNA-guided nuclease is a Cas endonuclease.

[0012] In some aspects, the siRNA molecules disclosed herein can specifically downregulate the gene expression of at least one variant of GPAM. In some aspects, the sgRNA molecules disclosed herein can specifically downregulate the gene expression of at least one variant of GPAM. In some aspects, at least one variant of GPAM is related to liver disease. Non-limiting examples of liver disease include fatty liver (FLD), alcohol-related liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic fatty hepatitis (NASH), end-stage liver disease (cirrhosis) caused by any cause, liver cancer, or any combination thereof. In some aspects, at least one variant of GPAM includes rs10787429C>T, rs7096937T>C, rs11446981T>TA, rs7898213T>C, rs2792759C>T, rs2297991T>C, rs1129555A>G, rs2254537T>A or any combination thereof.

[0013] In some aspects, the nucleic acid molecules disclosed herein can be conjugated with at least one targeting ligand. In some aspects, at least one targeting ligand includes a liver targeting ligand. In some aspects, the liver targeting ligand includes at least one N-acetylgalactosamine (GalNAc) conjugate. In some aspects, the nucleic acid molecule is conjugated with about one to about three GalNAc conjugates. In some aspects, the nucleic acid molecule comprises at least one chemical modification. In some aspects, the nucleic acid molecule includes a modification of at least one ribose moiety of its nucleotide sequence. In some aspects, at least one ribose moiety is modified by 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 aspects, less than about 10% to about 70% of the ribose moieties in the total nucleotide sequence are modified.

[0014] In some aspects, the disclosure also relates to a pharmaceutical composition comprising any of the compositions disclosed herein and at least one pharmaceutically acceptable carrier. In some aspects, the pharmaceutical composition further comprises nanoparticles. In some aspects, the pharmaceutical composition further comprises lipids.

[0015] In some aspects, the disclosure further relates to a method for treating a subject in need, the method comprising administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein. In some aspects, a subject in need is a human subject suffering from or suspected of having liver disease. In some aspects, liver disease includes fatty liver (FLD), alcohol-related liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) caused by any cause, liver cancer, or any combination thereof. In some aspects, the method of administration includes parenteral administration. In some aspects, compared with untreated subjects with the same disease condition and predicted results, administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein increases the life expectancy of the subject. In some aspects of the method, compared with untreated subjects with the same disease condition and predicted results, administering a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein improves the liver function of the subject. In certain aspects of the method, administration of a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein reduces liver fibrosis in a subject compared to an untreated subject with the same disease condition and predicted outcome. In certain aspects, administration of a therapeutically effective amount of a composition disclosed herein or a pharmaceutical composition disclosed herein prevents additional liver fibrosis in a subject compared to an untreated subject with the same disease condition and predicted outcome.

[0016] In some aspects, the present disclosure also relates to a kit, which includes: a container containing a composition disclosed herein or a pharmaceutical composition disclosed herein, a drug administration tool; and instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The following drawings constitute part of this specification and are used to further illustrate certain aspects of the present disclosure. Certain aspects of the present disclosure may be better understood by referring to the drawings in combination with the detailed description of specific embodiments herein.

[0018] Figure 1A Screening of 63 known NASH-associated genes is shown. The experimental scheme is shown in the figure.

[0019] Figure 1B Results from a screen of 63 known NASH-associated genes are shown. The most amplified clones were often associated with loss of lipogenic genes such as GPAM.

[0020] Figure 1C The screened popular genes are provided, including GPAM.

[0021] Figure 1DThe pathways in which these genes are involved are shown. The results of this screen suggest that genes that are mutated in human cirrhotic liver tissue may be good drug targets for treating NASH. GPAM is one such gene that is frequently mutated in human liver disease.

[0022] Figure 2A The researchers showed that during the development of NASH, GPAM mutant clones in the liver can outcompete wild-type clones. Red blood cells are wild-type cells, and their numbers decreased over time in the Western diet group.

[0023] Figure 2B The researchers showed that GPAM-mutated red blood cells increased in number over time in the Western diet group. In addition, when there were more GPAM-mutated clones in the liver, the NASH phenotype was improved (compare Figure 2A and Figure 2B This experiment demonstrated that the GPAM mutant clones were more fit and had reduced levels of steatosis in the liver.

[0024] Figure 3A Shown is a luciferase-based screen testing the relative knockdown efficacy of various human GPAM siRNAs.

[0025] Figure 3B Shown are GPAM protein levels detected by Western blot analysis after siRNA knockdown of GPAM.

[0026] Figure 4A Shows Gpam before and after Cre recombination f / f Genotyping of liver tissue. Asterisks are located next to mixed annealing bands of top and bottom PCR products.

[0027] Figure 4B qPCR detection of Gpam mRNA levels in the liver before and after Cre recombination is shown. Primer design is shown on the left. Two pairs of primers were used (n=3 mice per group).

[0028] Figure 4C Quantification of Tomato+ cells in LSL-tdTomato het liver sections (n=7, 11, 11 mice per group) is shown. Each dot represents one image field; two fields were analyzed per mouse liver. Statistical analysis was performed on the average image data of individual mice.

[0029] Figure 4D Gpam is shown f / f; Quantification of Tomato+ cells in LSL-tdTomato het liver sections (n=7, 15, 15 mice per group). Each dot represents one image area; two areas are shown for each mouse liver. Statistical analysis was performed on the average image data of individual mice.

[0030] Figure 4E Body weights of Gpam KO mice treated with diet or WD and corresponding control mice are shown (n=6, 16, 6, 12 mice per group).

[0031] Figure 4F Liver weights of whole-liver Gpam KO mice and corresponding control mice treated with diet or WD are shown (n=6, 16, 6, 12 mice per group).

[0032] Figure 4G Liver / body weight ratios of liver-specific Gpam WT and KO mice fed WD for 3 months are shown (n=6, 16, 6, 12 mice per group). These mice were injected with a high dose of AAV8-TBG-Cre to generate global hepatic Gpam deletion in nearly all hepatocytes.

[0033] Figure 4H Representative H&E staining results of Gpam WT and KO liver sections after 3 months of WD are shown.

[0034] Fig. 4I Liver function test results of plasma ALT are shown (n=6, 16, 6, 12 mice per group).

[0035] Figure 4J Liver function test results of plasma AST are shown (n=6, 16, 6, 12 mice per group).

[0036] Figure 4K NAFLD activity scores of H&E sections are shown. The right panel is the NAFLD activity score, which is the sum of the three scores in the left panel (n=13 and 14 mice per group).

[0037] Figure 4L The results of triglyceride measurements in liver tissues of WT and KO mice are shown (n=6, 16, 6, 12 per group).

[0038] Figure 4M The results of cholesterol measurements in liver tissues of WT and KO mice are shown (n=6, 16, 6, 12 mice per group).

[0039] Figure 4N Plasma cholesterol levels of WT and KO mice are shown (n=6, 16, 6, 12 per group).

[0040] Fig.4O Plasma triglycerides of WT and KO mice are shown (n=6, 16, 6, 12 per group).

[0041] The drawings do not limit the inventive concept to the specific embodiments disclosed and described herein. The drawings are not necessarily drawn to scale, emphasis instead being placed upon clearly illustrating the principles of certain embodiments of the inventive concept.

[0042] Detailed Description

[0043] The following detailed description refers to the accompanying drawings that illustrate various embodiments of the inventive concept. The drawings and description are intended to describe the various aspects and embodiments of the inventive concept in sufficient detail so that those skilled in the art can practice the inventive concept. Other components may also be used and modified without departing from the inventive concept. Therefore, the following description is not intended to be limiting. The scope of the inventive concept is defined solely by the appended claims and the full range of equivalents to which these claims are entitled.

[0044] With the increase 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 people, and there is increasing evidence that mutation load increases with age and chronic tissue damage. The present disclosure is based in part on a novel discovery that positive selection of somatic mutations in the liver of NASH patients can improve cell fitness and competitiveness by reducing lipid accumulation in NASH. In vivo genetic screening of somatic mutant genes in the liver identified other genes that can promote clonal fitness by reversing lipotoxicity when mutated, including GPAM (glycerol-3-phosphate acyltransferase, mitochondria). Therefore, the present invention provides compositions and methods of use for the treatment of liver diseases, especially metabolic liver diseases, including but not limited to non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0045] I. Terminology

[0046] The words and terms used herein are for descriptive purposes only and should not be construed as limiting. For example, the use of singular terms, such as "a", "an", "an", "an" and "an" does not imply a limitation on the number of items. In addition, the use of relative terms, such as "top", "bottom", "left", "right", "above", "below", "lower", "upper" and "side" in the description is for clarity when specifically referring to the drawings and is not intended to limit the scope of the inventive concept or the appended claims.

[0047] In addition, since the inventive concept is subject to a variety of different forms of implementation, the present disclosure should be regarded as an embodiment of the inventive concept principle, and is not intended to limit the inventive concept to the specific embodiments shown and described. Any feature of the inventive concept can be used alone or in combination with any other feature. The term "implementation" and / or similar terms mentioned in the description mean that the mentioned feature is included in at least one aspect of the description. The terms "implementation" and / or similar terms mentioned separately in the description do not necessarily refer to the same implementation, nor are they mutually exclusive, unless otherwise specified and / or it is easy for a person skilled in the art to see from the description. For example, the features, structures, processes, steps, actions, etc. described in one implementation may also be included in other implementations, but not necessarily. Therefore, the inventive concept may include various combinations and / or combinations of the embodiments described herein. In addition, all aspects of the disclosure described herein are not essential to its practice. Similarly, other systems, methods, features and advantages of the inventive concept will also be apparent to those skilled in the art after reading the drawings and the description. All of these additional systems, methods, features and advantages should be included in this specification, belong to the scope of the inventive concept, and are included in the claims.

[0048] As used herein, the term "about" can mean ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% relative to a recited value, such as an amount, dosage, temperature, time, percentage, etc.

[0049] The terms "include", "comprising", "involving" and "having" can be used interchangeably in this disclosure. The terms "include", "comprising", "involving" and "having" mean including but not necessarily limited to the stated contents.

[0050] As used herein, the terms "or" and "and / or" should be interpreted as inclusive or meaning any one or any combination. Thus, "A, B, or C" or "A, B, and / or C" means any of the following: "A", "B", or "C"; "A and B"; "A and C"; "B and C"; "A, B, and C". Exceptions to this definition will only occur if a combination of elements, functions, steps or acts are inherently mutually exclusive to some extent.

[0051] As used herein, "biomarker" refers to any biological molecule (such as nucleic acids, genes, peptides, proteins, lipids, hormones, metabolites, etc.) that can reflect the current state of a biological system or predict the future state, either alone or together. Therefore, as used herein, the presence or concentration of one or more biomarkers can be detected and associated with a known condition, such as a disease state. In some aspects, detecting the presence and / or concentration of one or more biomarkers herein can be used as an indication of the risk of liver disease in a subject. In other aspects, detecting the presence and / or concentration of one or more biomarkers herein can be used to treat and / or prevent chronic liver disease in a subject.

[0052] As used herein, the terms "treat," "treatment," and the like, unless otherwise indicated, may refer to reversing, alleviating, inhibiting, or preventing the process of the disease, disorder, or condition to which the term applies, or one or more symptoms of the disease, disorder, or condition, including the administration of any composition, pharmaceutical composition, or dosage form described herein to prevent the occurrence of symptoms or complications, or to alleviate symptoms or complications, or to eliminate the condition or disorder.

[0053] The term "biological macromolecule" as used herein refers to, but is not limited to, proteins, enzymes, antibodies, DNA, siRNA and small molecules. "Small molecules" as used herein may refer to chemicals, compounds, drugs, etc.

[0054] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless otherwise specified, the term includes nucleic acids containing known analogs of natural nucleotides, which have similar binding properties to the reference nucleic acid and are metabolized in a manner similar to natural nucleotides. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes conservatively modified variants thereof (such as degenerate codon substitutions), alleles, orthologs, SNPs and complementary sequences as well as the sequences explicitly indicated. Specifically, degenerate codon replacement 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)).

[0055] The terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, but there is no limit to the maximum number of amino acids that make up a protein or peptide sequence. Polypeptides include any peptide or protein that includes two or more amino acids linked to each other by peptide bonds. The term used herein refers to both short chains, which are also commonly referred to as peptides, oligopeptides and oligomers in the art, and long chains, which are commonly referred to as proteins in the art, and there are many types. "Polypeptides" include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, etc. Polypeptides include natural peptides, recombinant peptides or, or a combination thereof.

[0056] It should also be understood that, unless explicitly stated to the contrary, in any method claimed herein, if more than one step or action is included, the order of the steps or actions of the method is not necessarily limited to the order in which the steps or actions of the method are described.

[0057] II. Composition

[0058] (a)GPAM

[0059] In certain embodiments, the compositions used in the methods disclosed herein can modulate the GPAM (glycerol-3-phosphate acyltransferase, mitochondrial) gene. The GPAM gene encodes the mitochondrial enzyme Gpam, which catalyzes the acylation of glycerol-3-phosphate with acyl coenzyme A (CoA) to generate CoA and lysophosphatidic acid (LPA). This is the rate-limiting step in the synthesis of TAG. This enzyme has two forms, one present in the mitochondria and the other present in the endoplasmic reticulum. In addition, there are two alternative splicing transcript variants of the GPAM gene.

[0060] As used herein, a composition that "modulates" GPAM may include any biomolecule that can reduce GPAM gene expression, reduce GPAM protein expression, reduce GPAM activity, or a combination thereof. In some aspects, the biomolecule 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 GPAM genes, reduce the expression of GPAM proteins, or a combination thereof.

[0061] In certain embodiments, the compositions used for the methods disclosed herein may include nucleic acid molecules. As used herein, the term "nucleic acid molecule" refers to a molecule having nucleotides. Nucleic acids may be single-stranded, double-stranded, or multi-stranded, and may include modified or unmodified nucleotides or non-nucleotides or various mixtures and combinations thereof. In some embodiments, the nucleic acid molecules used herein may be double-stranded RNA. In certain embodiments, the double-stranded RNA suitable for use herein may be small sequence RNA, small nuclear RNA, small nucleolar RNA, short hairpin RNA, micro RNA, or the like. In certain embodiments, the double-stranded RNA suitable for use herein may be small interfering RNA (siRNA).

[0062] The term "siRNA" as used herein refers to a small inhibitory RNA duplex that induces the RNA interference (RNAi) pathway. The siRNA molecules disclosed herein can silence, reduce and / or inhibit the expression of a target gene (such as GPAM). The length of these molecules can vary (generally about 5-50 base pairs), and the degree of complementarity with their target mRNA in the antisense strand can also vary. Some siRNAs (but not all siRNAs) have unpaired overhanging end bases at the 5' or 3' end of the sense strand and / or antisense strand. The term "siRNA" as used herein includes a duplex of two independent chains, and also includes a single strand that can form a hairpin structure containing a double-stranded region.

[0063] In certain embodiments, the siRNA molecules disclosed herein can be any interfering RNA with a double-stranded length of about 2-60, about 5-50, or about 10-40 nucleotides, more typically about 2-30, about 5-25, or about 10-25 nucleotides. In some embodiments, the siRNA molecules disclosed herein can have a nucleotide sequence of about 2 to about 30 nucleotides, about 5 to about 25 nucleotides, or about 10 to about 20 nucleotides in length. In some embodiments, the siRNA molecules disclosed herein can have a length of about 10 nucleotides, a length of about 11 nucleotides, a length of about 12 nucleotides, a length of about 13 nucleotides, a length of about 14 nucleotides, a length of about 15 nucleotides, a length of about 14 nucleotides, a length of about 15 nucleotides, a length of about 16 nucleotides, a length of about 17 nucleotides, a length of about 18 nucleotides, a length of about 19 nucleotides, a length of about 20 nucleotides, a length of about 21 nucleotides, a length of about 22 nucleotides, a length of about 23 nucleotides, a length of about 24 nucleotides, or a length of about 25 nucleotides. Each complementary sequence of the double-stranded siRNA can be about 2-60, about 5-50, about 10-40, about 2-30, about 5-25, or about 10-25 nucleotides in length, but other non-complementary sequences may also 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 end of about 1 to about 4 or more nucleotides.

[0064] In certain embodiments, the siRNA molecules disclosed herein may have about 2-60, about 2-50, about 2-40, or about 2-30 consecutive nucleotides homologous to a target nucleotide sequence (such as GPAM). In certain aspects, the target nucleotide sequence herein may be a human GPAM nucleotide sequence or a variant thereof. In other aspects, the target nucleotide sequence herein may be human GPAM, reference sequence (Ref Seq): NM_001244949.2 or a variant thereof. In other aspects, the target nucleotide sequence herein may be the following SEQ ID NO: 1 or a variant thereof:

[0065] Human GPAM (SEQ ID NO: 1)

[0066]

[0067]

[0068] In certain embodiments, the siRNA molecules disclosed herein may have a nucleotide sequence of about 2-30 consecutive nucleotides in length and at least about 80% homology (e.g., about 80%, about 85%, about 90%, about 95%, or about 99%) with at least about 2 to about 30 consecutive nucleotides of the human GPAM cDNA sequence. In some embodiments, the siRNA molecules disclosed herein may have a nucleotide sequence of about 2-30 consecutive nucleotides in length and at least 80% homology (e.g., about 80%, about 85%, about 90%, about 95%, or about 99%) with at least about 2 to about 30 consecutive nucleotides of the human GPAM sequence reference sequence: NM_001244949.2 or a variant thereof. In some embodiments, the siRNA molecules disclosed herein may have a nucleotide sequence of about 2-30 consecutive nucleotides in length and at least 80% homology (e.g., about 80%, about 85%, about 90%, about 95%, or about 99%) to at least about 2 to about 30 consecutive nucleotides of SEQ ID NO: 1. In other embodiments, the siRNA molecules disclosed herein may have a nucleotide sequence of about 2-30 consecutive nucleotides in length and at least about 2 to about 30 consecutive nucleotides homologous to SEQ ID NO: 1.

[0069] The siRNA molecules disclosed herein can be synthesized in any of a variety of configurations. One skilled in the art can identify the type of siRNA configuration for a particular purpose. Examples of siRNA configurations include, but are not limited to: a double-stranded polynucleotide molecule assembled from two independent strand molecules, one of which is a sense strand and the other is a complementary antisense strand; a double-stranded polynucleotide molecule assembled from single-stranded molecules, wherein the sense region and the antisense region are connected by a nucleic acid-based or non-nucleic acid-based linker; a double-stranded polynucleotide molecule having a hairpin secondary structure, which has a complementary sense region and an antisense region; or a circular single-stranded polynucleotide molecule having two or more circular structures and a stem, the stem having a self-complementary sense region and an antisense region. For circular polynucleotides, the polynucleotides can be treated in vivo or in vitro to produce active double-stranded siRNA molecules.

[0070] In certain embodiments, the siRNA molecules disclosed herein can be double-stranded siRNA molecules. In certain aspects, the double-stranded siRNA molecules disclosed herein can have at least one sense sequence. In other aspects, the double-stranded siRNA molecules disclosed herein can have at least one antisense sequence. In other aspects, the double-stranded siRNA molecules disclosed herein can have at least one antisense sequence and at least one sense sequence. In some embodiments, the double-stranded siRNA molecules disclosed herein can have at least one antisense sequence selected from Table 1, at least one sense sequence selected from Table 1, or both.

[0071] Table 1

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] 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%) with 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%) with 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 a sense sequence of at least one of SEQ ID NOs: 2-83. In some embodiments, the double-stranded siRNA molecules disclosed herein may have an antisense sequence of at least one of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of at least one of SEQ ID NOs: 2-83 and an antisense sequence of at least one of SEQ ID NOs: 84-165. In some embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of at least one of SEQ ID NOs: 2-63. In some embodiments, the double-stranded siRNA molecules disclosed herein may have an antisense sequence of at least one of SEQ ID NOs: 84-145. In some embodiments, the double-stranded siRNA molecules disclosed herein may have a sense sequence of at least one of SEQ ID NOs: 2-63 and an antisense sequence of at least one of SEQ ID NOs: 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 other aspects, 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 other aspects, 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 other aspects, 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.

[0078] In some embodiments, the present disclosure also includes nucleic acid sequences encoding double-stranded siRNA molecules disclosed herein. In some embodiments, the nucleic acid sequence encoding the double-stranded siRNA disclosed herein can be double-stranded DNA, single-stranded DNA, plasmid vectors, viral vectors, such as retroviral vectors, lentiviral vectors, poxvirus vectors, adenoviral vectors, or adenovirus-associated vectors. 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%) with 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%) with any one of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid may 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 may encode at least one sense sequence of any one of SEQ ID NOs: 2-83. In some embodiments, the nucleic acid may encode at least one antisense sequence of any one of SEQ ID NOs: 84-165. In some embodiments, the nucleic acid may 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 may encode the sense sequence of SEQ ID NO: 9 and the antisense sequence of SEQ ID NO: 91. In some aspects, the nucleic acid can encode the sense sequence of SEQ ID NO: 13 and the antisense sequence of SEQ ID NO: 95. In some aspects, the nucleic acid molecule can encode the sense sequence of SEQ ID NO: 17 and the antisense sequence of SEQ ID NO:99.

[0079] The present disclosure also provides methods for making siRNA molecules disclosed herein. The manufacture of siRNA can be by chemical synthesis, or siRNA can be encoded and transcribed by a plasmid, or siRNA can be expressed by an engineered viral vector. siRNA can be a single-stranded molecule with complementary sequences that can self-hybridize into a duplex with a hairpin loop. siRNA can also be produced by cleaving the parent dsRNA using an appropriate enzyme, such as E. coli RNase III or Dicer. The parent dsRNA can be any double-stranded RNA duplex that can produce siRNA, such as a complete or partial mRNA transcript. The use of cell lysates or in vitro treatment may further involve the subsequent separation of short nucleotide siRNAs (e.g., about 2-25 nucleotides in length) from the lysate, making the entire process somewhat cumbersome and expensive. The method of chemical synthesis is to first make two single-stranded RNA-oligomers, and then anneal the two single-stranded oligomers into double-stranded RNA. There are many different methods for chemical synthesis, and different siRNAs can be easily synthesized.

[0080] In certain embodiments, the siRNA molecules disclosed herein can eliminate the gene expression of GPAM. RNA expression detection and quantification methods known in the art that are suitable for use herein include, but are not limited to, northern blotting and in situ hybridization, RNAse protection detection, polymerase chain reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative reverse transcription PCR (RT-qPCR or qPCR), gene expression analysis based on sequencing (such as continuous analysis of gene expression (SAGE)), gene expression analysis of massively parallel signature sequencing (MPSS), etc.

[0081] The siRNA molecules of the present invention can specifically downregulate the gene expression of GPAM (glycerol-3-phosphate acyltransferase, mitochondria) or its variants. The term downregulation can be interchangeably expressed as reducing, inhibiting, preventing, blocking or silencing. Here, "downregulating gene expression" refers to a decrease in the gene expression level compared to the normal expression level. For example, the reduced gene expression level can be about 70% to 0% of the normal expression level. In other words, compared to the normal expression level, about 30% to 100% of the gene expression is downregulated, reduced, blocked, inhibited, prevented or silenced. Specifically, the reduced gene expression level 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 reduction in the gene expression level can also be any percentage or range as described above. On the other hand, the term "specific" or "specifically" used in connection with downregulation refers to downregulation of expression of a target gene with little or no binding or downregulation of other nucleic acids or their expression.

[0082] In some embodiments, the siRNA molecules disclosed herein can reduce the gene expression of GPAM by at least about 50%. In some aspects, the siRNA molecules disclosed herein can reduce the 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 the 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%.

[0083] In some embodiments, the siRNA molecules disclosed herein can eliminate the expression of Gpam protein. Protein expression detection and quantitative methods known in the art that are applicable to this article include but are not limited to ELISA (enzyme-linked immunosorbent assay), immunoblotting assay, flow cytometry, immunohistochemistry assay, radioimmunoassay, protein blot (Westernblot) assay, immunofluorescence assay, chemiluminescence assay, mass spectrometry, matrix-assisted laser desorption / ionization time of flight (MALDI-TOF) mass spectrometry, liquid chromatography / quadrupole time of flight electrospray tandem mass spectrometry (LC / Q-TOF-ESI-MS / MS), two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), etc.

[0084] In some embodiments, the siRNA molecules disclosed herein can reduce Gpam protein expression by at least about 50%. In some aspects, the siRNA molecules disclosed herein can reduce Gpam protein expression 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 Gpam protein expression by about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.

[0085] In some embodiments, the siRNA molecules disclosed herein may have one or more chemical modifications. Non-limiting examples of chemical modifications may include end caps, phosphate backbone modifications, and the like. Examples of classes of end caps include, but are not limited to, inverted deoxy abasic residues, glycerol modifications, 4′,5′-methylene nucleotides, 1-(β-D-furanose) nucleotides, 4′-thionucleotides, carbocyclic nucleotides, 1,5-anhydrohexitol nucleotides, L-nucleotides, α-nucleotides, modified base nucleotides, threo-pentofuranosyl (threo-pentofuranosyl) nucleotides, and the like. pentofuranosyl) nucleotide, acyclic 3′,4′-seco nucleotide, acyclic 3,4-dihydroxybutyl nucleotide, acyclic 3,5-dihydroxypentyl nucleotide, 3′-3′-reverse nucleotide portion, 3′-3′-reverse abasic portion, 3′-2′-reverse nucleotide portion, 3′-2′-reverse abasic portion, 5′-5′-reverse nucleotide portion, 5′-5′-reverse abasic portion, 3′-5′-reverse deoxy abasic portion, 5′ -aminoalkyl phosphate, 1,3-diamino-2-propyl phosphate, 3-aminopropyl phosphate, 6-aminohexyl phosphate, 1,2-aminododecyl phosphate, hydroxypropyl phosphate, 1,4-butanediol phosphate, 3'-phosphoramidate, 5'-phosphoramidate, hexyl phosphate, aminohexyl phosphate, 3'-phosphate, 5'-amino, 3'-phosphorothioate, 5'-phosphorothioate, phosphorodithioate, and bridged or non-bridged methylphosphonate or 5'-sulfhydryl moiety. Non-limiting examples of phosphate backbone modifications (i.e., resulting in modified internucleotide connections) include phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidate, carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, methylal, thiomethylal, and alkylsilicon substitution. These chemical modifications can occur at the 5' end and / or the 3' end of the sense strand, the antisense strand, or both strands of the siRNA.

[0086] Chemical modification of siRNA molecules disclosed herein may include modification of at least one ribose moiety in its nucleotide sequence. The ribose moiety may be 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 aspects, the proportion of the siRNA molecules disclosed herein to the total nucleotide sequence is less than about 10% to about 70% (such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%) ribose moieties may be modified. Chemical modification of siRNA molecules disclosed herein may include connecting a conjugate to the siRNA molecule. The type of conjugate used and the degree of conjugation to the siRNA may be evaluated for improving the pharmacokinetic characteristics, bioavailability and / or stability of the siRNA while maintaining its activity. Therefore, those skilled in the art can utilize any of various well-known in vitro cell culture or in vivo animal models, including the above-mentioned negative control expression studies, to screen siRNA molecules connected with various conjugates to identify siRNA conjugates with improved properties. The conjugate can be connected to the 5' end and / or 3' end of the sense strand and / or antisense strand of the siRNA by a covalent linker (such as a nucleic acid or non-nucleic acid linker). The conjugate can be connected to the siRNA by a carbamate group or other linking group (for example, see U.S. Patent Publication Nos. 20050074771, 20050043219 and 20050158727, the entire contents of which are incorporated herein by reference). The conjugate can be added to the siRNA for a variety of purposes. For example, the conjugate can be a molecular entity that promotes the delivery of siRNA to cells, or it can be a molecule containing a drug or a label. Examples of conjugate molecules suitable for attachment to the siRNA of the invention include, but are not limited to, steroids such as cholesterol, glycols such as polyethylene glycol (PEG), human serum albumin (HSA), fatty acids, carotenoids, terpenoids, bile acids, folic acid (such as folic acid, folic acid analogs and derivatives thereof), carbohydrates (such as galactose, galactosamine, N-acetylgalactosamine, glucose, mannose, fructose, fucose, etc.), phospholipids, peptides, cell receptor ligands capable of mediating cellular uptake, and combinations thereof. Other examples include, for example, lipophilic moieties, vitamins, polymers, peptides, proteins, nucleic acids, small molecules, oligosaccharides, carbohydrate clusters, intercalating agents, small groove binders, cleaving agents, and cross-linking agent conjugate molecules described in U.S. Patent Publication Nos. 20050119470 and 20050107325, the contents of each of which are incorporated herein by reference in their entirety. Other examples include 2'-O-alkylamines, 2'-O-alkoxyalkylamines, polyamines, C5-cationic modified pyrimidines, cationic peptides, guanidine groups, amidine groups, cationic amino acid conjugate molecules, and the like.Other examples of conjugate molecules include hydrophobic groups, membrane active compounds, cell penetrating compounds, cell targeting signals, interaction modifiers, or steric stabilizers as described in US Patent Publication No. 20040167090, the entire contents of which are incorporated herein by reference.

[0087] In certain embodiments, the siRNA molecules disclosed herein can be conjugated with at least one targeting ligand. The targeting ligands considered herein include ligands suitable for targeting siRNA molecules to the liver, liver tissue and / or hepatocytes. Non-limiting examples of targeting ligands suitable for use herein may include galactose, galactosamine, N-formyl-galactosamine, N-acetyl-galactosamine, N-propionyl-galactosamine, N-n-butyryl-galactosamine, N-isobutyryl-galactosamine, galactose clusters and N-acetyl-galactosamine trimers, and may optionally have a pharmacokinetic modulator selected from the following: a hydrophobic group with 16 or more carbon atoms, a hydrophobic group with 16-20 carbon atoms, a palmitoyl group, a hexadecene-8-enoyl group, an oleoyl group, (9E, 12E)-octadecene-9,12 dienoyl group, a dioctanoyl group, a C16-C20 acyl group, and cholesterol. In some aspects, the liver targeting ligand suitable for use herein can be an N-acetylgalactosamine (GalNAc) conjugate. In some embodiments, the siRNA molecules disclosed herein can be conjugated with at least one GalNAc conjugate. In some embodiments, the siRNA molecules disclosed herein can be conjugated with 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 can be conjugated with about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 GalNAc conjugates.

[0088] Any siRNA molecule 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 may include rs10787429C>T, rs7096937T>C, rs11446981T>TA, rs7898213T>C, rs2792759C>T, rs2297991T>C, rs1129555A>G, rs2254537T>A, or any combination thereof. In some aspects, the siRNA molecules disclosed herein can specifically downregulate the gene expression of GPAM or its variants, such as rs10787429C>T, rs7096937T>A, rs11446981T>TA, rs7898213T>C, rs2792759C>T, rs2297991T>C, rs1129555A>G, rs2254537T>A or any combination thereof. The GPAM gene and / or its variants may be associated with liver disease, liver dysfunction, liver injury and / or liver damage. Liver disease may include fatty liver disease (FLD), alcohol-related liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) caused by any cause, liver cancer or any combination thereof. Liver damage may include cirrhosis, 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, 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.

[0089] In certain embodiments, the methods and compositions provided herein may include a vector containing any of the siRNA molecules disclosed herein. In some embodiments, the vector used herein may be a viral vector. As used herein, the term "viral vector" may refer to a nucleic acid vector construct, which includes at least one element of viral origin, has the ability to be packaged into viral vector particles, and encodes at least one exogenous polynucleotide. In certain embodiments, vectors and / or particles can be used to transfer any nucleic acid to cells in vitro or in vivo. There are many viral vectors known in the art. The term virion may refer to a single infectious viral particle. "Viral vector", "viral vector particle" and "viral particle" also refer to complete viral particles with DNA or RNA core and protein capsid present outside the cell. Non-limiting examples of viral vectors used herein may include adenovirus, adeno-associated virus (AAV), herpes virus, retrovirus, lentivirus, integrase defective lentivirus (IDLV), etc. In some embodiments, the viral vector disclosed herein may 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), sheep myelin shedding virus and progressive pneumonia virus, baboon pseudovirus, 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 (such as CRISPR). In certain embodiments, the methods provided herein may include generating cells expressing any 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.

[0090] In some aspects, the current disclosure also relates to the use of gene editing systems, such as CRISPR-based systems to eliminate or downregulate the gene expression of GPAM. Therefore, the current disclosure also relates to compositions specifically targeting GPAM or its variants, which include clustered regularly interspaced short palindromic repeats (CRISPR) system-related nucleic acid sequences, such as single guide RNA (sgRNA), CRISPR-RNA (crRNA), trans-activating crRNA (tracrRNA), and further encoding clustered regularly interspaced short palindromic repeats (CRISPR) system-related nucleic acid sequences plasmid DNA (pDNA) or viral vectors. In some aspects, the gene editing system includes at least sgRNA targeting GPAM and RNA-guided nucleases, such as Cas9. In some aspects, the sgRNA target sequence has at least about 80% (e.g., about 80%, about 85%, about 90%, about 95%, or about 99%) homology with any one of SEQ ID NO: 177-276. In some aspects, the sgRNA target sequence is at least about 80% identical to any one of SEQ ID NOs: 177-276 provided in Table 2. In some aspects, the sgRNA target sequence 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 aspects, the sgRNA targets 20 consecutive nucleotides in any one of SEQ ID NOs: 277-376. None of the gRNAs provided in Table 2 are predicted to target the last exon or have a 10 bp off-target match.

[0091] Table 2

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101]

[0102]

[0103]

[0104]

[0105] (b) Pharmaceutical Compositions

[0106] The siRNA molecules targeting GPAM disclosed herein for use in the methods described herein may be provided per se and / or may be provided as part of a pharmaceutical composition, wherein the modulator and / or inhibitor may be mixed with a suitable carrier or excipient.

[0107] As used herein, "pharmaceutical composition" refers to a preparation of one or more 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. Here, the term "active ingredient" refers to any siRNA molecule disclosed herein. The term "active ingredient" as used herein may also include any vector, culture medium, microorganism or cell culture that synthesizes, expresses and / or contains siRNA molecules, such as genetically modified cells, viral vectors, plasmids, bacteria, yeast, fungi and cultures or culture media thereof.

[0108] (i) Pharmaceutically acceptable carriers and excipients

[0109] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" are used interchangeably 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 given compound. Adjuvants are also included in these phrases.

[0110] In certain embodiments, the compositions disclosed herein may further include one or more pharmaceutically acceptable diluents, excipients and / or carriers. Pharmaceutically acceptable diluents, excipients or carriers used herein refer to materials suitable for administering medication to a subject without causing undesirable biological effects or interacting with any component of the contained composition in a harmful manner. Pharmaceutically acceptable diluents, carriers and excipients may include, but are not limited to, physiological saline, Ringer's solution, phosphate solution or buffer, buffered physiological saline, and other carriers known in the art.

[0111] In some embodiments, the pharmaceutical composition herein may also include stabilizers, antioxidants, colorants, other drugs or medicaments, carriers, adjuvants, preservatives, stabilizers, wetting agents, emulsifiers, solution promoters, salts, solubilizers, defoamers, antioxidants, dispersants, surfactants or any combination thereof. Here, the term "excipient" refers to an inert substance added to the pharmaceutical composition to further promote the use of active ingredients. Examples of excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars and starch types, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols. The formulation and administration techniques of the drug can be found in the latest version of "Remington's Pharmaceutical Sciences" of Mike Publishing Company, Easton, Pennsylvania, which is incorporated herein by reference.

[0112] 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 adjuvants, so as to facilitate the processing of genetically modified endothelial progenitor cells into pharmaceutically usable preparations. In some embodiments, any well-known techniques, carriers and excipients can be used, as long as they are suitable and / or consistent with the understanding of the art.

[0113] In certain embodiments, the pharmaceutical composition described herein may be an aqueous suspension comprising one or more polymers as suspending agents. In some embodiments, the polymers that may comprise the pharmaceutical composition described herein include: water-soluble polymers, such as cellulose polymers, for example, hydroxypropyl methylcellulose; water-insoluble polymers, such as cross-linked carboxyl-containing polymers; mucoadhesive polymers, selected from, for example, carboxymethylcellulose, carbomer (acrylic acid polymer), polymethyl methacrylate, polyacrylamide, polycarbophil, acrylic acid / butyl acrylate copolymer, sodium alginate and dextran, etc.; or combinations thereof. In some embodiments, the pharmaceutical composition disclosed herein may comprise 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% of the polymer as a suspending agent, based on the total weight of the composition. In some embodiments, the pharmaceutical composition disclosed herein may comprise a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the polymer as a suspending agent, based on the total weight of the composition.

[0114] In certain embodiments, the pharmaceutical compositions disclosed herein may include viscous preparations. In certain embodiments, the viscosity of the compositions described herein may be increased by adding one or more gelling or thickening agents. In some embodiments, the compositions disclosed herein may include one or more gelling or thickening agents in an amount that can provide a sufficiently viscous preparation to remain on the treated tissue. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition. In some embodiments, the thickener suitable for use herein can be hydroxypropyl methylcellulose, hydroxyethylcellulose, polyvinyl pyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, sodium hyaluronate. In other aspects, the viscosity increasing agent can be gum arabic, agar, magnesium aluminum silicate, sodium alginate, sodium stearate, bladder gum, bentonite, carbomer, carrageenan, Carbopol, xanthan gum, cellulose, microcrystalline cellulose (MCC), carob gum, chitosan, carboxymethyl chitosan, chondroitin, glucose, furcellaran, gelatin, gati gum, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, karaya gum, xanthan gum, tragacanth gum, ethylcellulose, ethylhydroxy Ethylcellulose, ethylmethylcellulose, methylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxypropylcellulose, polyhydroxyethyl methacrylate, oxypolygelatin, pectin, polygelatin, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropylcellulose, hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), (glucose, maltodextrin and sucralose), or any combination thereof.

[0115] In certain embodiments, the pharmaceutical compositions disclosed herein may include additional agents or additives selected from surfactants, detergents, solvents, acidifiers, alkalizers, buffers, tonicity modifiers, ionic additives that can effectively increase the ionic strength of the solution, antibacterial agents, antibiotics, antifungal agents, antioxidants, preservatives, electrolytes, defoamers, oils, stabilizers, enhancers, and the like. In certain embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more agents by weight of the total composition. In certain embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of one or more agents by weight of the total composition. In some embodiments, one or more of these agents may be added to improve the performance, efficacy, safety, shelf life, and / or other properties of the muscarinic antagonist compositions disclosed herein. In some embodiments, the additive may be biocompatible, non-irritating, non-abrasive, and / or non-allergenic.

[0116] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more acidifiers. "Acidifier" as used herein refers to a compound used to provide an acidic medium. For example, such compounds include, but are not limited to, acetic acid, amino acids, citric acid, fumaric acid, and other α-hydroxy acids, such as hydrochloric acid, ascorbic acid, and nitric acid, as well as other compounds known to those of ordinary skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic acid may be used. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more acidifiers, based on the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of one or more acidifiers, based on the total weight of the composition.

[0117] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more alkalizing agents. "Alkalizing agents" used herein are compounds for providing alkaline media. For example, such compounds include, but are not limited to, ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, trihydroxyethylamine, triethanolamine, and other compounds known to those of ordinary skill in the art. In some embodiments, any pharmaceutically acceptable organic or inorganic base may be used. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more alkalizing agents, accounting for a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0118] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more antioxidants. As used herein, "antioxidants" are agents that inhibit oxidation, and therefore can be used to prevent preparations from deteriorating due to oxidation processes. For example, such compounds include, but are not limited to, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, sulfoxylate, monothioglycerol, propyl gallate, sodium ascorbate, sodium bisulfite, sodium formaldehyde sulfinate, sodium pyrosulfite, and other compounds known to those of ordinary skill in the art. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0119] In certain embodiments, the pharmaceutical compositions disclosed herein may include a buffer system. As used herein, a "buffer system" is a composition consisting of one or more buffers, wherein a "buffer" is a compound used to resist pH changes when diluting or adding an acid or base. Buffers include, for example, but are not limited to potassium metaphosphate, potassium phosphate, monobasic sodium acetate, sodium citrate anhydrate and dihydrate, and other compounds known to those of ordinary skill in the art. In certain embodiments, any pharmaceutically acceptable organic or inorganic buffer may be used. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more buffers of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of one or more buffers of the total weight of the composition.

[0120] In some embodiments, the amount of one or more buffers depends on the desired pH of the composition. In some embodiments, the pH of the pharmaceutical compositions disclosed herein may be from about 6 to about 9. In some embodiments, the pH of the pharmaceutical compositions disclosed herein may be greater than about 8, greater than about 7.5, greater than about 7, greater than about 6.5, or greater than about 6.

[0121] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more preservatives. "Preservatives" as used herein refer to agents or combinations of agents that can inhibit, reduce or eliminate bacterial growth in pharmaceutical dosage forms. Non-limiting examples of preservatives include parabens, propylparabens, isopropanol, and combinations thereof. In some embodiments, any pharmaceutically acceptable preservative may be used. In some embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more preservatives of the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of one or more preservatives of the total weight of the composition.

[0122] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more surfactants or detergents. In certain embodiments, the surfactants or detergents may be synthetic, natural or semi-synthetic. In some embodiments, the compositions disclosed herein may include anionic detergents, cationic detergents, zwitterionic detergents, zwitterionic detergents, acid-base amphoteric detergents, non-ionic detergents with steroid skeletons, or combinations thereof. In certain embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more surfactants or detergents. In some embodiments, the pharmaceutical compositions disclosed herein may include a total amount of about 5% to about 99%, about 10%, about 95%, or about 15% to about 90% of the total weight of the composition.

[0123] In certain embodiments, the pharmaceutical compositions disclosed herein may include one or more stabilizers. "Stabilizer" as used herein refers to a compound used to stabilize an active agent to counteract the effects of physical, chemical or biochemical processes, otherwise the therapeutic activity of the active agent will be reduced. For example, suitable stabilizers include, but are not limited to, succinic anhydride, albumin, sialic acid, creatinine, glycine and other amino acids, nicotinamide, sodium acetyltryptophanate, zinc oxide, sucrose, glucose, lactose, sorbitol, mannitol, glycerol, polyethylene glycol, sodium caprylate and sodium saccharin, and other compounds known to those of ordinary skill in the art. In certain embodiments, the pharmaceutical compositions disclosed herein may include 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% of one or more stabilizers of the 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.

[0124] In some embodiments, the pharmaceutical compositions disclosed herein may include one or more tension agents. "Tensity agent" as used herein refers to a compound that can be used to adjust the tension of a liquid formulation. Suitable tension agents include, but are not limited to, glycerol, lactose, mannitol, glucose, sodium chloride, sodium sulfate, sorbitol, trehalose, and other compounds known to those of ordinary skill in the art. The osmotic pressure in the composition can be expressed in milliosmole / liter (mOsm / L). The osmotic pressure can be measured using methods generally known in the art. In some embodiments, the osmotic pressure of the composition disclosed herein is calculated using the vapor pressure reduction method. In some embodiments, the amount of one or more tension agents included in the pharmaceutical compositions disclosed herein can make the composition osmotic pressure be about 150mOsm / L to about 500mOsm / L, about 250mOsm / L to about 500mOsm / L, about 250mOsm / L to about 350mOsm / L, about 280mOsm / L to about 370mOsm / L, or about 250mOsm / L to about 320mOsm / L. In some embodiments, the osmotic pressure of the compositions herein ranges from about 100 mOsm / kg to about 1000 mOsm / kg, from about 200 mOsm / kg to about 800 mOsm / kg, from about 250 mOsm / kg to about 500 mOsm / kg, or from about 250 mOsm / kg to about 320 mOsm / kg, or from about 250 mOsm / kg to about 350 mOsm / kg, or from about 280 mOsm / kg to about 320 mOsm / kg. In some embodiments, the osmotic pressure of the pharmaceutical compositions described herein may be 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 may include 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%, based on the total weight of the composition. In some embodiments, the pharmaceutical compositions disclosed herein may 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.

[0125] (ii) Dosage Formulation

[0126] In certain embodiments, the present disclosure provides compositions formulated for one or more routes of administration. For example, suitable routes of administration may include oral, rectal, mucosal, nasal, intestinal and / or parenteral administration. In certain embodiments, the compositions formulated herein may be used for parenteral administration. In some embodiments, the compositions formulated herein may be formulated for intramuscular, subcutaneous, intramedullary, intravenous, intraperitoneal and / or intranasal injection.

[0127] In certain embodiments, the compositions herein can be administered in a local or systemic manner, for example, by direct local injection of the pharmaceutical composition into a tissue area of ​​the patient. In some embodiments, the pharmaceutical compositions disclosed herein can be administered parenterally, for example, by intravenous injection, intraventricular injection, intracisterna magna injection, intraparenchymal injection, or a combination thereof. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to a subject in a manner disclosed herein. In some embodiments, the pharmaceutical compositions disclosed herein can be used in human patients. In some embodiments, the pharmaceutical compositions disclosed herein can be administered to human patients by at least two routes of administration. In some embodiments, the combination of routes of administration can be intraventricular injection and intravenous injection; intrathecal injection and intravenous injection; intracisterna magna injection and intravenous injection; and / or intraparenchymal injection and intravenous injection.

[0128] In certain embodiments, pharmaceutical compositions of the present disclosure can be manufactured by processes well known in the art, such as by conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0129] In certain embodiments, the pharmaceutical compositions used in accordance with the present disclosure can be formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants) that aid in processing the active ingredient into a pharmaceutically acceptable formulation. The correct formulation depends on the selected route of administration. For injection, the active ingredient of the pharmaceutical composition herein can be formulated in an aqueous solution, preferably in a physiologically compatible buffer, such as Hank's solution, Ringer's solution, physiological saline buffer, or any combination thereof.

[0130] In certain embodiments, pharmaceutical compositions as described herein can be prepared in the form of nanoparticles.Nanoparticles can have a monolayer surrounding a nanoparticle core, wherein the siRNA molecule is placed in the nanoparticle core.In one embodiment, the nanoparticle core includes solid lipids (i.e., lipids that maintain a solid state at room temperature and body temperature) or liquid lipids (i.e., oils that maintain a liquid state at room temperature and body temperature, such as vegetable oils or lipids extracted from human adipose tissue).Particularly, embodiments of the present disclosure include nanoparticles and compositions for controlling and / or sustained release (e.g., releasing at a predetermined rate, maintaining a certain concentration within a certain time) reagents (e.g., small interfering RNA (siRNA) from nanoparticles).

[0131] In certain embodiments, the pharmaceutical compositions described herein may be formulated for parenteral administration, for example, by bolus injection or continuous infusion. The injection formulations herein may be presented in unit dose form, such as in ampoules or multi-dose containers, with the optional addition of preservatives. In some embodiments, the compositions herein may be suspensions, solutions or emulsions in oily or aqueous carriers, and / or may contain formulation agents such as suspending agents, stabilizers and / or dispersants.

[0132] In certain embodiments, the pharmaceutical composition formulated for parenteral administration herein may include an aqueous solution of a water-soluble active agent (such as an siRNA molecule). In some embodiments, the composition comprising an active agent suspension may be prepared into an oily or aqueous injection suspension. Suitable lipophilic solvents and / or carriers used in the text may 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, the composition comprising an aqueous injection suspension herein may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, and / or dextran. In certain embodiments, the composition comprising a suspension may also contain one or more suitable stabilizers and / or agents that increase the solubility of the active ingredient (such as an siRNA molecule) to prepare a preparation of a high concentration solution.

[0133] In some embodiments, the compositions herein may contain the active ingredient in powder form for constitution with a suitable vehicle (eg, sterile, pyrogen-free, water-based solution) before use.

[0134] Pharmaceutical compositions suitable for the present disclosure may include compositions that may contain an amount of active ingredients effective to achieve the intended purpose. In some embodiments, a therapeutically effective amount refers to an amount of active ingredient (such as siRNA molecules) that effectively prevents, slows down, alleviates or improves symptoms of a disorder (such as liver disease) or prolongs the survival of a subject being treated.

[0135] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0136] For any formulation used in the disclosed methods, a therapeutically effective amount or dosage can be initially estimated based on the in vitro and cell culture assays and / or screening platforms disclosed herein. For example, a dosage can be formulated in an animal model to achieve a desired concentration or titer. This information can be used to more accurately determine an effective dosage in humans.

[0137] In some embodiments, the toxicity and therapeutic effect of the active ingredients disclosed herein can be determined in vitro, in cell culture or in experimental animals by standard pharmaceutical procedures. In some embodiments, the data obtained from these in vitro and cell culture assays and animal studies can be used to formulate a dosage range for human subjects. In some embodiments, the dosage used in this regimen may vary depending on the dosage form and route of administration used. The specific dosage form, route of administration and dosage can be selected by the doctor according to the patient's condition. (See Fingl et al., 1975, "The Pharmacological Basis of Therapeutics", Chapter 1).

[0138] In certain embodiments, dosage and / or dosing interval can be adjusted individually to the brain or blood level of the active ingredient sufficient to induce or inhibit biological effects (minimum effective concentration, MEC). In some embodiments, the MEC of active ingredient (such as siRNA molecules or compositions disclosed herein) may vary due to each preparation, but can be estimated based on in vitro data. In some embodiments, the dosage required to reach MEC herein may depend on individual characteristics and route of administration. Detection analysis can be used to determine plasma concentration.

[0139] In certain embodiments, depending on the severity and responsiveness of the condition to be treated, the compositions herein may be administered single or multiple times, with courses of treatment lasting from several days to several weeks or until a cure or a reduction in the disease state is achieved.

[0140] In certain embodiments, the dosage of the composition of the present invention depends on the subject to be treated, the severity of the affliction, the mode of administration, the judgment of the prescribing physician, etc. In some embodiments, the effective dose can be extrapolated from a dose-response curve derived from an in vitro or in vivo test system.

[0141] III. How to use

[0142] The present disclosure provides methods for treating, alleviating and preventing liver disease in subjects in need thereof. In several embodiments, the method for treating, alleviating or preventing liver disease in a subject may include administering to a subject (including a human subject) an effective amount of one or more siRNA molecules targeting GPAM disclosed herein or a nucleic acid encoding a siRNA molecule targeting GPAM disclosed herein. In several embodiments, the method for treating, alleviating or preventing liver disease in a subject may include administering to a subject (including a human subject) an effective amount of a nucleic acid encoding a suitable sgRNA, or a suitable sgRNA targeting GPAM and an RNA-guided endonuclease.

[0143] Methods disclosed herein may include treating a subject in need by administering a therapeutically effective amount of one or more siRNA molecules or pharmaceutical compositions disclosed herein. Subjects may be human subjects suffering from or suspected of suffering from liver disease, liver damage, liver dysfunction or liver injury risk. The terms "liver disease", "liver damage" or "abnormal liver function" may be used interchangeably, referring to any injury of the liver, including but not limited to hardening of the liver, scarring of the liver, decreased or abnormal biliary function, abnormal liver enzyme activity, cirrhosis, physiological abnormalities 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 methods disclosed herein may include fatty liver (FLD), alcohol-related liver disease (ARLD), non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), end-stage liver disease (cirrhosis) caused by any cause, liver cancer HCC or any combination thereof. In some embodiments, compared with untreated subjects with the same disease status and predicted results, subjects benefit from the extension of life expectancy. In other embodiments, treatment improves liver function in the subject compared to an untreated subject with the same disease status and predicted outcome. In other embodiments, treatment reduces liver fibrosis in the subject compared to an untreated subject with the same disease status and predicted outcome. In some embodiments, treatment prevents the subject from developing additional liver fibrosis compared to an untreated subject with the same disease status and predicted outcome.

[0144] Subjects suitable for the treatment of liver diseases disclosed herein can be selected according to the diagnosis of the subject. In some embodiments, the diagnostic method can detect one or more serum markers indicating liver disease. Non-limiting examples of serum markers indicating liver disease (such as NAFLD or NASH) may include alpha-fetoprotein (AFP) (such as AFP levels of 20ng / mL or higher), de-γ-carboxy prothrombin, lentil agglutinin reactive AFP (AFP-L3), etc. The diagnostic method may also include assessing at least one clinical symptom associated with liver disease. Non-limiting examples of clinical symptoms associated with liver disease may include mild to moderate upper abdominal pain, weight loss, early satiety or palpable masses in the upper abdomen, paraneoplastic syndrome, hypoglycemia, polycythemia, hypercalcemia, intractable diarrhea and related electrolyte disorders (such as hyponatremia, hypokalemia, metabolic alkalosis), skin manifestations (such as dermatomyositis, pemphigus foliaceus, seborrheic keratosis, pityriasis circumflexus), intraperitoneal hemorrhage, jaundice, fever, suppurative liver abscess, etc. Other aspects of the diagnosis may include diagnosing and / or determining the severity of the following diseases: cirrhosis, 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, 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.

[0145] In some embodiments, the risk of a subject suffering from liver disease (such as NASH or NAFLD) can be diagnosed and / or predicted to be high or low by histological or imaging-based examinations, such as contrast-enhanced multiphase CT, ultrasound and / or MRI. Imaging features for diagnosis may include growth of liver size, dynamics, contrast-enhanced patterns, and continuous imaging, wherein size may be measured as the maximum cross-sectional diameter of the most clearly visible lesion on the image. NASH or NAFLD biopsy histology may include fatty degeneration, inflammation, and fibrosis.

[0146] The methods and compositions disclosed herein can be used to treat subjects with fatty liver-related disorders such as NAFLD and / or NASH. The subject's biliary function may be normal or substantially normal. Any suitable method known in the art can be used to determine whether a subject has normal or abnormal biliary function. In general, the preferred tests for biliary function in NASH patients can be divided into two categories: physiological-based tests and biochemical-based tests. Physiological tests include, but are not limited to, abdominal ultrasound, abdominal CT scan, endoscopic retrograde cholangiopancreatography (ECRP), percutaneous transhepatic cholangiography (PTCA), or magnetic resonance cholangiopancreatography (MRCP). Biochemical tests 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 urine bilirubin assays. In some cases, one or more tests may be used to characterize biliary function. In some cases, a combination of multiple tests may be used to assess biliary function in NASH subjects.

[0147] In some embodiments, any method disclosed herein may further include monitoring the occurrence of one or more adverse reactions of the subject. Adverse reactions may include, but are not limited to, liver damage, hematotoxicity, nervous system toxicity, skin toxicity, gastrointestinal toxicity, or a combination thereof. When one or more adverse reactions are observed, the method disclosed herein may further include reducing or increasing the dosage of one or more treatment regimens according to the adverse reactions of the subject. For example, when moderate to severe liver damage is observed in the subject after treatment, the concentration or frequency of the composition used to treat the subject may be reduced.

[0148] In certain embodiments, the treatment given 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 condition (such as NAFLD or NASH) and predicted results. "Patient life expectancy" used herein is defined as the time when 50% of the subjects survive and 50% of the subjects die. In some embodiments, after treatment according to the methods disclosed herein, the patient's life expectancy can be indefinite. In other aspects, compared to untreated subjects with the same disease condition and predicted results, the patient's life expectancy can increase 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. In some embodiments, the patient's life expectancy can 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, at least about 100% as compared to an untreated subject with the same disease condition and predicted outcome. In some embodiments, the patient's life expectancy can be 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 about 50% or more, at least about 51% or more to at least about 52% or more, at least about 53% or more to at least about 54% or more, at least about 55% or more to at least about 56% or more, at least about 57% or more to at least about 58% or more, at least about 59% or more to at least about 60% or more, at least about 61% or more to at least about 62% or more, at least about 63% or more to at least about 64% or more, at least about 65% or more to at least about 66% or more, at least about 67% or more to at least about 68% or more, at least about 69% or more to at least about 70% or more, at least about 71% or more to at least about 72% or more, at least about 73% or more to at least about 74% or more, at least about 75% or more to at least about 76% or more, at least about 77% or more to at least about 78% or more. More to at least about 50% or more, at least 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, at least about 95% or more to at least about 100%.

[0149] In some embodiments, treating a liver disease, such as NAFLD or NASH, according to the methods disclosed herein can reduce, shrink, decrease, or prevent liver fibrosis compared to the starting size of liver fibrosis. In some embodiments, the extent of reducing or shrinking the area of ​​liver fibrosis compared to the starting size of liver fibrosis can be from 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. More, at least 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, at least about 95% or more to at least about 100% (referring to complete disappearance of liver fibrosis after treatment).

[0150] In some embodiments, liver disease, such as NAFLD or NASH, can be treated according to the methods disclosed herein to improve liver function. Liver function can be detected by conventional biochemical methods. Biochemical tests may include, but are not limited to, GGT detection, liver function tests, bilirubin detection, alkaline phosphatase (ALP) detection, liver enzyme detection, amylase blood test, lipase blood test, prothrombin time and urine bilirubin determination. In some cases, one or more tests can be used to characterize biliary function. In some cases, a variety of test combinations can be used to assess biliary function. Improvement in liver function can also be assessed by relief of symptoms in the subject, such as relief of symptoms such as fatigue, weight loss and weakness in the subject. Other relieved symptoms may include reduced fluid retention, muscle atrophy, intestinal bleeding, and any combination thereof.

[0151] IV. Kit

[0152] The present disclosure provides a kit for using the method disclosed herein. In some aspects, the present disclosure provides a kit for treating liver diseases disclosed herein (such as NASH or NAFLD) and diagnosing liver diseases. Such a kit may include a tool for accommodating and / or administering such siRNA compositions or pharmaceutical compositions.

[0153] In some embodiments, the kits disclosed herein may have a medical container to contain the composition in a safe, stable and durable manner. In some embodiments, the kits disclosed herein may also include a tool for applying the composition, such as a needle or a spatula.

[0154] Any kit may further comprise instructions for how to use the kit for treatment. The manual may be written with physicians or liver specialists as the target audience.

[0155] Having described several embodiments, those skilled in the art will recognize that various modifications, alternative structures and equivalents may be used without departing from the spirit of the inventive concept. In addition, in order to avoid unnecessarily obscuring the inventive concept, some well-known processes and elements are not described. Therefore, this description should not be regarded as limiting the scope of the inventive concept.

[0156] Those skilled in the art will appreciate that the presently disclosed embodiments are taught by way of example and not by way of limitation. Therefore, the contents contained in this description or shown in the accompanying drawings should be interpreted as illustrative and not restrictive. The following claims are intended to cover all generic and specific features described herein, as well as all statements of the scope of methods and components that can be said to fall therein in a language. Example

[0157] The following examples are intended to illustrate preferred embodiments of the present disclosure. It will be appreciated by those skilled in the art that the techniques disclosed in the following examples represent techniques that the inventors have discovered to work well in the practice of the present disclosure, and therefore can be considered to constitute preferred modes for the practice of the present disclosure. However, it will be appreciated by those skilled in the art that, based on the present disclosure, many changes may be made to the disclosed specific embodiments without departing from the spirit and scope of the present disclosure, and still the same or similar results may be obtained.

[0158] Example 1.

[0159] To broadly understand how mutations in candidate NASH genes affect clonal competition in vivo in an unbiased manner, a method was developed to generate mutant cell pools within Cas9-expressing mouse tissues. To generate a virus that integrates into the genome to enable in vivo screening, a hybrid adeno-associated virus (AAV) was created that carries the Sleeping Beauty transposase (SB100), Cre, a single guide (sg) RNA, and its payload is flanked by transposon integration sequences (SB-IR). This AAV was used to deliver sgRNAs into the liver of doxorubicin (dox)-inducible Cas9-expressing mice for in vivo CRISPR screening. Briefly, a hybrid screen was performed for 63 NASH genes identified in GWAS (i.e., APOB, TM6SF2, GCKR), exome sequencing (i.e., PNPLA3, MBOAT7, HSD17B13), somatic mutation sequencing (i.e., PPARGC1B, FOXO1, GPAM, ACVR2A), or biochemical studies. A pool of hepatocytes with mosaic deletions of these NASH genes was established after injection of the AAV to induce Cas9. After two weeks of Cas9 activity, doxorubicin was stopped so that no further cutting would occur. These chimeric mice were then given either normal chow (NC) or a Western diet (WD: high sugar, high fat, high cholesterol) to understand the clonal dynamics under both conditions. After 6 months, sgRNA deep sequencing identified the genes with the greatest impact on clonal fitness, specifically in the NASH setting. To rule out pure proliferative effects unrelated to NASH, sgRNAs that shared enrichment or depletion under NC and WD conditions were excluded. Six genes, when deleted, were most associated with clonal expansion: Acvr2a, Irs1, Srebp1, Gpam, Dgat2, and Pparg ( Figure 1A-Figure 1D ).

[0160] Glycerol-3-phosphate acyltransferases (GPATs), including GPAT1 and GPAM (glycerol-3-phosphate acyltransferase, mitochondrial), catalyze the initial and key steps in glyceride biosynthesis and are thought to play a key role in the regulation of cellular triacylglycerol and phospholipid levels. Notably, GPAM mutant clones are more competitive and play a beneficial role in NAFLD / NASH. For example, in NASH livers, recurrent and convergent loss-of-function mutations in genes that promote lipogenesis were found (Ng et al., Nature, 2021). Abnormal accumulation of lipid droplets in hepatocytes ultimately leads to lipotoxicity, cell death, and cirrhosis. Observations of loss-of-function mutations in metabolic enzymes that generate hepatic lipids, such as GPAM, suggest that some somatic mutations can improve fitness by reversing the driving etiology of the disease.

[0161] Top results from the in vivo NASH mouse screen were analyzed in chimeric liver disease models. To study GPAM mutations in the context of liver-specific loss or clonal mosaicism, a CRISPR approach was used to generate systemic GPAM knockout mice and GPAM floxed mice targeting exon 3, which was predicted to result in a frameshift and stop codon causing premature termination. GPAMfloxed mice were confirmed to achieve GPAM loss following Cre recombination. To investigate how chimeric GPAM wild-type and knockout clones compete in the liver under normal and NAFLD conditions, low-dose AAV-Cre was administered to induce 10-20% hepatocyte loss, and chimeric mice were then placed on normal chow or WD conditions. This resulted in increased survival of GPAM knockout clones and reduced NASH severity after 6 months ( Figure 2A-2B ). The data mirror human genetic data, indicating that GPAM mutant clones are selected in human livers with NASH.

[0162] To further identify which genes increase clonal expansion by affecting metabolic fitness, liver-specific GPAM knockout mice were used to assess the extent to which ideal siRNA knockdown would affect liver and systemic metabolism in the context of NASH. Body weight, liver weight, histology, steatosis, fibrosis, serum assays (liver function tests, cholesterol, TG, non-esterified free fatty acids (NEFA)) were measured for each conditional CRISPR KO model. After 3 months of NASH diet, liver-specific GPAM knockout mice were protected from the effects of hepatic triglyceride accumulation.

[0163] Example 2.

[0164] To address whether mouse siRNAs targeting GPAM could prevent and / or reverse NASH in mouse models, a siRNA tool compound for mouse was used to investigate translational aspects of GPAM biology. Eight siRNAs were tested against mouse GPAM in Cos7 cells, and the siRNA sequence with the highest knockdown efficiency was determined based on a mouse GPAM reporter assay. The siRNA was then modified and conjugated with GalNAc. Briefly, to minimize nucleolytic degradation and immune response in vivo, each 2'-position was modified with a 2'-O-methyl group. To determine whether these stabilizing modifications affected silencing activity, the in vitro activity of the siRNAs was determined prior to GalNAc conjugation. GalNAc conjugation allowed for efficient delivery to hepatocytes without lipid nanoparticle packaging, according to industry standard methods. The optimal in vivo dosing regimen for GalNAc-siGPAM was determined. Starting at 4 weeks of age, WTB6 mice were injected subcutaneously (SC) with three doses of siRNA (1, 5, 10, 15 mg / kg) in PBS every two weeks. After siRNA treatment, mice were sacrificed and subjected to GPAM qPCR, Western blot and histological examination (n=5 mice / group). The maximum tolerated dose was determined based on the absence of severe toxicity (such as weight loss of more than 10%, organ (heart / kidney / liver) failure and death). In all the following siRNA experiments, GalNAc-siLuc was used as a control.

[0165] To determine whether GalNAc-siGPAM can prevent NASH, the optimized dose defined above was used. It was then determined whether GalNAc-siRNA could mimic the GPAM KO model. In parallel with the genetic experiments, GalNAc-siRNA SC injections began at 6 weeks of age and the NASH diet began at 8 weeks of age for 12 weeks (a total of 6 siRNA doses). Mice were euthanized at 20 weeks of age, a time point at which steatosis, inflammation, and fibrosis were detectable. Without being bound by theory, the siRNA approach mimicked the genetic NASH prevention model.

[0166] To determine whether GalNAc-siGPAM could reverse NASH, siRNA administration was initiated in mice that had been on a WD diet for 24 weeks (30 weeks of age), and WD and siRNA were continued for a total of 12 weeks. The liver was then evaluated for NASH pathological features. Assessments included hepatocyte ballooning, inflammation, and fibrosis, which, as mentioned above, are hallmarks of NASH. These siRNA experiments have tremendous translational value and pave the way for clinical treatment.

[0167] Example 3.

[0168] Specific siRNA sequences targeting human GPAM were designed for the treatment of NASH. Based on human somatic sequencing data that have identified recurrent loss-of-function GPAM mutations in NASH livers, screening of genetic screens according to the exemplary methods described herein, and the biological properties of GPAM in mouse models of NASH and insulin resistance, GPAM represents a promising therapeutic target for human NASH.

[0169] Optimized siRNA sequences corresponding to the human sequence of human GPAM were identified. This required screening many candidate siRNAs for each gene target using an in vitro luciferase reporter-based assay. Briefly, the full-length GPAM cDNA (without the 3'UTR) was cloned into the psicheck2 plasmid backbone containing the luciferase gene. This generated 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. Therefore, the ratio of Renilla and Firefly signals can reflect changes in transfection efficiency and cell viability. The assay was performed in 96-well plates with technical replicates at 48 hours as the assay readout. The reporter gene and siRNA were co-transfected into Cos7 monkey kidney cells using lipofectamine. After 48 hours, the cells were lysed and the signals were captured using a luminometer with firefly and Renilla substrates. Effective siRNAs against the target gene showed a reduction in the ratio of Renilla and Firefly signals compared to non-targeted controls. A total of 39 siRNAs targeting the entire GPAMORF were designed and tested ( Figure 3A-3B At least 21 effective siRNAs against human GPAM were identified (Table 3). The optimized siRNAs were modified by conjugation with acetylgalactosamine (GalNAC) in a standard manner and tested in vivo.

[0170] Table 3

[0171]

[0172]

[0173] Example 4.

[0174] Some human siRNAs for GPAM disclosed herein were tested in a human cell line model of NASH. The test used optimized siRNA and / or its conjugate with acetylgalactosamine (GalNAc) to improve liver targeting. The experimental design is divided into three steps. First, the target gene of interest, i.e., GPAM, is knocked down using siRNAs that have been determined in human liver cancer cells (such as Huh7 or HepG2). Specifically, Huh7 cells are grown to 60-80% confluence and then transfected with siRNA. Opti-MEM (Thermo Fisher 31985062) containing siRNAs and liposomes (Lipofectamine) are mixed and incubated, and then cells are added for transfection (to 6-well plates, 400 microliters / well; or to 100 mm plates, 2.4 ml). After 6 hours, the transfection medium is replaced with medium containing 100 μM FA. Then, cells are fed lipids and labeled with lipid dyes. Finally, fluorescence activated cell sorting (FACS) is performed to quantify lipid accumulation. FACS purifies specific cell populations based on phenotypes detected by flow cytometry. This method allows the characterization of a single cell population without the influence of other cells. Cells were stained with 10 μg BODIPY493 / 503 (BD) (Thermo Fisher D3922) in 1 ml PBS for 15 minutes at room temperature, and then FACs were measured. Early studies have shown that in cells stained with lipophilic fluorophores, the fluorescence intensity measured by FC reflects lipid levels (Wolins, 2018).

[0175] Aliquots of cells grown under each condition were assayed for triglycerides (TG). Briefly, cells were trypsinized, washed once with PBS, resuspended in PBS containing 10 mM EDTA, and counted. Cells were loaded into 13 × 100 mm glass tubes in triplicate in a final volume of 200 μl. Triolein standards (Sigma T7140) were prepared in parallel in a final volume of 200 μl PBS / 10 mM EDTA and loaded into 13 × 100 mm glass tubes. TGs were extracted and quantified. Briefly, 2 ml of isopropanol: hexane: water (40:10:1) was added to cells or standards, and the samples were then vortexed, covered, and incubated at room temperature for 30 min. Then, 500 μl of a 1:1 mixture of hexane: ether was added to the samples, which were then vortexed and incubated at room temperature for 10 min. Then, add 1 ml of water to the sample, vortex the tube, and keep it capped for 30-45 min at room temperature to allow the layers to separate. Use a Pasteur pipette to transfer the top layer to a 12 × 75 mm glass tube and incubate at N 2Dry until complete at 400 μl. After the drying step, add 400 μl Infinity triglyceride reagent (Thermo Scientific TR22421) to each test tube and vortex. Cover the test tube, incubate at 37°C for 90 minutes, and shake at 250 rpm. Finally, transfer 300 μl of each sample to a 96-well plate and measure the absorbance at a wavelength of 540 nm using a microplate reader. To determine whether fixed cells affect the measurement of TG, TG quantification was performed on fixed and unfixed cells from the same batch of Huh7 cells cultured in 2 m MFA.

[0176] Example 5.

[0177] Mutations in Gpam were investigated further because it was a hit in the functional NASH screen and human cell mutation sequencing efforts described above. GPAM catalyzes the acylation of glycerol-3-phosphate with acyl-CoA (CoA) to produce CoA and lysophosphatidic acid (LPA), which is the rate-limiting step in the synthesis of triacylglycerols. Since conditional KO mice required to study clonal dynamics were not available, GPAM floxed mice targeting exon 3 (whose deletion is expected to result in a frameshift and premature stop codon) were generated as provided in Example 2. One week after injection, saturating doses of AAV8-TBG-Cre resulted in deletion of exon 3 and almost complete depletion of GPAM mRNA ( Figure 4A and Figure 4B Using these floxed mice, WT and KO clones were compared under normal and WD conditions. An increase in the abundance of GPAM KO clones was observed under a Western diet (WD) environment ( Figure 4C and Figure 4D ). These data demonstrate diet- and steatosis-dependent beneficial effects of GPAM loss-of-function mutations.

[0178] To determine why GPAM mutant clones were more fit, whole livers of GPAM KO mice were induced with AAV8-TBG-Cre. Modest differences in body and liver weights were observed after 3 months of WD ( Figure 4E and Figure 4F , Table 4), but KO mice showed a decreased trend in liver / body weight ratio ( Figure 4G ), decreased fatty degeneration ( Figure 4H ), the trend of improvement of transaminase ( Fig. 4I and Figure 4J ).

[0179] Table 4

[0180]

[0181]

[0182]

[0183]

[0184]

[0185] Steatosis and liver damage were quantified using a NAFLD activity scoring system tailored for rodent histology. Results showed that macrovesicular steatosis, microvesicular steatosis, and hepatocyte hypertrophy were reduced in KO livers ( Figure 4K In the KO group fed WD, hepatic triglyceride levels decreased, but cholesterol levels did not decrease ( Figure 4L and Figure 4M ), which is consistent with the role of GPAM as the rate-limiting enzyme in triglyceride synthesis. Interestingly, plasma cholesterol was reduced in the KO group, but triglycerides were not reduced ( Figure 4N and Fig.4O Together, these data suggest that GPAM loss-of-function mutations lead to clonal expansion in NASH and that GPAM is a promising therapeutic target in NASH.

[0186] Experimental methods and experimental subjects used in the examples

[0187] Mouse strains and breeding

[0188] All mice were handled in accordance with the guidelines of the University of Texas Southwestern Medical Center Institutional Animal Care and Use Committee. All experiments were performed in an age- and sex-controlled manner unless otherwise stated. All mice used in this study were male, except for the mice in Figure S8F-J. All experiments used C57BL / 6 background mice. tm1a(KOMP)Wtsi / H mice are from KOMP, originally produced by the Wellcome Trust Sanger Institute. Gpam f / fMice were generated at the CRI Mouse Genome Engineering Core at the University of Texas Southwestern Medical Center. 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 fertilized eggs, 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. Homozygous mice for Rosa-rtTA and TetO-Cas9 were used to ensure high expression levels of Cas9 in the liver. The Western diet (WD) used for the NAFLD / NASH model was described in 45. It consists of high-fat solid food (ENVIGO#TD.120528) and high-sugar water containing 23.1 g / L of d-fructose (Sigma-Aldrich#F0127) and 18.9 g / L of d-glucose (Sigma-Aldrich#G8270).

[0189] Lineage tracing of floxed mice

[0190] For whole-liver deletion of Mboat7 or Gpam, 5*10 of the commercially produced AAV8-TBG-Cre (Addgene#107787) or control AAV8-TBG-GFP (Addgene#105535) were injected into the liver of 8-week-old mice. 10 Genome copies (in 100 μl saline) were retrogradely injected into Mboat7 f / f or Gpam f / f In vivo mice. For chimeric deletion of Mboat7 or Gpam and Tomato labeling of hepatocytes, 0.125*10 of AAV8-TBG-Cre in 100 μl of saline were injected into 8-week-old mice. 10 Post-orbital injection of genomic copies into Mboat7 f / f ;LSL-tdTomato het, Gpam f / f ;LSL-tdTomato het or control LSL-tdTomato het mice. One week after low-dose AAV8-TBG-Cre injection, 7 mice from each group were collected to determine the initial Tomato labeling percentage in the liver. The remaining mice were divided into feed or WD groups and followed for another 4 or 6 months.

[0191] Fluorescence imaging and image processing

[0192] For fluorescent imaging, the liver was fixed in buffered formalin (Fisherbrand #245-685) for 24 hours and gently shaken at 4°C, then transferred to a 30% sucrose (w / v) solution and shaken for another 24 hours at 4°C. The liver was then embedded and frozen in cryogel (Cryo-Gel) (Leica #39475237) with a slice thickness of 16 μm. Images were taken using the Zeiss Axionscan Z1 system in the UTSW Whole Brain Microscope Facility to observe Tomato clones. For statistical analysis of the percentage of "Tomato+" cells, black and white fluorescent images were taken on the same slide at 4x magnification using an Olympus IX83 microscope. Two different areas were taken from each liver. The percentage of Tomato+ cells (bright areas) was analyzed using ImageJ.

[0193] H&E, immunohistochemistry (IHC), immunofluorescence (IF), TUNEL and Sirius red staining

[0194] Liver sections were fixed in buffered formalin (Fisherbrand #245-685) for 24 h at 4°C with gentle shaking and then transferred to 70% EtOH for an additional 24 h at 4°C with shaking. Paraffin embedding, liver sectioning (4 μm thick) and H&E staining were performed at the University of Texas Southwestern Medical Center Tissue Management Shared Resource Core. IHC was performed as described previously. Briefly, paraffin-embedded sections were dewaxed in xylene and hydrated with an ethanol gradient. Slides were then boiled in antigen retrieval buffer (10 mM sodium citrate, 0.05% Tween 20, pH 6.0) for 20 min and soaked in 3% hydrogen peroxide (methanol) for 10 min. After blocking with 5% goat serum for 1 h at room temperature, slides were incubated with primary antibodies overnight at 4°C. After washing, slides were incubated with secondary antibodies for 0.5 h at room temperature. The secondary antibody was conjugated to HRP using the ABC-HRP kit (Vector Laboratories, #PK-6101). The slides were developed using the DAB kit (Vector laboratories, #SK-4100). The following primary antibodies were used for IF staining: RFP (Rockland #600-401-379, IF1:500); Ki67 (Invitrogen #14-5698-82, IF1:500); HNF4a (Abcam #ab41898, IF1:500): goat anti-rat IgG (H&L) Alexa Fluor Plus 488 (Invitrogen #A-48262, IF1:500); donkey anti-rabbit IgG (H&L) Alexa Fluor 594 (Invitrogen #A-21207, IF1:500); goat anti-mouse IgG2a Alexa Fluor 647 (Invitrogen #A-21241, IF1:500). IF was performed on paraffin-embedded mouse liver sections using the same protocol as IHC, except that Alexa Fluor-conjugated antibodies were used instead of secondary antibodies. Paraffin-embedded liver sections were stained for TUNEL using the In Situ Cell Death Detection Kit, Fluorescein (Roche #C755B40) following the manufacturer's protocol. Paraffin-embedded liver sections were stained for Sirius Red using the PicroSirius Red staining kit (Abcam #ab150681) following the manufacturer's protocol. QuPath software was used to quantify TUNEL staining and IHC staining for Ki67. Sirius Red staining was quantified using ImageJ.

[0195] Plasma and liver metabolic assays

[0196] After killing mice, heparinized tubes were used to collect fluid from the inferior vena cava immediately, and then transferred to 1.5ml tubes, and centrifuged at 4 ° C for 15 minutes at 2000g. The supernatant (plasma) after centrifugation was analyzed by fully automatic OCD Vitros 350 dry chemical analyzer at the metabolic typing core of the University of Texas Southwestern Medical Center, according to the scheme provided by the kit manufacturer (Ortho Clinical Diagnostics, Raritan, NJ) for AST, ALT, cholesterol and triglyceride (the manufacturer's reference number is 8433815, 1655281, 1669829 and 1336544, respectively) of the supernatant (plasma) after centrifugation. At the metabolic phenotype core of the University of Texas Southwestern Medical Center, the liver 100-150mg of each mouse was weighed for lipid extraction and quantification. In brief, multiple automatic tissue disruptors (TissueLYser II, Qiagen, Germantown, MD) were used to homogenize the quick-frozen tissue samples with a chloroform-methanol mixture (v / v) of 2:1. The organic extract was transferred to a 5 ml graduated vial to a total volume of 5 ml. 100 μl and 25 ml of lipid extract were used for triplicate total cholesterol and triglyceride analyses, respectively. The concentrations of total cholesterol and triglyceride were determined by a commercial enzymatic colorimetric method following the protocol described by the manufacturer (INFINITY TM Cholesterol Liquid Stable Reagent #TR13421; INFINITY TM Triglycerides Liquid Stable Reagent #TR22421; Matrixplus TM Chemistry Reference Kit (Chemistry Reference Kit #NC9592194).

[0197] Chimera reagent construction

[0198] The chimeric plasmid was based on the pX602 plasmid. The sequences between the two AAV ITRs were removed using NsiI and NotI restriction enzymes. The following fragments were cloned between the two AAV ITRs: the first SB100 that binds to the IR, the U6-driven sgRNA scaffold, the CAG promoter-driven SB100-P2A-Cre fusion cDNA (with a β-globin poly(A) signal), and the second SB100 that binds to the IR. For the construction of the in-vivo library, all mouse candidate genes for in vivo screening were generated using mouse homologs of human genes. Some genes were not included in the mouse gene list due to the lack of homologous genes or belonging to known tumor suppressor genes. The individual sgRNA sequences corresponding to the mouse candidate genes were extracted from the Brie library or obtained from the GUIDES server and synthesized by CustomArray. Most genes had 5 different sgRNAs, 4 of which were from Brie and 1 from GUIDES. Some genes had 4 targeting sgRNAs due to the overlap of sgRNA sequences between Brie and GUIDES. The sgRNA sequences of GPAM are shown in Table 5. The library construction protocol was the same as the established protocol in the field. In brief, the synthetic oligonucleotide library was amplified by PCR, purified with a PCR purification kit (Qiagen, #28104), and assembled into a BsaI-digested MOSAICS vector using a DNA assembly kit (NEB, #E5520A). 1 μl of the assembled vector was then electroporated into 25 μl competent cells (Lucigen, #60052-2). After 1 hour of recovery in SOC medium, the bacteria were plated on 245*245 mm LB agar plates and cultured overnight at 37°C. The bacteria were then harvested using the HiSpeed ​​Plasmid Max i kit (Qiagen, #12663) to prepare plasmids. During the construction process, each sgRNA maintained a representation greater than 1000 times. For cloning of a single sgRNA, the forward and reverse primers were annealed and fused to the BsaI-digested MOSAICS plasmid using T4 ligase. Primers associated with sgRNA are shown in Table 5.

[0199] Table 4

[0200]

[0201] AAV production and purification

[0202] AAV8 was cultured using AAV-Pro 293T cells (Takara #632273) in one or more 15 cm dishes. The cells were plated at 50% confluency the day before transfection so that the confluency of the cells could reach 80-90% the next day. When transfecting a 15 cm dish, 10 μg of chimeric vector, 10 μg of pAAV2 / 8 (Addgene #112864) and 20 μg of pAdDeltaF6 (Addgene #112867) plasmids were mixed with 1 ml of Opti-MEM medium in one test tube. In another test 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 the two test tubes were then mixed and incubated for 10 minutes before being added to the cell culture. 48 hours after transfection, cells were scraped from the culture dishes and collected by centrifugation at 500g for 10 minutes. The supernatant was discarded after disinfection, and the cell pellet was lysed in 1.5ml / 15cm culture dish lysis buffer (PBS, supplemented with NaCl powder to a final concentration of 200mM, and CHAPS powder to a final concentration of 0.5% (w / v)). The cell suspension was placed on ice for 10 minutes, vortexed intermittently, and then centrifuged at 20,000g for 10 minutes at 4°C. The supernatant containing AAV was collected. To set up a gravity column for purifying AAV, 0.5ml of AAV8-binding slurry magnetic beads (ThermoFisher #A30789) was loaded into an empty column (Bio-Rad #731-1550), which was sufficient to purify AAV in three 15cm culture dishes. After the magnetic beads were tightly filled at the bottom, they were washed with 5ml of wash buffer (PBS, supplemented with NaCl powder to a final concentration of 500mM). The supernatant containing AAV is then loaded onto the column. After all supernatants flow through, the magnetic beads are washed twice with 10ml washing buffer. The AAV is then eluted with 3ml elution buffer (100mM glycine, 500mM NaCl aqueous solution, pH 2.5), and the eluate is immediately neutralized with 0.12ml of 1M Tris-HCl (pH 7.5-8.0). AAV is concentrated by centrifugation at 2000g for 3-5 minutes at 4°C using a 100k Amicon ultracentrifugal filter unit (Millipore#UFC810024). After centrifugation, the volume of AAV should be equal to or less than 0.5ml. The concentrated AAV is diluted with 4-5ml AAV dialysis buffer (PBS, and supplemented with powder to a final concentration of 212mM NaCl and 5% sorbitol (w / v)), then centrifuged at 2000g for 3-5 minutes at 4°C. The dilution and centrifugation process is repeated 3 times. The final concentrated AAV was transferred to a 1.5 ml tube and centrifuged at 20,000 g for 5 min to remove debris.The supernatant was aliquoted, snap frozen in liquid nitrogen, and stored at -80°C.

[0203] Genomic DNA extraction, sgRNA amplification, and amplicon library construction

[0204] To extract genomic DNA containing the integrated sgRNA, the entire liver (except for a small piece for sectioning and H&E staining) was cut into approximately 1 mm pieces using a razor blade. 3of the liver and weighed them. Small nodules observed in some epigenetic factor screening livers given WD (5 of 8 livers had ≤3 nodules) were excluded from samples processed for genomic DNA extraction. The minced liver in two volumes (w / v) of homogenization buffer (100mM NaCl, 25mM EDTA, 0.5% SDS, 10mM Tris-HCl, pH8) was transferred to a glass Wheaton Dounceue tissue grinder and stirred 50 times or until no lumps of tissue were visible. After homogenization, 200μl of liver lysate from the diet or 300pl of liver lysate from the WD was transferred to a 15ml test tube and genomic DNA was extracted using the Blood and 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. In brief, 10 ml of kit buffer G2, 100 pl proteinase K (Roche # 03115828001, or Qiagen kit proteinase K) and 100 pl ribonuclease A (Invitrogen # 12091-021) were added to a 15 ml test tube containing lysate and digested overnight in a 50 ° C water bath. The next day, the test tube was centrifuged at 4000 g for 10 minutes and the upper lipid layer was discarded. The remaining supernatant was loaded onto the column, washed, and then genomic DNA was eluted / precipitated according to the manufacturer's protocol. The precipitated DNA was resuspended in 100 pl 10 m M Tris (pH 8.0) and shaken on a shaking table at 55 ° C for 2 hours to help it dissolve. To prepare the amplicon library, 5pg genomic DNA, 5pl universal forward primer mix (5pM), 5pl barcode-specific reverse primer (5pM), 1pl Q5 DNA polymerase, 10pl Q5 buffer, 10pl high GC buffer, 1pl dNTP and water were mixed and 50pl PCR reactions were performed, with two reactions for each genomic DNA sample. The PCR cycle was 95°C 3min-(95°C 30s-60°C 30s-72°C 20s)*n-72°C 2min. The number of PCR cycles was pre-optimized using the same PCR reaction in a smaller volume. The number of cycles that produced faint but clear bands on the DNA gel was used. In the final PCR reaction, 23 cycles were used to prepare the NASH gene, transcription factor and epigenetic factor screening, and 30 cycles were used to prepare the guide mini-pool validation screening. After PCR, the two tubes containing the same genomic DNA template were combined (total 100pl) and 70pl were resolved on the DNA gel. The 250 bp band corresponding to the amplicon was excised and purified using the QIAquick Gel Extraction Kit (Qiagen #28704).DNA concentration was determined using the Qubit kit (Invitrogen #Q32853) and high-throughput sequencing was performed using the Illumina NextSeq500 system at the CRI, UT Southwestern Medical Center Sequencing Facility.

[0205] Bioinformatics analysis of chimera screening results

[0206] Reads from the above amplicon library sequencing were trimmed using cutadapt (version 1.9.1) to remove excess adapter sequences, thereby retaining only sgRNA sequences. 5' sequences were trimmed with options -O 32-discard-untrimmed-gCTTTATATCTTGTGGAAAGGACGAAACACCG. 3' sequences were trimmed with options -O 12-aGTTTTAGAGCTAGAAATAGCA. The abundance of each sgRNA was calculated using the count function (default options) in MAGeCK (version 0.5.6). The trimmed fastq files were assigned to feed-fed and WD-fed groups and uploaded to a server pre-installed with MAGeCK together with the library files containing the sgRNA sequences and target gene names. The enrichment of each sgRNA was calculated using the test function in MAGeCK.

[0207] Transcriptome analysis of human NASH liver

[0208] Dataset 1 (NAFLD / NASH cohort): RNA sequencing transcriptome profiles of biopsied liver tissue were downloaded (GEO: GSE130970) and analyzed for 72 NAFLD / NASH patients with a range of disease severity (NASH activity score 1 to 6) and associated histological features, namely steatosis, inflammation, fibrosis, and hepatocellular ballooning. Raw sequence reads were aligned to the GENCODE human reference genome (GRCh37, p13) using the STAR aligner (version 2.6.1b), and gene-level count data were generated by the Subread package (version 1.6.1 ) Feature counting function and GENCODE genome annotation (GRCh37, v19 ) Generated. Count data were normalized using 'Relative Log Expression' normalization (RLE) implemented in the DESeq2 software package Dataset 2 (HCV Cirrhosis Cohort): Microarray gene expression profiles of formalin-fixed needle biopsy specimens of the liver from 216 patients with hepatitis C-related early (Child-Pugh grade A) cirrhosis (GEO: GSE156540) were analyzed. The cohort was prospectively followed up for a median of 10 years at a single Italian center, and relevant time-to-event outcomes including childhood, death, HCC, and decomposition were collected. Both male and female patients were included in these studies.

[0209] Quantitative and statistical analysis

[0210] Data in most figures reflect multiple experiments performed on different days using mice from different litters. Differences in all figures are expressed as mean ± SD with standard deviation. Two-tailed unpaired Student's t-test was used to test the significance of differences between groups. Unless otherwise stated, statistical significance is indicated as ns (not significant, or p>=0.05), * (p<0.05), ** (p<0.01), *** (p<0.001), **** (p<0.0001). Quantitative image analysis was blinded.

Claims

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

2. The composition of claim 1, wherein the nucleic acid is siRNA, a clustered regularly interspaced short palindromic repeats (CRISPR)-related 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 the nucleic acid encodes a siRNA molecule.

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

5. The composition according to claim 3 or 4, wherein the siRNA molecule comprises a nucleotide sequence of 2 to 30 nucleotides in length and at least 80% homologous to at least 2 to 30 consecutive nucleotides in the human GPAM cDNA sequence. The composition according to claim 5 , wherein the human GPAM cDNA sequence is SEQ ID NO:

1.

7. The composition according to any one of claims 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 of any one of claims 3-7, 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 3-8, wherein the siRNA molecule comprises a nucleotide sequence in SEQ ID NO: 2-165, or any combination thereof.

10. The composition of claim 9, wherein the at least one sense sequence comprises SEQ ID NOs: 2-83.

11. The composition of claim 9, wherein the at least one antisense sequence comprises SEQ ID NOs: 84-165.

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

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

14. The composition of claim 12 or 13, wherein the sgRNA target sequence 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 according to any one of claims 3-11, wherein the siRNA molecule specifically downregulates gene expression of at least one variant of GPAM.

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

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

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

20. A composition according to any one of claims 16-19, wherein at least one variant of the GPAM comprises rs10787429C>T, rs7096937T>C, rs11446981T>TA, rs7898213T>C, rs2792759C>T, rs2297991T>C, rs1129555A>G, rs2254537T>A or any combination thereof.

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

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 one to about three GalNAc conjugates.

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

26. The composition of any one of claims 25, wherein the nucleic acid molecule comprises a modification of at least one ribose moiety of its nucleotide sequence.

27. The composition of claim 26, wherein the at least one ribose 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. The composition of claim 26 or 27, wherein less than about 10% to about 70% of the ribose moieties in the total nucleotide sequence are modified.

29. A pharmaceutical composition comprising the composition of any one of claims 1-28 and at least one pharmaceutically acceptable carrier.

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

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

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

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

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

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

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

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

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

39. The method of any one of claims 32-38, wherein administration of a therapeutically effective amount of the composition of any one of claims 1-28 or the pharmaceutical composition of any one of claims 29-31 prevents the subject from developing additional liver fibrosis compared to an untreated subject with the same disease condition and predicted outcome.

40. A kit comprising: (i) a container containing the composition according to any one of claims 1 to 28 or the pharmaceutical composition according to any one of claims 29 to 31; (ii) a drug administration device; and (iii) Instructions.

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