Inhibition of hepatic glycerol metabolism as a treatment for hypertriglyceridemia and fatty liver disease

Abstract

This disclosure provides methods for inhibiting glycerol kinase (GK) to treat or prevent a disease associated with elevated levels of serum triglycerides (TAGs) metabolic dysfunction- associated steatotic liver disease (MASLD). Also provided are novel nucleic acids capable of inhibiting GK.

Description

INHIBITION OF HEPATIC GLYCEROL METABOLISM AS A TREATMENT FOR HYPERTRIGLYCERIDEMIA AND FATTY LIVER DISEASECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 583,891, filed September 20, 2023, which is hereby incorporated by reference in its entirety.REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (SeqList-070439-01844.xml; Size: 128,848 bytes; and Date of Creation: September 9, 2024) is herein incorporated by reference in its entirety.FIELD OF THE INVENTION

[0003] The present invention relates to methods of preventing or treating conditions associated with high serum triglycerides (TAGs) using a glycerol kinase (GK) inhibitor.BACKGROUND OF THE INVENTION

[0004] Hypertriglyceridemia or high serum triglycerides (TAGs) is a common human disorder affecting more than 100 million people in the US, contributing to an increased incidence of acute myocardial infarctions, stroke, and peripheral vascular disease. TAGs are synthesized from smaller molecules in a process termed de novo lipogenesis. Quantitatively, most TAGs are synthesized in the liver and transported by serum lipoproteins to peripheral adipose tissues for chronic storage. Excess hepatic TAG synthesis (particularly from a high carbohydrate diet) can overwhelm the TAG transport system, resulting in excess pathogenic storage of TAG and TAG accumulation in the liver, termed non-alcoholic fatty liver disease (NAFLD) or metabolic dysfunction-associated steatotic liver disease (MASLD). Additionally, diabetes and obesity lead to TAG accumulation in the liver.

[0005] However, limited options are available to treat hypertriglyceridemia, and no FDA- approved therapy is available to treat MASLD. Consequently, there remains a significant need to develop effective therapies for hypertriglyceridemia and MASLD.SUMMARY OF THE INVENTION

[0006] In one aspect, this disclosure provides a glycerol kinase (GK) inhibitor. In another aspect, this disclosure provides a method of treating or preventing a disease associated with elevated levels of serum triglycerides (TAGs) and MASLD, wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a GK inhibitor. In some embodiments, the disease is hypertriglyceridemia or MASLD.

[0007] In some embodiments, the GK inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, or a nucleic acid. In some embodiments, the GK inhibitor comprises a nucleic acid.

[0008] In some embodiments, the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said nucleic acid comprises the nucleic acid sequence pairs as set forth by SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 9 and SEQ ID NO: 10

[0009] In some embodiments, the nucleic acid comprises a chemical modification. In some embodiments, the chemical modification comprises one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof conjugated to the nucleic acid, directly or indirectly via a linker. In some embodiments, the chemical modification is conjugated to the 3' end of the first strand.

[0010] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 7, 9, 10, and 11 from the 5' end of the first strand are modified with 2’-deoxy-2’-fluoro (2’F); b) the nucleotides at positions 1-6, 8, and 12-21 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); c) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2 ’-deoxy-2’ -fluoro (2’F); d) the nucleotides at positions 1, 3-5, 8-13, 15, and 17-23 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); ande) the nucleotides at position 7 is from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0011] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 1-4, 6, 10, 11, and 13-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5, 7-9, and 12 from the 5' end of the first strand are modified with 2 ’-deoxy-2’ -fluoro (2’F); c) the nucleotides at positions 1, 3, 4, 5, 9-13, 15-19, and 21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); d) the nucleotides at positions 2, 6, 14, and 20 from the 5' end of the second strand are modified with 2’-deoxy-2’-fluoro (2’F); e) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0012] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 1-4, 6, 10, and 11-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5 and 7-9 from the 5' end of the first strand are modified with 2’ -deoxy-2’ -fluoro (2’F); c) the nucleotides at positions 1, 3, 4, 5, 9-13, 15, and 17-21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); d) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’-deoxy-2’-fluoro (2’F); e) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0013] In some embodiments, the nucleic acid comprises at least one phosphorothioate internucleotide linkage. In some embodiments, the nucleic acid comprises two consecutive phosphorothioate intemucleotide linkages at both the 3' and 5' ends of the second strand and two consecutive phosphorothioate intemucleotide linkages at the 5' end of the first strand.

[0014] In some embodiments, the nucleic acid or pharmaceutical composition thereof is administered at a dose of about 100 mg to about 800 mg. In some embodiments, the nucleic acid or pharmaceutical composition thereof is administered subcutaneously. In some embodiments, administration of the nucleic acid or pharmaceutical composition thereof results in about a 70% decrease in serum triglyceride levels.

[0015] In some embodiments, the nucleic acid or pharmaceutical composition thereof is administered in combination with another therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group comprising fibrates, metformin, statins, and niacin.

[0016] In some embodiments, provided is a method for reducing an activity or an expression level of glycerol kinase (GK) in a subject in need thereof comprising administering to a subject in need thereof the nucleic acid or pharmaceutical composition thereof as disclosed herein.

[0017] In some embodiments, administration of the nucleic acid or pharmaceutical composition inhibits mTORCl (mTOR complex 1).

[0018] In some embodiments, provided is a method of reducing cholesterol levels in a subject in need thereof, wherein the method comprises administering the nucleic acid or pharmaceutical composition thereof as disclosed herein.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 is a schematic of the TAG Synthesis Pathway.

[0020] FIG. 2 illustrates Hematoxylin and Eosin (H&E) staining of liver sections of hepatic GK knockout (LGKKO) mice. RD indicates regular diet. HFD indicates high-fat diet.

[0021] FIG. 3 is a Western blot of lipogenic protein expression in LGKKO mice and wildtype (WT) mice.

[0022] FIG. 4 is a series of four graphs illustrating cellular metabolites that were13C isotope labeled in primary mouse hepatocytes after incubation of cultures in physiological fasting concentrations of glycerol, pyruvate / lactate, L-glutamine and glucose (Gro / PL / Gln / Glu). In each graph, only one of the substrates is13C labeled and shown with an asterisk. The exception is the pyruvate / lactate condition where both substrates are13C labeled due to rapidinterconversion. Error bars indicate SEM.13C isotopologues of cellular metabolites are indicated (m + 0 to m + 6) and color-coded n = 9 biological replicates. The major labeled species in each condition was m+3 unless otherwise denoted. DHAP, dihydroxyacetone phosphate; GAP, glyceraldehyde 3-phosphate; Gin, L-glutamine.

[0023] FIGS. 5 A and 5B illustrate the body weight and GK expression in the kidney of liverspecific GK knockout mice (LGKKO) and wild-type (WT) mice on RD (regular diet). FIG. 5A is a graph that shows the body weight of the mice. FIG. 5B is a graph that depicts GK expression in eWAT (epidydimal adipose tissue), small intestine (slnt), kidney, liver, pancreas, and muscle. RP17 (Ribosomal Protein L7) was used as reference control to standardize expression. ## indicates p < 0.01 from a t-test, comparing WT and LGKKO mice. Bars on the left represent WT mice and bars to the right represent LGKKO mice.

[0024] FIGS. 6A-6I demonstrate that hepatic glycerol kinase deficiency decreased glucose levels and decreased glucose fasting in LGKKO mice. FIG. 6A shows graphs of GK expression in the kidney and liver. # indicates p < 0.05 from a T-test comparing WT or LGKKO mice under RD and HFD conditions. FIG. 6B is a Western blot showing GK protein levels in WT and LGKKO mice. PPIB (peptidylpropyl isomerase B) served as a loading control. FIG. 6C is a graph of glucose levels in WT, LGKKO, and albumin-cre (alb- CRE) mice after intraperitoneal (IP) injection of glycerol at 9mM / kg (glycerol tolerance test). FIG. 6D shows body weight in WT and LGKKO mice fed a regular diet (RD) or a high-fat diet (HFD). FIG. 6E shows graphs of lean and fat mass in RD- and HFD-fed LGKKO and WT mice. ## indicates p < 0.01 from a t-test, comparing WT and LGKKO mice. FIG. 6F is a graph of glucose levels in WT and LGKKO mice fed a RD or HFD after a 12 hour fast. FIG. 6G shows graphs of glucose levels in RD- and HFD-fed LGKKO and WT mice. The Glycerol tolerance test (Glycerol TT) involved IP injection of glycerol at 9mM / kg and subsequent blood glucose measurements. The Pyruvate tolerance test (Pyruvate TT) involved IP injection of sodium pyruvate at 9mM / kg and subsequent blood glucose measurements. FIG. 6H shows graphs of circulating glycerol levels and non-esterified free fatty acid (NEFA) in WT and LGKKO mice fed a RD or HFD. FIG. 61 is a graph of fastinginsulin levels in RD- and HFD-fed LGKKO and WT mice. # indicates p < 0.05 from a t-test, comparing WT and LGKKO mice.

[0025] FIGS. 7A-7G demonstrate that hepatic glycerol kinase deficiency decreased triglyceride storage in the liver in LGKKO mice. FIG. 7A is a graph of the liver weight (g) of RD- and HFD-fed LGKKO and WT mice. # indicates p < 0.05 from a t-test, comparing WT and LGKKO mice. FIG. 7B shows graphs of the weight of muscle and eWAT of RD- and HFD- fed LGKKO and WT mice. FIG. 7C is a graph of the steatosis grade of RD- and HFD-fed LGKKO and WT mice. # indicates p < 0.05 from a t-test, comparing WT and LGKKO mice and ## indicates p < 0.01 from a t-test, comparing WT and LGKKO mice. FIG. 7D is a graph of the hepatic triglyceride levels of RD- and HFD-fed LGKKO and WT mice. FIG. 7E is a graph of serum alanine transaminase (ALT) activity levels of RD- and HFD-fed LGKKO and WT mice. FIG. 7F shows graphs of the levels of indicated de novo lipogenesis (DNL) proteins in RD- and HFD-fed LGKKO and WT mice. Srebflc = the gene encoding the sterol regulatory element (SRE) binding transcription factor 1 (SREBP1). Pparg = the gene encoding peroxisome proliferator-activated receptor gamma. Acly= the gene encoding ATP citrate lyase. Acaca= the gene encoding acetyl-coenzyme A carboxylase alpha. Fasn= the gene encoding fatty acid synthase. FIG. 7G shows graphs of ACLY (ATP Citrate Lyase), ACC (AcetyLCoA carboxylase), and FAS (fatty acid synthase) protein levels (from the previously mentioned genes) in RD- and HFD-fed LGKKO and WT mice. # indicates p < 0.05 from a t-test, comparing WT and LGKKO mice and ## indicates p < 0.01 from a t-test, comparing WT and LGKKO mice.

[0026] FIGS. 8A-8D demonstrate hepatitis GK enhances re-esterification of triglycerides in the liver in LGKKO mice. FIG. 8A is a schematic of hepatic triglyceride production. FIG. 8B shows graphs of CD36 and Slc27a5 (FATP5 gene) expression in RD- and HFD-fed LGKKO and WT mice. FIG. 8C shows graphs of Fabpl (fatty acid binding protein 1), Acsll (acyl- CoA synthetase long chain family member 1), and Acsl3 (acyl-CoA synthetase long chain family member 3) expression in RD- and HFD-fed LGKKO and WT mice. FIG. 8D shows graphs of Gpam (glycerol -3 -phosphate acyltransferase), Gpat4 (glycerol -3 -phosphate acyltransferase 4), Agpat2 (l-acylglycerol-3 -phosphate O-acyltransferase 2), Mogatl(monoacylglycerol O-acyltransferase 1), Dgatl (diacylglycerol O-acyltransferase 1), and Dgat2 (diacylglycerol O-acyltransferase 2) expression in RD- and HFD-fed LGKKO and WT mice.

[0027] FIGS. 9 A and 9B illustrate that in vitro administration of GalNAc-siRNAs against GK reproduced the LGKKO mouse phenotype. FIG. 9A is a graph of GK mRNA levels in WT primary mouse hepatocytes treated with either lOnM or 100 nM purified GalNAc-siRNAs against mouse GK (mGKl (SEQ ID NO: 1), and mGK2 (SEQ ID NO:3)). FIG. 9B is a graph of blood glucose levels after a glycerol tolerance test in WT mice injected with 3 mg / kg (SQ) of the indicated GalNAc-siRNAs against GK (mGKl (SEQ ID NO: 1), and mGK2 (SEQ ID NO:3)). Ctrl (control) is a non-specific siRNA. * P<0.04 compared to Ctrl.DETAILED DESCRIPTION OF THE INVENTION

[0028] This disclosure relates to an unexpected discovery that agents capable of inhibiting glycerol kinase (GK) are effective in treating or alleviating conditions associated with elevated triglyceride (TAG) levels. GK converts glycerol to glycerol -3 -phosphate (glycerol 3-P). The consensus view is that glycerol 3-P can be formed in a number of ways, and hepatic levels do not depend on an intact GK pathway.

[0029] Glycerol 3-P is the backbone for triglyceride (TAG) synthesis (FIG. 1). Elevated TAG levels have implications for many diseases or disorders, such as hypertriglyceridemia or MASLD. TAGs are composed of three long-chain fatty acids (acyl groups) in ester linkage with a glycerol backbone molecule (the process of attaching fatty acids to glycerol is called esterification). In this configuration, TAGs are non-polar and rapidly assemble as lipid droplets in adipocytes. During fasting, lipase acts on TAGs in peripheral adipocytes to release fatty acids and glycerol into the circulation. Glycerol that returns to the liver must first be activated by glycerol kinase (producing glycerol 3-P) to initiate esterification.

[0030] The GK inhibitor may be used in a subject in need thereof, for example, to treat or prevent a disease associated with elevated levels of serum triglycerides (TAGs) and MASLD. In some embodiments, the disease is hypertriglyceridemia or MASLD. In some embodiments, the GK inhibitor reduces an activity or an expression level of GK in a subject in need thereof. In further embodiments, administration of the GK inhibitor to a subject inneed thereof results in about a 70% decrease in serum triglyceride levels. For example, the GK inhibitor can decrease serum triglyceride levels by at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or any value in-between.

[0031] Additionally, the GK inhibitor can be used to reduce cholesterol levels in a subject in need thereof. In some embodiments, the GK inhibitor can reduce cholesterol levels by at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or any value in-between.

[0032] In some embodiments, administration of the GK inhibitor inhibits mTORCl. In some embodiments, the GK inhibitor inhibits mTORCl such that mTORCl expression or activity levels is reduced by at least about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, or any value in-between.GK Inhibitors

[0033] In some embodiments, the agents comprise GK inhibitors. In some embodiments, the GK inhibitor comprises an antibody or antigen-binding fragment thereof, a small molecule, or a nucleic acid.

[0034] In some embodiments, the nucleic acid is an siRNA. In further embodiments, the nucleic acid is a GalNAc-siRNA conjugate.

[0035] As used herein, the term “nucleic acid” refers to two strands comprising nucleotides that are able to interfere with gene expression of GK. The resulting inhibition of GK can be complete or partial. The term “nucleic acid” can also include nucleic acid conjugates.As used herein, the term “GalNAc” refers to 2-(Acetylamino)-2-deoxy-D-galactopyranose, also referred as N-acetyl galactosamine. The terms “GalNAc” or “N-acetyl galactosamine” includes both the P-form: 2-(Acetylamino)-2-deoxy- -D-galactopyranose and the a-form: 2- (Acetylamino)-2-deoxy-a-D-galactopyranose. Both the P-form: 2-(Acetylamino)-2-deoxy-P- D-galactopyranose and a-form: 2-(Acetylamino)-2-deoxy-a-D-galactopyranose may be used interchangeably.

[0036] The nucleic acid comprises two separate polynucleotide strands. In some embodiments, the nucleic acid is an siRNA (short interfering RNA) molecule. siRNAs can inhibit the expression of a target gene (i.e., GK) through the RNA interference (RNAi) pathway).Specifically, inhibition of the target gene occurs through targeted degradation of mRNA transcripts of the target gene following transcription. The siRNA is incorporated into the RNA-induced silencing complex (RISC). The RISC complex RNA by sequence complementarity with the target sequence.

[0037] In some embodiments, each nucleic acid strand comprises an overhang of two base pairs. The overhangs (5’ or 3’) can be on either end of the first or second strands. In some embodiments, both ends of the nucleic acid have overhangs. As used herein, “overhang” refers to a single stranded portion of a nucleic acid that extends beyond the terminal nucleotide of a complementary strand in a double strand nucleic acid. A member of the Argonaute protein family recognizes the overhangs and selects a nucleic acid strand to become the guide strand (also termed the antisense strand). The second strand is referred to as the passenger strand, which is degraded.

[0038] In some embodiments, the nucleic acid is 18-29 nucleotides in length. In some embodiments, the first strand of nucleic acid is 18-29 nucleotides in length. In some embodiments, the first strand of nucleic acid is 19-25 nucleotides in length. In some embodiments, the second strand of nucleic acid is 18-29 nucleotides in length. In some embodiments, the second strand of nucleic acid is 20-27 nucleotides in length.

[0039] In further embodiments, the first strand is 21 nucleotides and the second strand is 23 nucleotides in length. In further embodiments, the first strand is 19 nucleotides and the second strand is 21 nucleotides in length.

[0040] In some embodiments, the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said nucleic acid comprises the nucleic acid sequence pairs as set forth in Table 1.Table 1. Example nucleic acid sequences that inhibit GK mRNA expression.= 2’F-dC, 3 = 2’F-dA, 4 = 2’-OMe-rA, 5 = 2’-OMe-rG, 6 = 2’-OMe-rC, 7 =2’-OMe-rU, 8 =GNA (glycol nucleic acid), 9=2’F-dG

[0041] The nucleic acid may comprise ribonucleotides, modified ribonucleotides, deoxynucleotides, deoxyribonucleotides, or nucleotide analogues that are able to mimic nucleotides and can pair with the corresponding base on the target sequence or complementary strand. In some embodiments, the nucleic acid comprises at least one modified nucleotide. In some embodiments, all of the nucleotides in the first and second strands are modified nucleotides.

[0042] The modified nucleotides can improve resistance to degradation and / or improve stability of the nucleic acid. Examples of modified nucleotides include, but are not limited to 2’- deoxy-2’ -fluoro (2’F), 2’-O-methyl (2’-0me), glycol nucleic acid (GNA), unlocked nucleic acid (UNA), and locked nucleic acid (LNA). GNA is an acyclic nucleic acid analogconnected via phosphodiester bonds. The modified nucleotides do not change the identity of the nucleic acid. In other words, modification of the backbone of the nucleic acid do not change the identity of the nucleotide because the nucleic acid base itself remains the same as in the reference sequence.

[0043] In some embodiments, the nucleic acid may comprise modified nucleosides. Examples of such modified nucleosides include, but are not limited to 2-thiouridine (s2U), pseudouridine ( ), and dihydrouridine (D). The modified nucleosides can modulate the silencing potency of the nucleic acid and affect its thermodynamic stability (Sipa et al. RNA. 2007. 13(8): 1301- 1316).

[0044] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 7, 9, 10, and 11 from the 5' end of the first strand are modified with 2 ’-deoxy-2’ -fluoro (2’F); b) the nucleotides at positions 1-6, 8, and 12-21 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); c) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’-deoxy-2’-fluoro (2’F); d) the nucleotides at positions 1, 3-5, 8-13, 15, and 17-23 from the 5’ end of the second strand are modified with 2’-O-methyl (2’-0me); and e) the nucleotides at position 7 is from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0045] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 1-4, 6, 10, 11, and 13-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5, 7-9, and 12 from the 5' end of the first strand are modified with 2’-deoxy-2’-fluoro (2’F); c) the nucleotides at positions 1, 3, 4, 5, 9-13, 15-19, and 21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); d) the nucleotides at positions 2, 6, 14, and 20 from the 5' end of the second strand are modified with 2’-deoxy-2’-fluoro (2’F);e) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0046] In some embodiments, the nucleic acid is modified as follows: a) the nucleotides at positions 1-4, 6, 10, and 11-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5 and 7-9 from the 5' end of the first strand are modified with 2’ -deoxy-2’ -fluoro (2’F); c) the nucleotides at positions 1, 3, 4, 5, 9-13, 15, and 17-21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); d) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’-deoxy-2’-fluoro (2’F); e) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

[0047] The “duplex region” of the nucleic acid comprises nucleotide base pairings between the first and second strands, generally based on Watson-Crick base pairing. The duplex region can comprise a modified nucleotide or the modified nucleotide can be outside of the duplex region within a single stranded region.

[0048] In some embodiments, the nucleic acid comprises two or more nucleotides that are linked to each other by natural phosphodiester linkages. In some embodiments, the nucleic acid comprises two or more nucleotides that are linked to each other by any other linkage such as a phosphorothioate (PS) or a phosphorodithioate linkage. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Phosphorothioates have one of the non-bridging oxygens replaced with sulfur. In some embodiments, the nucleic acid comprises at least one phosphorothioate internucleotide linkage. In further embodiments, the nucleic acid comprises two consecutive phosphorothioate intemucleotide linkages at both the 3' and 5' ends of the second strand and two consecutive phosphorothioate intemucleotide linkages at the 5' end of the first strand. The PS intemucleotide linkages can improve the stability of the nucleic acid.

[0049] The nucleic acid can comprise at least one chemical modification. Tn some embodiments, the chemical modification comprises one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof conjugated to the nucleic acid, directly or indirectly via a linker. GalNAc binds to the asialoglycoprotein receptor (ASGPR), which is predominantly expressed on liver hepatocytes. GalNAc-siRNA conjugates are thus delivered to liver hepatocytes.

[0050] The chemical modification can be conjugated to the 5’ or 3' end of the first or second strand. In some embodiments, the chemical modification is conjugated to the 3’ end of the first strand. In further embodiments, the chemical modification is conjugated to the 3' end of the first strand using a triantennary GalNAc conjugation. Conjugates having three GalNAc ligands and comprising phosphate groups are described in Dubber et al. Bioconjug. Chem. 2003. 14(l):239-46. Additionally, triantennary cluster glycosides are described in US Patent No. 5,885,968.

[0051] In some embodiments, the nucleic acid is conjugated to a chemical modification comprising a compound of formula (I): [S— X]3-Y-Z— (I) wherein:S is GalNAc;X is a linker to the branching unit;Y is a branching unit;Z represents a bridging unit; and wherein a nucleic acid as described herein is conjugated to Z via a phosphate or modified phosphate.

[0052] In some embodiments, formula (I) has the following structure:

[0053] In the above formula (I), branching unit “A” branches into three to accommodate three saccharides. The branching unit is covalently attached to the remaining tethered portions of the chemical modification and the nucleic acid. The branching unit may comprise a branched aliphatic group comprising groups selected from amide, alkyl, disulphide, polyethylene glycol, ether, thioether and hydroxyamino groups. The branching unit may also comprise groups selected from alkyl and ether groups.

[0054] The “X3” bridging unit can be linear and covalently bound to the branching unit and the nucleic acid.

[0055] The saccharide in formula (I) can be selected from N-acetyl galactosamine, mannose, galactose, glucose, glucosamine and fucose. In some embodiments, the saccharide is N- acetyl galactosamine (GalNAc).

[0056] In some embodiments, the nucleic acid is conjugated to a chemical modification and has the following structure:wherein Z is a nucleic acid as described herein.

[0057] The nucleic acids described herein can be synthesized by methods to one of ordinary skill in the art (Hu et al. Signal Transduction and Targeted Therapy. 2020. 5(101): doi.org / 10.1038 / s41392-020-0207-x). For example, the nucleic acid can be synthesized via chemical synthesis or in vivo expression. In one embodiment, the nucleic acid of interest is produced via an expression vector within a host organism or cell.Methods of Treatment

[0058] Further aspects of the present disclosure include methods to treat a disease via administration of the GK inhibitor described herein. In some embodiments, the GK inhibitor is used in a method of treating or preventing a disease associated with elevated levels of serum TAGs and MASLD. In some embodiments, the disease is hypertriglyceridemia or MASLD. MASLD commonly progresses to steatohepatitis, cirrhosis, and hepatocellular carcinoma. Additionally, diseases associated with MASLD include, but are not limited to metabolic syndrome, type 2 diabetes mellitus, and dyslipidemia.

[0059] In some embodiments, the GK inhibitor is used in a method of treating or preventing high elevated cholesterol levels (e.g., hyperlipidemia or hypercholesterolemia).

[0060] In some embodiments, the GK inhibitor treats a disease by inhibiting mTORCl. mTORCl is one of two complexes (mTORC2) that comprise mTOR. mTOR functions as a nutrient sensor and regulates cellular functions linked to survival, proliferation, and growth. mTORCl regulates cell growth and metabolism. mTORCl activation occurs when it is recruited to the surface of lysosomes. Dysregulated mTORCl signaling is associated with several disease including diabetes (Orozco et al. Nat. Metab. 2020. 2(9): 893-901). Inhibiting mTORCl blocks both fatty acid and cholesterol synthesis. In further embodiments, the GK inhibitor described herein can treat both diseases associated with high serum TAGs and elevated cholesterol levels.

[0061] As used herein, the term “treating” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (z.e., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the subject. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g, stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0062] The terms “prevent,” “preventing,” “prevention,” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder (or disease) or condition.

[0063] As used herein, the term “subject” refers to a mammal. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and farm animals. Mammals, include, but are not limited to, a human or non-human mammal, such as a canine, bovine, equine, ovine, or feline, etc. Individuals and patients are also subjects herein.

[0064] As used herein, the term “therapeutically effective dose” generally refers to a particular amount of a GK inhibitor results in or more of: a) reduces the activity or an expression level of GK, b) treats or prevents a disease associated with elevated levels of TAGs and MASLD, c) inhibits mTORCl, and / or d) reduces cholesterol levels in a subject in need thereof.

[0065] As used herein, the term “hyperlipidemia” refers to the presence of abnormally elevated levels of lipids in the blood. Hyperlipidemia can appear in at least three forms: (1) hypertriglyceridemia, i.e., an elevated triglyceride level; (2) hypercholesterolemia, i.e., an elevated cholesterol level; and (3) combined hyperlipidemia, i.e., a combination of hypercholesterolemia and hypertriglyceridemia.Administration

[0066] The GK inhibitor described herein can be delivered to a subject via various modes of delivery. For example, the GK inhibitor can be administered to a subject subcutaneously, intravenously, orally, parenterally, intraperitoneally, or systemically. In particular embodiments, the GK inhibitor can be administered subcutaneously.

[0067] In some embodiments, the GK inhibitor can be combined with a delivery vehicle such as liposomes or any delivery method of nucleic acids as known in the art.

[0068] In some embodiments, the GK inhibitor is administered to a subject via a pharmaceutical composition that comprises the GK inhibitor described herein. The pharmaceutical composition can comprise a pharmaceutically acceptable excipient such as a buffer, diluent, preservative, and stabilizer. In further embodiments, the pharmaceutical composition can be in lyophilized form, formulated as a sterile injectable aqueous solution, or is a galenic carrier substance (e.g., gels, tablets, pellets, etc.). In some embodiments, the pharmaceutical composition is formulated for improved stability.

[0069] The GK inhibitor described herein can be administered to a subject in need thereof at a therapeutically effective dose. In some embodiments, the GK inhibitor is an siRNA conjugate administered at a dose of about 100 mg to about 800 mg. For example, the GK inhibitor can be administered at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, or any amount in-between.

[0070] In some embodiments, the GK inhibitor is a small molecule inhibitor that is administered at a dose of about 0.001 to about 1.0 milligrams per kilogram body weight of the patient. For example, the GK inhibitor can be administered at about 0.01 mg / kg, about 0.02 mg / kg, about 0.03 mg / kg, about 0.04 mg / kg, about 0.05 mg / kg, about 0.5 mg / kg, about 1 mg / kg or any value in-between.

[0071] In some embodiments, the GK inhibitor is administered to a subject in need thereof for a certain period of time at one therapeutically effective dose, followed by administration at a different therapeutically effective dose. For example, the GK inhibitor can be administered at about 100 mg for at least one week followed by administration at about 500 mg. In some embodiments, the GK inhibitor is administered to a subject in need thereof at a constant dose. The dose of the GK inhibitor can be varied should a subject experience intolerable adverse events as evaluated by an attending physician.

[0072] In some embodiments, the GK inhibitor is administered to a subject in need thereof at least once a week, at least once a month, or at least once a year. For example, the GK inhibitor can be administered every week, every two weeks, every three weeks, every four weeks, every five weeks, every six weeks, every seven weeks, every eight weeks, or annually. The GK inhibitor is administered to a subject until an attending physician determines that administration of the GK inhibitor is no longer needed.Combination Therapy

[0073] Some aspects of the present disclosure include combining the GK inhibitor with other therapeutic agents for a synergistic effect. In some embodiments, the GK inhibitor is administered in combination with a second therapeutic agent. For example, the second therapeutic agent can be selected from the group comprising fibrates, metformin, statins, and niacin (vitamin B3), which are used to treat high TAG levels. In one embodiment, Fibrates and niacin can be used if TAGs are elevated in isolation. In one embodiment, metformin can be used if TAGs are elevated due to diabetes. In one embodiment, statins are used if both cholesterol and TAGs are elevated.

[0074] Fibrates manage and treat dyslipidemia. Examples of fibrates, include, but are not limited to gemfibrozil (LOPID®) and fenofibrate (Lipofen, FIBRICOR®, or TRIGLIDE™).

[0075] Metformin (Riomet, GLUCOPHAGE®, or GLUMETZA®) is used to treat high blood sugar levels due to type 2 diabetes.

[0076] Statins lower cholesterol levels. Examples of statins include, but are not limited to Atorvastatin (LIPITOR™), Fluvastatin (LESCOL® XL), Lovastatin (ALTOPREV®), Pitavastatin (LIVALO® or ZYPITAMAG®), Pravastatin (PRAVACHOL®), Rosuvastatin (CRESTOR® or Ezallor), and Simvastatin (ZOCOR®).

[0077] In some embodiments, the second therapeutic agent is administered on the same day as with the GK inhibitor. In some embodiments, the second therapeutic agent is administered separately from the GK inhibitor (e.g., on a different day).

[0078] In some embodiments, at least two therapeutic agents are administered concurrently in combination with the GK inhibitor. In some embodiments, at least two therapeutic agents are administered separately from the GK inhibitor (e.g., on a different day).Additional Definitions

[0079] As used herein and in the appended claims, the singular forms “a,” “and” and “the” include plural references unless the context clearly dictates otherwise.

[0080] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0081] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0082] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0083] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearlydictates otherwise. All publications mentioned herein are incorporated herein by reference in their entireties.EXAMPLES

[0084] It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments or examples disclosed, but it is intended to cover modifications that are within the spirit and scope of the present invention as defined by the appended claims.Example 1. Materials and Methods

[0085] This Example details the materials and methods used in Example 2.Primary hepatocyte isolation

[0086] Age-matched C57BL / 6J albino males between 3 and 4 months were used for PH culture isolation. All mice were anesthetized using a ketamine / xylazine mixture (Henry Schein) before isolation. The hepatic portal vein was cannulated and perfused with Kreb’s Ringer Bicarbonate Buffer (Sigma- Aldrich) containing EGTA for 10 min at 37 °C. After the first wash, a second Kreb’s Ringer wash containing CaCE and Liberase (Roche Holding AG) was applied for 10 min at 37 °C. Hepatocytes were filtered through a gauze mesh and resuspended in plating media: William’s Media E (Sigma- Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 200 nM dexamethasone (Sigma-Aldrich), 1 x penicillin / streptomycin (Thermo Fisher Scientific), and 2 mM L-glutamine (Thermo Fisher Scientific). Cells were plated at a density of 3 x io5on a six-well collagen (Sigma-Aldrich)-coated plate. Hepatocytes were allowed to recover overnight and all experiments were started 24 h post isolation, unless otherwise indicated.In vitro isotope labeling experiments

[0087] After recovery at 37 °C and 5% CO2 from isolation and viral treatment, if applicable, PH were serum-starved for 3 h in a basal medium (no-glucose Dulbecco’s modified Eagle’s medium supplemented with 3.52 mg / ml Hepes, 1 x penicillin / streptomycin, and 2 mM L- Gln). For in vitro glucose production, the cells were washed once with 1 x PBS and treatedwith glucose production medium (no-glucose Dulbecco’s modified Eagle’s medium with 3.52 mg / ml Hepes and 1 x penicillin / streptomycin) and substrates with or without 20 nM glucagon. For the physiological-concentration substrate scheme, 0.5 mM L-glutamine, 0.33 mM glycerol, 0.25 mM sodium pyruvate, and / or 2.5 mM sodium lactate were used. Culture medium was collected every 2 h for the measurement of glucose production and labeling patterns. To compensate the loss of substrates and maintain the physiological concentration, 0.22 pmol glycerol, 0.33 pmol pyruvate, and 0.05 pmol L-glutamine per ml culture were added every 2 h after the sampling of media.

[0088] 13C-labeled substrate experiments, the same conditions were used, but substrates were substituted one at a time with13C3-sodium pyruvate / lactate,13C5-L-glutamine,13C.3- Glycerol, or13Ce-D-glucose (Cambridge Isotope Lab, Inc) as indicated. The medium metabolites were directly extracted in 100 volumes of ice-cold 40:40:20 methanol:acetonitrile:water solution with 0.1% formic acid. The cells were washed once with prewarmed 1 x PBS and extracted with ice-cold 40:40:20 meth- anol:acetonitrile:water solution with 0.1% formic acid (2 ml per million cells). Both media and cell extracts were incubated on ice for 5 min and neutralized with 0.7% NH4HCO3, followed by centrifugation for 10 min at 16,000g. The supernatant was then transferred to another clean tube for mass spectrometry (LC-MS) analysis (See Chiles, E, et al. Anal. Biochem. 2019. 575: 40-43).Animals and animal care

[0089] The glucose homeostasis varies throughout the estrus cycle in female mice (See Santiago, A.M. et al. Physiol. Behav. 2016. 167: 248-254). To avoid the variation caused by estrus cycles, only male mice were used in this study. All mice were maintained on a C57BL / 6J- albino background (lackson Laboratory; B6(Cg)-Tyrc-2J / I). Floxed Gk Mice were bred to Albumin-Cre transgenic mice, yielding control (WT) or LGKKO mice. All mice described herein were housed in a pathogen-free barrier facility with a 12-h light / dark cycle. All animal protocols were approved by the Institutional Animal Care and Use Committee of Rutgers University.Protein extraction and Western blotting

[0090] Protein preparations were conducted with both hepatocytes that were lysed in Laemmli buffer with P-mercaptoethanol and tissue samples. For tissue protein preparations, the tissues had been snap-frozen in liquid nitrogen following the sacrifice of the animal and were mechanically homogenized in ice-cold RIPA buffer (Sigma-Aldrich) with 1 x protease inhibitor (Roche Holding AG) and 1xphosphatase inhibitor (Thermo Fisher Scientific) using Bullet Blender (Next Advance, Inc). The homogenate was then centrifuged at 16,000g at 4 °C and the supernatant was collected. Protein concentrations were measured using the Pierce BCA assay (Thermo Fisher Scientific). Western blotting was performed using standard procedures utilizing the standard V3 Western Workflow (Bio-Rad Laboratories). All standard Western blotting reagents were obtained from Bio-Rad Laboratories. Antibodies obtained from Cell Signaling Technology: ACLY (Cat# 4332), ACC (Cat# 3676), anti-rabbit IgG, HRP-linked (Cat# 7074S), anti-mouse IgG, HRP-linked (Cat# 58802)), FAS (Cat# 4233), or PPIB (Cat# 43603). Antibody obtained from Abeam: GK (Cat# abl26599).Metabolic tests

[0091] For the pyruvate and glycerol tolerance tests, mice were fasted overnight (12 h) and then injected i.p. with sodium pyruvate (9 mmol / kg; Sigma- Aldrich) or glycerol (9 mmol / kg; Sigma-Aldrich). Blood glucose measurements were obtained via a small nick in the lateral tail vein using a glucometer (Bater Contour).Statistical analysis

[0092] All analysis and graphs were done on GraphPad Prism 7.0 software (graphpad.com / scientific-software / prism / ). Statistics were performed using either Student’s t test or one-way ANOVA where appropriate.Example 2. Hepatic GK Knockout (LGKKO) Mice are Protected Against Hypertriglyceridemia and MASLD Induced by a High-Fat Diet (HFD)

[0093] Glycerol metabolism in the liver is considered an accessory pathway and not predicted to be the main determinant of TAG synthesis and storage. Hepatic GK knockout (L-GkKO) mice unexpectedly demonstrate that approximately 90% of hepatic glycerol 3-P is derived from the GK pathway (FIG. 4).

[0094] Moreover, the LGKKO mice are protected against hypertriglyceridemia and MASLD induced by a high-fat diet (HFD) (FIG. 2, clear spaces mark TAG deposition). One might expect that serum non-esterified fatty acids (NEFAs) would be elevated in the setting of a defect in liver esterification. This was not the case in LGKKO mice, where serum NEFAs were 60% lower and statistically different from HFD fed mice (FIG. 3). The mice also exhibited a defect in de novo lipogenesis linked to impaired activation of the nutrient sensor mechanistic target of rapamycin complex 1 (mTORCl). FIG. 3 illustrates the resulting defect in lipogenic protein expression (ACLY (ATP Citrate Lyase), ACC (Acetyl-CoA carboxylase), and FAS) in LGKKO mice, suggesting that a defect in glycerol metabolism also reduces fatty acid synthesis.

[0095] A unique GalNAc-siRNA conjugate against GK in primary mouse hepatocytes reproduced the GK knockout effect in the liver. Several unmodified and modified GalNAc- siRNA conjugates against mouse GK were synthesized and tested in primary mouse hepatocytes. These modifications improve liver-targeting (3x GalNAc) or improve siRNA stability (changes in the ribose or its linkages). The modifications used are listed in Table 1 (SEQ ID NO: 1 and SEQ ID NO: 2 (mGKl and mGKl-2) and SEQ ID NO: 3 and SEQ ID NO:4 (mGK2 and mGK2-2)). Two unique modified GalNAc-siRNA conjugates were selected that lacked non-specific effects and completely eliminated GK mRNA expression (Table 1 and FIG. 3). Treatment with these conjugates reproduced the GK knockout effect in the liver resulting in undetectable hepatic GK expression 3-4 weeks after treatment. Human GK GalNAc-siRNA conjugates were also synthesized for testing in human primary hepatocytes. A knockdown of hepatic GK expression using GalNAc-siRNA conjugates may serve as an effective treatment for hypertriglyceridemia and MASLD in humans.

[0096] Altered glycerol metabolism in humans also suggests its critical role in TAG synthesis. For example, obese male and female subjects, who frequently exhibit hypertriglyceridemia and MASLD, have statistically significantly higher serum fasting glycerol and NEFA levels, reflecting increased basal lipolysis in peripheral adipose tissue (Arner et al. Obes Facts. 2015. 8(2): 147-55). These substrates can return to the liver and be esterified to TAGs. Conversely,adults with germline GK mutations that reduce glycerol metabolism have been described as asymptomatic, but their lipid metabolism remains unknown.

[0097] A systematic review of adult GK genetic deficiency suggested a lower prevalence of cardiovascular disease, obesity, and diabetes versus control subjects (Lamiquiz-Moneo et al. Atherosclerosis, 2020 315: 24-32). As expected, serum glycerol levels were markedly elevated, but serum TAGs (measured directly) and GGT (a marker of obesity-related liver disease) were statistically lower than control subjects.Example 3. Phase II Clinical Trial of GK inhibitor

[0098] This study is a phase II study evaluating 3xGalNAc conjugated siRNA against GK at multiple-ascending-doses in patients with hypertriglyceridemia and at increased risk for cardiovascular disease.Study Objectives

[0099] The primary objective of the multiple-ascending-doses is to assess safety, tolerability, and pharmacokinetics (PK) of 3xGalNAc conjugated siRNA against GK (specifically, 1. SEQ ID NO: 5 and SEQ ID NO: 6, 2. SEQ ID NO: 7 and SEQ ID NO: 8, and 3. SEQ ID NO: 9 and SEQ ID NO: 10) when administered once or twice at three different doses and compared to a placebo control injection.

[0100] The secondary objective of this study is to determine if 3xGalNAc siRNA is effective at reducing serum triglyceride levels compared to a placebo control injection.Study Design

[0101] This study is a phase II study evaluating 3xGalNAc siRNA at ascending-doses. Patients are randomly assigned to receive a single dose of placebo or 200, 300, or 500 mg of 3xGalNAc siRNA or two doses (at days 1 and 90) of placebo or 100, 200, or 300 mg of 3xGalNAc siRNA. A total of 20 patients are expected to be enrolled in each of the six 3xGalNAc siRNA treatment groups and in each of the two placebo groups (one or two doses of placebo).

[0102] The expected duration of the patients’ participation is approximately 180 days, which includes several scheduled follow-up periods. Additional follow-up may be necessary and if so, the expected maximum duration of the study is 270 days.

[0103] Patients are treated as follows:

[0104] Single dose (on Day 1):• Screening: day -14 to -1• Randomization, initiation of study drug: day 1• Treatment phase: day 1• Follow-up: days 2, 14, 90, and 180

[0105] Two doses (one injection each on day 1 and day 90):• Screening: day -14 to -1• Randomization, initiation of study drug: day 1• Follow-up: days 2, 14, 90, 92, 104, and 180Inclusion Criteria

[0106] Patients are included in the study if they meet the following criteria:1. Male or female subjects >18 years of age.2. History of atherosclerotic cardiovascular disease or atherosclerotic cardiovascular diseaserisk equivalents (Type 2 diabetes, familial hypercholesterolemia, including subjects whose 10-year risk of a cardiovascular event assessed by Framingham Risk Score* or equivalent).3. Serum triglyceride>150 mg / dL for atherosclerotic cardiovascular disease subjects or >100 mg / dL for atherosclerotic cardiovascular disease-risk equivalent subjects at screening.4. Calculated glomerular filtration rate >30 mL / min by estimated glomerular filtration rate using standardized local clinical methodology.5. Subjects on lipid-lowering therapies (such as statin and / or ezetimibe) are to be on a stable dose for >30 days before screening with no planned medication or dose change during study participation.6. Willing and able to give informed consent before initiation of any study-related procedures and willing to comply with all required study procedures.*By Framingham Risk Score >20%.Exclusion Criteria

[0107] Patients are excluded from the study for the following reasons:1. Any uncontrolled or serious disease, or any medical or surgical condition, that may either interfere with participation in the clinical study and / or put the subject at significant risk (according to investigator’s judgment) if he / she participates in the clinical study.2. An underlying known disease, or surgical, physical, or medical condition that, in the opinion of the investigator, might interfere with interpretation of the clinical study results.3. New York Heart Association (NYHA) class II, III or IV heart failure or last known left ventricular ejection fraction 180 mmHg or diastolic blood pressure >110 mmHg prior to randomization despite anti-hypertensive therapy.4. Cardiac arrhythmia within 3 months prior to randomization that was not controlled by medication or via ablation.5. Any history of hemorrhagic stroke.6. Major adverse cardiac event within 6 months prior to randomization.7. Uncontrolled severe hypertension: systolic blood pressure >180 mmHg or diastolic blood pressure >110 mmHg prior to randomization despite anti-hypertensive therapy.8. Poorly controlled Type 2 diabetes, i.e., glycated hemoglobin Ale (HbAlc) >10.0% prior to randomization.9. Active liver disease defined as any known current infectious, neoplastic, or metabolic pathology of the liver or unexplained alanine aminotransferase (ALT), aspartate aminotransferase (AST), elevation >2x the upper limit of normal (ULN), or total bilirubin elevation >1.5x ULN at screening confirmed by a repeat measurement at least 1 week apart.10. Serious comorbid disease in which the life expectancy of the subject is shorter than the duration of the trial (e.g., acute systemic infection, cancer, or other serious illnesses). This includes all cancers with the exception of treated basal -cell carcinoma occurring >5 years before screening.11. Females who are pregnant or nursing, or who were of childbearing potential and unwilling to use at least two methods of contraception (oral contraceptives, barrier methods, approved contraceptive implant, long-term injectable contraception, intrauterine device ortubal litigation for the entire duration of the study). Women who were >2 years postmenopausal defined as >1 year since last menstrual period and if <55 years old with a negative pregnancy test within 24 hours of randomization or surgically sterile are exempt from this exclusion.12. Males who were unwilling to use an acceptable method of birth control during the entire study period (z.e., condom with spermicide).13. Known history of alcohol and / or drug abuse with the last 5 years.14. Treatment with other investigational medicinal products or devices within 30 days or five half-lives, whichever is longer.15. Use of other investigational medicinal products or devices during the course of the study.16. Any condition that according to the investigator could interfere with the conduct of the study, such as but not limited to: a. Inappropriate for this study, including subjects who were unable to communicate or to cooperate with the investigator. b. Unable to understand the protocol requirements, instructions and study-related restrictions, the nature, scope, and possible consequences of the study (including subjects whose cooperation is doubtful due to drug abuse or alcohol dependency). c. Unlikely to comply with the protocol requirements, instructions, and study-related restrictions (e.g., uncooperative attitude, inability to return for follow-up visits, and improbability of completing the study). d. Have any medical or surgical condition, which in the opinion of the investigator would put the subject at increased risk from participating in the study. e. Involved with, or a relative of, someone directly involved in the conduct of the study. f. Any known cognitive impairment (e.g., Alzheimer’s disease).Data Analysis

[0108] Data are collected at baseline, day 90 and day 180 of the study. The primary end point analyzed is the percentage reduction from baseline in triglyceride level at day 180. Thesecondary end point analyze is the percentage reduction from baseline in serum cholesterol level at day 180.

[0109] Fasting blood samples are obtained at each study visit and serum triglyceride and LDL cholesterol levels are measured by standard methods. A survey is performed at each visit to measure the side effect profile of 3xGalNAc siRNA.Results

[0110] It is expected that treatment with 3xGalNAc siRNA will target hepatic GK expression and reduce serum triglyceride levels to < 50% of baseline values versus a placebo injection (primary end-point). A reduction in serum LDL cholesterol may also be observed (secondary end-point).Example 4. Generation of liver-specific glycerol kinase knockout mice

[0111] As systemic GK knockout mice demonstrate severe defects in metabolism, development and postnatal lethality, liver-specific GK knockout mice (LGKKO) were generated using Gk floxed and albumin-Cre lines. To generate Gk floxed mice, loxp sites were introduced before exon 2 and after exon 3 of Gk using CRISPR-Cas9 technology (see below sequence):

[0112] SEQ ID NO: 11- Murine nucleic acid sequence containing loxp sites before and after exon 3 of GkCCTTGAAATGCTGACAGAGTGAATCTTGTGTAGGCTCTGTCCAGGGAAGCTCAGCCACA GTGAGTTTATGAAGGCAACAACCATGCCATGTCCCGAATAACTTCGTATAGCATACATT ATACGAAGTTATGACCTTTTGTGCCATAGCTACTCATCCTTCAGCTCTTGCACTGCCTTC ATCCCCTCTTTGATGTTCCCTGAGTCTTGGAGGGATGCTTAGAAGGCAGTTAACAGCATG TCCTTTTAGCAAAACACTAGTAGTTGGTTTCTTTCTCAGCGTATCGTTCCCAGACATAGGC CCCTGACCAGGTTTGCATCTATGAGAAATTCTTCTTTCATTTTTTAAAAAAAATATTTTTA TTTTGTGTTTGGGTGTTTTATCCATATGTATGTCTGTGCAATACATGCATGCCTGCTGTTC ATGGAGGCTAGAGGTGTCCATTTTGGAACTGGAGTAACAGAGGGTTGTGGGTGCTGCAA ATGGAACCCAGAAACATCCAAGCCATCTCTCCAGCTTGCTTGTTTGTTCTTTCTTTCTTTC TTTCTTTCTTTCTTTCTTTCTTTCTTTTCTTTCTTTTCTTTCTTTTCTTTCTTTTCTTTTCTTTT CTTTTCTTTTCTTTTCTTTTCTTTCTTTCTTTTTGAAAAATTGTCTATAATTTTTATAGACAG GATGCCACATTGTAGCTCAGGTTAGCCTCAAAGTCTGGATTCTTCTGCCCAAGCCTCCAGAGCACTATGCTCTCAGATGTGAGGCCCCATCAGTCCTGGTTATGAGAAGTTATTTCTGGT GTAATATTGCAATTTTTAAGATACATTTTCACATTTTCATATGTATGTATGCTCTCTCTCT CTCTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGT TCGTTTGGAGTTACAGGAAGTTATGAGGCAGCTGAAGTGGCTGCTGGGAAATGAACTTG GATCTTAACCACTGAATCATCTCTACAGCCACCCACACTGTAATGCTTTGTGATTCTGTCT GAAGAATAAGTCAAACATCATTGGTCATCATTCCATATGATTCAGTCTTTTCTACATGTT AATAAGAGCTTTATTTGCTCCATAACTTCAAACAGATTTGATACCAACTTGCATTTTTATACTGATATGTTTTTCAGGTTTTCAATTCAAAAACAGCTGAACTTCTTAGTCATCATCAAGTAGAAATAAAACAGGAATTCCCAAGAGAAGGGTATGTTTCTTAATTTACTATGTAAAGACATATCACATTGATTGGTTTGTCTCACCCCATGTATGCTGTGGATTGATTGGTAGAGTAAACTTAGCCAGACACCTTCTGGAGAAGGCCTGAGAGAATGGGAGTGGTCAAGAAATGAAGCTAGAATGCAGTAATTCAGCAAAAATAGGGGAGCAATGGGTAGCTTAGGCATTCATTGACAGAAAGCAAAGTATAAGTAATGTAACACACACACATAAAGTCTGTATTACAACAAAGGAATATTAACCATCAGAGTCTGCATTAAGTCTGAAGTTTGAGGTGATTTCCTTCTGTGTCCCTGTTACTAGCAGTGCTAAAATGATTACTTTTCAGGAGTGTGTTCTATTTTACCTTTGTTTTTGCCT TTGGAACCACGATCATATCAATGTACAGACCAGGCTCCCTCTCAAGGGCTGGGATTTGAAGTGTGTGAGCCACTGGTTAATGACCACCCTTAGAAGAAGGATGGATTATTATTAGTCTGTGAAGAATAAGAAAAATGTATAGGAAATAGGTTTTTTTTTATCCCTTTAGTATGTAGAAAACAGATATAATGTATTTATGGAAACATGTTATGTTTGTATTGAATTCTTGA A A ACT A AC ATTTATCCTTTTGTTCC A A AGA TGGGTA GAACAA GACCCGAA GGAAATTCT GCAGTCTGTTTATGAGTGTATAGAGAAAACGTGTGAGAAACTTGGACAGCTCAATATTGATA777CCAACA7’tAAAG’GTATTTTTATTGAGAATTTTCCTCTATAGTCCAGCTCTGCCAATCCCATTCATGAATTCATTCTTTCAGCTTATAATAATTGAGTCTTTATAAGCTGCAAGCCTTTTTGGTTTTTGTTTGTTTGTTTGTTTGTTTGTTGGTGAGACTTTGTGGGAAGAACAATAAACAAGATTCTCAGTTCCCGTTCACTGTGACATTTTATTCTGATGCATATGAGAACAATAAAAGACGACTATACAAGTTAATCTTACATACTAACAAATGCCATGTGGGAAATTTTAAAATGAGGAATATCATAGGGGTTCCTACATGGCTTAGACACAGTAGGTGGGCACAGGAGGGCTTCTCTTCTCTGAAGAGGTGATTTTTTTTCCTGAGATTGCAAGGGACTTGAGGGGTCAATCATGTGATTGGAGAAAAATATTTGAAGAACAAATAAACAGCCTATGCTGCCATGGTTGGTTGATGTCCCTGGGAGGCCCATTCTTTTCTGAAGGGAAAGGGGGAGGAGTGGGGGAGTGGCTGGGAGGGGAGGAGAGAGGGGAAACTGCAGTCAGGATGTAATATATAAGAGAAAAATCAATTTAAAAGTTAATAAATAAAAATGTAACATTAAAAAATAAAAATAAAAGTTACTGGATCTTTTGTTTATATAGTAGAAATTTAAGTTGGGTTTTAGTCATTCTTTGTTTTCATTTCCCTTGAATTTCTTTATAGGAATGCTTAAAACTATCCAGTTCAGTAGAATATAGTGAGCTGGAGCTCAGGTGGCACTTGAGGGACTTTGAAACAGAACAATGGAAGAGATCTGTGCTTCTCCATTT TT TTC TA TT TT CC CT CT TA TG TA CA TT TT AT CC AT TG TA TT CG TG CA TAA ATT TT TC TTC GA CC ATT TT AC TC GT CG CA TA TTG TT AT TT CTA AA TTT GTT TA TGC GC ATT CG AT AT TA TGGTTTCTGTTCCTAATTTTATTTTACTTTTCTTTATTTGGCTTCATGTACTCTTTCCTTATTTATTTTTAAATTACTTCGTAGCATTTGACTTTGTCTTTTTAACTATTGCATTTTAATCTTTTTATTAATTAAATTTATTCTCTGACAATATCATAAAATACACATCCCATGaATAACTTCGTATAGCATACATTATACGAAGTTATCGAAGATCCCAAGGTGGGAATATAAATTGATACAGTCTAGAGTCACGAAGAAGACTTGTAAAAACAAGTTGAGTGGGCAGAGTGGGTAAGTAAGGAGTTTCATATAACAGAGATTGGAGAAATCAGTGAGGCCTTGTCAGTCATATTTAAGAG TTTGAATGTTAGTTTTAGTATCAGAAGGGACAGGTCATGACTCACAATTTAGAGATGGCT ACATACCTG

[0113] Note: Bold indicates loxp sites. Bold and underlined sequence indicates exon 2. Bold and italicized sequence indicates exon 3.

[0114] LGKKO mice showed efficient, liver-specific GK deletion (FIG. 6A), while body weight (FIG. 5A) and GK expression in kidney (FIGS. 5B and 6A) and other peripheral tissues such as epidydimal adipose tissue (eWAT) and small intestine (slnt) were unaffected (FIG.5B). Accordingly, hepatic GK levels were undetectable in LGKKO compared to control WT mice (FIG. 6B)

[0115] It was also determined whether a liver-specific deletion of GK affected blood glucose levels after glycerol administration since other organs and tissues (e.g., kidney, small intestine, and macrophages / neutrophils) also express GK. Interestingly, LGKKO mice showed significantly reduced blood glucose levels after glycerol administration, demonstrating that direct hepatic conversion of glycerol mediates this effect (FIG. 6C). Taken together, the LGKKO mouse is a novel animal model to study the role of hepatic GK in glycerol -mediated gluconeogenesis (GNG) and lipogenesis.Example 5. Hepatic glycerol kinase deficiency decreased fasting glucose, decreased triglyceride storage in the liver, and suppresed de novo lipogenesis

[0116] To determine whether hepatic glycerol metabolism regulated glucose production in mice, LGKKO mice (generated as in Example 4) were fed with either a regular diet (RD) or a high- fat diet (HFD). HFD feeding increased body weight and fat mass in WT and LGKKO mice without affecting lean mass (FIGS. 6D and 6E). However, both RD- and HFD-fed LGKKO mice showed significantly reduced fasting (FIG. 6F) and glycerol-stimulated blood glucose levels (FIG. 6G), suggesting impaired hepatic glucose production. Pyruvate-induced blood glucose levels were also reduced in HFD-fed LGKKO mice compared to control WT mice (FIG. 6G), reflecting an important role for hepatic glycerol metabolism in generating the triose phosphate pool. As the liver is a major site for glycerol metabolism, circulating glycerol levels were significantly increased in LGKKO mice, but non-esterified free fatty acid (NEFA) levels were unchanged compared to WT mice (FIG. 6H). Fasting insulin levels were also significantly reduced in RD- and HFD-fed LGKKO compared to WT mice (FIG. 61). These results indicate that liver-specific deletion of GK suppressed hepatic GNG, results in reduced fasting glucose and insulin levels.

[0117] WT and LGKKO mice fed an HFD for 16 weeks showed similar increases in body weight and total fat mass without differences in lean mass as in FIGS.6-6I. Predictably, liver weights of HFD-fed WT mice were significantly increased compared to RD-fed WT mice. However, liver weights of HFD-fed LGKKO mice were significantly reduced compared toHFD-fed WT mice (FIG. 7A). There were no differences in the weight of epidydimal adipose tissue, and muscle (FIG. 7B). To determine whether a liver-specific GK deletion blocked fat accumulation in the liver, TG liver content was measured. As expected, H&E staining showed increased fat accumulation in HFD-fed WT livers compared to RD-fed WT mice. However, H&E staining of HFD-fed LGKKO mice showed reduced hepatic fat accumulation and a significantly decreased steatosis grade compared to HFD-fed WT mice (FIG. 7C). TG measurements confirmed reduced hepatic TG content in HFD-fed LGKKO mice compared to HFD-fed WT mice (FIG. 7D). Consistent with increased TG storage and liver inflammation in HFD-fed WT mice, serum alanine transaminase (ALT) activity levels were increased (FIG. 7E). In contrast, serum ALT levels in HFD-fed LGKKO mice were reduced compared to HFD-fed control WT mice (FIG. 7E), suggesting that a liver-specific GK deletion mitigates TG accumulation and lipotoxicity induced by HFD-feeding.

[0118] To determine if suppressed de novo lipogenesis (DNL) explained reduced hepatic TG accumulation in LGKKO mice, RNA and protein expression of DNL genes was measured. Interestingly, the RNA expression of master regulators of DNL, Srebflc and Ppara, was significantly reduced in HFD-fed LGKKO mice compared to HFD-fed WT mice (FIG. 7F). Accordingly, RNA expression of DNL genes such as Acly, Acaca and Fasn were also significantly suppressed in both RD- and HFD-fed LGKKO compared to WT mice fed the same diet (FIG. 7F). The protein levels of DNL genes in the liver were also determined. GK protein expression was increased in HFD-fed mice and predictably absent in LGKKO mice (FIG. 7G). ACLY, ACC, and FAS expression were also increased in HFD-fed WT mice compared to RD-fed WT mice, while expression in HFD-fed LGKKO mice was unchanged (FIG. 7G), suggesting that GK, in part, mediates the paradoxical increase in DNL found in insulin-resistant states due to HFD feeding.

[0119] Hepatic TG production is also mediated by the re-esterification of preexisting fatty acids (FIG. 8A). Thus, the gene expression of key enzymes in fatty acid transport, fatty acyl-CoA synthesis, and re-esterification were measured. Interestingly, the expression of fatty acid transporters such as Cd36 and Slc27a5 (FATP5 gene) and re-esterification enzymes including Gpam, Agpat2, Mogat and Dgat2 were significantly suppressed in HFD-fedLGKKO mice compared to HFD-fed WT. However, fatty acyl-CoA synthesis showed little difference in HFD-fed LGKKO. These results indicate that hepatic GK enhances re- esterification of TG in liver.Example 6. In Vitro Administration of GalNAc-siRNAs against Gk reproduced the LGKKO mouse phenotype

[0120] Thirteen -week old WT mice (four mice per group) were injected (3 mg / kg S.C.) with purified GalNAc-siRNAs against mouse Gk (GK1, 2, and 3). A control GalNAc-siRNA was also injected using the same method. After 5 weeks, hepatic Gk mRNA levels were determined (FIG. 9A). GK1 and GK3 were most effective at reducing hepatic GK mRNA levels. Glycerol tolerance testing (FIG. 9B) confirmed that these siRNAs also reduced glucose production after glycerol to a similar extent as observed in LGKKO mice (FIG. 6G).

[0121] Sequence Listing (note all sequences are shown 5’ to 3’)SEQ ID NO: 1 - mGKlCUUAGUCAUCAUCAAGUAGAASEQ ID NO: 2 - mGKl-2UUCUACUUGAUGAUGACUAAGAASEQ ID NO: 3- mGK2CCUGUGUAUUAUGCGUUGGAASEQ ID NO: 4 - mGK2-2UUCCAACGCAUAAUACACAGGUUSEQ ID NO: 5 - hGKlGAACAGGACCCUAAGGAAASEQ ID NO: 6 - hGKl-2UUUCCUUAGGGUCCUGUUCCUSEQ ID NO: 7 hGK2AGAGAAAACAUGUGAGAAASEQ ID NO: 8 - hGK2-2UUUCUCACAUGUUUUCUCUAUSEQ ID NO: 9 -hGK3GGAAGAAAGCUGUGAUGAASEQ ID NO: 10 - hGK3-3UUCAUCACAGCUUUCUUCCAUSEQ ID NO: 11 -Murine nucleic acid sequence containing loxp sites before and after exon 3 of GkCCTTGAAATGCTGACAGAGTGAATCTTGTGTAGGCTCTGTCCAGGGAAGCTCAGCCACAGTGAGTTTATGAAGGCAACAACCATGCCATGTCCCGAATAACTTCGTATAGCATACATTATACGAAGTTATGACCTTTTGTGCCATAGCTACTCATCCTTCAGCTCTTGCACTGCCTTCAT CCCCTCTTTGATGTTCCCTGAGTCTTGGAGGGATGCTTAGAAGGCAGTTAACAGCATGTC CTTTTAGCAAAACACTAGTAGTTGGTTTCTTTCTCAGCGTATCGTTCCCAGACATAGGCCCCTGACCAGGTTTGCATCTATGAGAAATTCTTCTTTCATTTTTTAAAAAAAATATTTTTATTTTGTGTTTGGGTGTTTTATCCATATGTATGTCTGTGCAATACATGCATGCCTGCTGTTCATGGAGGCTAGAGGTGTCCATTTTGGAACTGGAGTAACAGAGGGTTGTGGGTGCTGCAAATGGAACCCAGAAACATCCAAGCCATCTCTCCAGCTTGCTTGTTTGTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTCTTTTCTTTCTTTTCTTTCTTTTCTTTCTTTTCTTTTCTTTTC TTTTCTTT CTTTTCTTTTCTTTCTTTCTTTTTGAAAAATTGTCTATAATTTTTATAGACAGGATGCCACATTGTAGCTCAGGTTAGCCTCAAAGTCTGGATTCTTCTGCCCAAGCCTCCAGAGCACTATGCTCTCAGATGTGAGGCCCCATCAGTCCTGGTTATGAGAAGTTATTTCTGGTGTAATATTGCAATTTTTAAGATACATTTTCACATTTTCATATGTATGTATGCTCTCTCTCTCT CTCTCTCTCTCTCTGTGTGTGTGTGTGTGTGTATGTGTGTGTGTGTGTGTGTGTGTGTGTTCGTTTGGAGTTACAGGAAGTTATGAGGCAGCTGAAGTGGCTGCTGGGAAATGAACTTGGATCTTAACCACTGAATCATCTCTACAGCCACCCACACTGTAATGCTTTGTGATTCTGTCTGAAGAATAAGTCAAACATCATTGGTCATCATTCCATATGATTCAGTCTTTTCTACATGTTAATAAGAGCTTTATTTGCTCCATAACTTCAAACAGATTTGATACCAACTTGCATTTTTATACTGATATGTTTTTCAGGTTTTCAATTCAAAAACAGCTGAACTTCTTAGTCATCATCAAGTAGAAATAAAACAGGAATTCCCAAGAGAAGGGTATGTTTCTTAATTTACTATGTAAAGACATATCACATTGATTGGTTTGTCTCACCCCATGTATGCTGTGGATTGATTGGTAGAGTAAACTTAGCCAGACACCTTCTGGAGAAGGCCTGAGAGAATGGGAGTGGTCAAGAAATGAAGCTA GAATGCAGTAATTCAGCAAAAATAGGGGAGCAATGGGTAGCTTAGGCATTCATTGACAGAAAGCAAAGTATAAGTAATGTAACACACACACATAAAGTCTGTATTACAACAAAGGAATATTAACCATCAGAGTCTGCATTAAGTCTGAAGTTTGAGGTGATTTCCTTCTGTGTCCCTGTTACTAGCAGTGCTAAAATGATTACTTTTCAGGAGTGTGTTCTATTTTACCTTTGTTTTTGC CTTTTGGAACCACGATCATATCAATGTACAGACCAGGCTCCCTCTCAAGGGCTGGGATTTGAAGTGTGTGAGCCACTGGTTAATGACCACCCTTAGAAGAAGGATGGATTATTATTAGT CTGTGAAGAATAAGAAAAATGTATAGGAAATAGGTTTTTTTTTATCCCTTTAGTATGTAGAAAACAGATATAATGTATTTATGGAAACATGTTATGTTTGTATTGAATTCTTGAAAACTAACATTTATCCTTTTGTTCCAAAGATGGGTAGAACAAGACCCGAAGGAAATTCTGCAGTCTGTTTATGAGTGTATAGAGAAAACGTGTGAGAAACTTGGACAGCTCAATATTGATATTTCCAACATCAAAGGTATTTTTATTGAGAATTTTCCTCTATAGTCCAGCTCTGCCAATCCCATTCATGAATTCATTCTTTCAGCTTATAATAATTGAGTCTTTATAAGCTGCAAGCCTTTTTGGTTTTTGTTTGT TGTTTGTTTGTTTGTTGGTGAGACTTTGTGGGAAGAACAATAAACAAGATTCTCAGTTCCCGTTCACTGTGACATTTTATTCTGATGCATATGAGAACAATAAAAGACGACTATACAAGTTAATCTTACATACTAACAAATGCCATGTGGGAAATTTTAAAATGAGGAAT ATCATAGGGGTTCCTACATGGCTTAGACACAGTAGGTGGGCACAGGAGGGCTTCTCTTCT CTGAAGAGGTGATTTTTTTTCCTGAGATTGCAAGGGACTTGAGGGGTCAATCATGTGATT GGAGAAAAATATTTGAAGAACAAATAAACAGCCTATGCTGCCATGGTTGGTTGATGTCC CTGGGAGGCCCATTCTTTTCTGAAGGGAAAGGGGGAGGAGTGGGGGAGTGGCTGGGAG GGGAGGAGAGAGGGGAAACTGCAGTCAGGATGTAATATATAAGAGAAAAATCAATTTA AAAGTTAATAAATAAAAATGTAACATTAAAAAATAAAAATAAAAGTTACTGGATCTTTT GTTTATATAGTAGAAATTTAAGTTGGGTTTTAGTCATTCTTTGTTTTCATTTCCCTTGAATT TCTTTATAGGAATGCTTAAAACTATCCAGTTCAGTAGAATATAGTGAGCTGGAGCTCAGG TGGCACTTGAGGGACTTTGAAACAGAACAATGGAAGAGATCTGTGCTTCTCCATTTCATTCTTAGAATTTCTGATGGAATTTCCACTTTCCTGAAGTTTTAATTTACCTTGTTAGTTTTTTT CCCTTTCTTACATTTCTCTAATTTTGCATATGCCTTTTATCATTGTTGGACAATTGTTTCTG TTCCTAATTTTATTTTACTTTTCTTTATTTGGCTTCATGTACTCTTTCCTTATTTATTTTTAA ATTACTTCGTAGCATTTGACTTTGTCTTTTTAACTATTGCATTTTAATCTTTTTATTAATTA AATTTATTCTCTGACAATATCATAAAATACACATCCCATGaATAACTTCGTATAGCATAC ATTATACGAAGTTATCGAAGATCCCAAGGTGGGAATATAAATTGATACAGTCTAGAGTC ACGAAGAAGACTTGTAAAAACAAGTTGAGTGGGCAGAGTGGGTAAGTAAGGAGTTTCAT ATAACAGAGATTGGAGAAATCAGTGAGGCCTTGTCAGTCATATTTAAGAGTTTGAATGTT AGTTTTAGTATCAGAAGGGACAGGTCATGACTCACAATTTAGAGATGGCTACATACCTG

Claims

CLAIMSWhat is claimed is:

1. A method of treating or preventing a disease associated with elevated levels of serum triglycerides (TAGs) and metabolic dysfunction-associated steatotic liver disease (MASLD), wherein the method comprises administering to a subject in need thereof a therapeutically effective amount of a glycerol kinase (GK) inhibitor.

2. The method of claim 1, wherein the disease is hypertriglyceridemia or MASLD.

3. The method of claim 1, wherein the GK inhibitor comprises an antibody or antigenbinding fragment thereof, a small molecule, or a nucleic acid.

4. The method of claim 3, wherein the GK inhibitor comprises a nucleic acid.

5. The method of claim 4, wherein the nucleic acid comprises at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said nucleic acid comprises the nucleic acid sequence pairs as set forth by SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 9 and SEQ ID NO: 10.

6. The method of claim 5, wherein the nucleic acid comprises a chemical modification.

7. The method of claim 6, wherein the chemical modification comprises one or more N- acetyl galactosamine (GalNAc) moieties or derivatives thereof conjugated to the nucleic acid, directly or indirectly via a linker.

8. The method of claim 6, wherein the chemical modification is conjugated to the 3' end of the first strand.

9. The method of claim 6, wherein the nucleic acid comprises at least one modified nucleotide.

10. The method of claim 9, wherein all of the nucleotides in the first and second strands are modified nucleotides.

11. The method of claim 10, wherein: a) the nucleotides at positions 7, 9, 10, and 11 from the 5' end of the first strand are modified with 2’ -deoxy -2’ -fluoro (2’F);b) the nucleotides at positions 1-6, 8, and 12-21 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); c) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’ -deoxy-2’ -fluoro (2’F); d) the nucleotides at positions 1, 3-5, 8-13, 15, and 17-23 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); and e) the nucleotides at position 7 is from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

12. The method of claim 11, wherein the nucleic acid comprises at least one phosphorothioate internucleotide linkage.

13. The method of claim 12, wherein the nucleic acid comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' ends of the second strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the first strand.

14. The method of claim 5, wherein the nucleic acid or pharmaceutical composition thereof is administered at a dose of about 100 mg to about 800 mg.

15. The method of claim 15, wherein the nucleic acid or pharmaceutical composition thereof is administered subcutaneously.

16. The method of claim 15, wherein administration of the nucleic acid or pharmaceutical composition thereof results in about a 70% decrease in serum triglyceride levels.

17. The method of claim 5, wherein the nucleic acid or pharmaceutical composition thereof is administered in combination with another therapeutic agent.

18. The method of claim 17, wherein the second therapeutic agent is selected from the group comprising fibrates, metformin, statins, and niacin.

19. A nucleic acid comprising at least one duplex region that comprises at least a portion of a first strand and at least a portion of a second strand that is at least partially complementary to the first strand, wherein said nucleic acid comprises the nucleic acid sequence pairs as set forth by SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 andSEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or SEQ ID NO: 7 and SEQ ID NO: 8, or SEQ ID NO: 9 and SEQ ID NO: 10.

20. The nucleic acid of claim 19, wherein the nucleic acid comprises a chemical modification.

21. The nucleic acid of claim 20, wherein the chemical modification comprises one or more N-acetyl galactosamine (GalNAc) moieties or derivatives thereof conjugated to the nucleic acid, directly or indirectly via a linker.

22. The nucleic acid of claim 20, wherein the chemical modification is conjugated to the 3' end of the first strand.

23. The nucleic acid of claim 20, wherein the nucleic acid comprises at least one modified nucleotide.

24. The nucleic acid of claim 23, wherein all of the nucleotides in the first strand and second strands are modified nucleotides.

25. The nucleic acid of claim 24, wherein: a) the nucleotides at positions 7, 9, 10, and 11 from the 5' end of the first strand are modified with 2’ -deoxy -2’ -fluoro (2’F); b) the nucleotides at positions 1-6, 8, and 12-21 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); c) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’ -deoxy-2’ -fluoro (2’F); d) the nucleotides at positions 1, 3-5, 8-13, 15, and 17-23 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); and e) the nucleotides at position 7 is from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

26. The nucleic acid of claim 24, wherein: a) the nucleotides at positions 1-4, 6, 10, 11, and 13-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5, 7-9, and 12 from the 5' end of the first strand are modified with 2’ -deoxy-2’ -fluoro (2’F);d) the nucleotides at positions 1, 3, 4, 5, 9-13, 15-19, and 21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); e) the nucleotides at positions 2, 6, 14, and 20 from the 5' end of the second strand are modified with 2’ -deoxy-2’ -fluoro (2’F); f) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

27. The nucleic acid of claim 24, wherein: a) the nucleotides at positions 1-4, 6, 10, and 11-19 from the 5' end of the first strand are modified with 2’-O-methyl (2’-0me); b) the nucleotides at positions 5 and 7-9 from the 5' end of the first strand are modified with 2’ -deoxy-2’ -fluoro (2’F); c) the nucleotides at positions 1, 3, 4, 5, 9-13, 15, and 17-21 from the 5' end of the second strand are modified with 2’-O-methyl (2’-0me); d) the nucleotides at positions 2, 6, 14, and 16 from the 5' end of the second strand are modified with 2’ -deoxy-2’ -fluoro (2’F); e) the nucleotides at position 7 from the 5' end of the second strand is modified with glycol nucleic acid (GNA).

28. The nucleic acid of claim 24, wherein the nucleic acid comprises at least one phosphorothioate internucleotide linkage.

29. The nucleic acid of claim 28, wherein the nucleic acid comprises two consecutive phosphorothioate internucleotide linkages at both the 3' and 5' ends of the second strand and two consecutive phosphorothioate internucleotide linkages at the 5' end of the first strand.

30. A method of treating or preventing a disease associated with elevated levels of serum triglycerides (TAGs) and metabolic dysfunction-associated steatotic liver disease (MASLD), wherein the method comprises administering to a subject in need thereof the nucleic acid or pharmaceutical composition thereof of claim 19.31 . A method for reducing an activity or an expression level of glycerol kinase (GK) in a subject in need thereof comprising administering to a subject in need thereof the nucleic acid or pharmaceutical composition thereof of claim 19.

32. The method of claim 30 or claim 31, wherein the nucleic acid or pharmaceutical composition thereof is administered subcutaneously.

33. The method of claim 30, wherein the disease is hypertriglyceridemia or MASLD.

34. The method of claim 30 or claim 31, wherein administration of the nucleic acid or pharmaceutical composition inhibits mTORCl.

35. A method of reducing cholesterol levels in a subject in need thereof, wherein the method comprises administering the nucleic acid or pharmaceutical composition thereof of claim 19.

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