Nucleic acid constructs for inhibiting GYS2 expression

Abstract

This disclosure relates to a nucleic acid construct useful for reducing GYS2 expression, particularly in hepatocytes. The disclosure also relates to the use of nucleic acid constructs directed to GYS2 to treat Metabolic Syndrome, Type 2 Diabetes, and related conditions.

Description

[0001] NUCLEIC ACID CONSTRUCTS FOR INHIBITING GYS2 EXPRESSION SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety.

[0003] FIELD OF THE INVENTION

[0004] The present application relates to nucleic acid therapeutics and uses thereof, particularly uses relating to the treatment of Metabolic Syndrome, Type 2 Diabetes, and associated conditions.

[0005] BACKGROUND OF THE INVENTION

[0006] Metabolic syndrome is a term used to define a cluster of related medical conditions and pathologies. Typically, the syndrome is diagnosed by the presence of at least three of the five following medical conditions: abdominal obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, and low levels of high-density lipoprotein (HDL) levels. An individual with Metabolic Syndrome is at higher risk of developing cardiovascular disease and Type 2 Diabetes. Presently, a third of the U.S. population is thought to have Metabolic Syndrome and one or more of the listed pathologies. Additional conditions associated with Metabolic Syndrome include hyperuricemia, fatty liver (which may progress to nonalcoholic fatty liver disease), polycystic ovarian syndrome (in women), erectile dysfunction (in men), and acanthosis nigricans. In addition, although metabolic dysfunction-associated steatohepatitis (MASH) is not part of the cluster of disorders which define Metabolic Syndrome, it is also a frequent hepatic complication.Type 2 Diabetes (T2D) is a chronic disease which affects over 500 million adults globally. T2D is primarily characterized by elevated plasma glucose levels due to abnormal glucose metabolism and impaired glycemic control. T2D can present as a component of Metabolic Syndrome, or independently. The human liver significantly influences plasma glucose concentrations by releasing glucose through both glycogen breakdown (glycogenolysis) and de novo synthesis of glucose (gluconeogenesis). In individuals with T2D, elevated plasma glucose levels primarily stem from increased hepatic glucose production, resulting from both gluconeogenesis and glycogenolysis in an insulin resistant liver.

[0007] Glycogen synthase 2 (GYS2) is involved in metabolism and is the enzyme responsible for glycogen synthesis in the liver. It catalyzes the rate-limiting step in the extension of glycogen chains through glycosyl residue transfer. As the primary hepatic isoform of glycogen synthase, strategies for targeting the GYS2 gene to treat diseases associated with altered glucose metabolism are needed.

[0008] SUMMARY OF THE DISCLOSURE

[0009] In one aspect, the disclosure provides a composition including a nucleic acid construct having a first RNA oligonucleotide and a second RNA oligonucleotide, where the first RNA oligonucleotide includes the sequence set forth in SEQ ID NO: 1, and the second RNA oligonucleotide includes the sequence set forth in SEQ ID NO: 2. In certain aspects, the disclosure provides a pharmaceutical composition including the above composition and a pharmaceutically acceptable carrier.

[0010] In yet another aspect, the disclosure provides a method of treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof,the method including administering to the subject any of the above compositions. In another aspect, the disclosure provides a method of treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof, the method including administering to the subject a nucleic acid construct directed to GYS2 (e.g., an siRNA, ASO, or a CRISPR system (e.g., a CRISPR system including a CRISPR (guide) RNA directed to the genomic sequence of GYS2 (e.g., GenBank accession number NG 016167.1))). In certain aspects, the above nucleic acid constructs are directed to a nucleic acid having the sequence set forth in SEQ ID NO: 7.

[0011] In certain of the above methods of treatment, the method further includes administration of a Cas protein or a nucleic acid encoding a Cas protein. In yet another aspect, the above methods include the administration of a Cas protein or a nucleic acid encoding a Cas protein and the CRISPR (guide) RNA in a single composition. Examples of such Cas proteins include a Type II Cas9 protein, Type I Cas3 protein, Type III Cas 10 protein, Type V Cas 12 protein or Type VI Cas 13 protein.

[0012] In some aspects, the disclosure provides a method of treating Metabolic Syndrome. In another aspect, the disclosure provides a method of treating Metabolic Syndrome when Metabolic Syndrome is diagnosed by the presence of at least three of five following conditions in the subject: elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, liver fibrosis, and low levels of high-density lipoprotein (HDL) levels.

[0013] In yet another aspect, the disclosure provides a method of treating a subject with Type 2 Diabetes. In one aspect, the disclosure provides a method of treating a subject with Type 2 Diabetes that occurs in the presence of Metabolic Syndrome. In yet another aspect, the disclosure providesa method of treating a subject with Type 2 Diabetes that occurs in the absence of Metabolic Syndrome.

[0014] BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is a graph depicting fasting blood glucose levels in both wild-type (WT) and diabetic-obese (db / db) mice on day 28 following 5 weekly doses of either PBS (phosphate buffered saline) or 5 mg / kg GalXC-GYS2.

[0016] Figure 2 is a graph depicting Gys2 mRNA levels in both wild-type and db / db mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0017] Figure 3A is a graph depicting hepatic glycogen levels in wild-type and db / db mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0018] Figure 3B is an image of a western blot depicting glycogen synthase (GS) levels in both wild-type and db / db mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0019] Figure 4A and Figure 4B are graphs depicting serum insulin levels in both wild-type and db / db mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0020] Figure 4C and Figure 4D are graphs depicting a homeostatic model assessment measurement for insulin resistance (HOMA-IR) in both wild-type and db / db mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.Figure 5 is a graph depicting a time course of fasted blood glucose levels in both normal chow diet fed mice and a diet-induced obesity mouse model (DIO) following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0021] Figure 6 is a graph depicting Gys2 mRNA levels in both chow fed mice and DIO mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0022] Figure 7 is a graph depicting hepatic glycogen levels in both chow fed mice and DIO mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0023] Figure 8A is a graph depicting insulin levels in both chow fed mice and DIO mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0024] Figure 8B is a graph depicting HOMA-IR levels in both chow fed mice and DIO mice on day 56 following 8 weekly doses of either PBS or 5 mg / kg GalXC-GYS2.

[0025] Figure 9 is a graph depicting a time course of GYS2 mRNA levels in non-human primates (NHPs) at day 84 following 3 doses (days 0, 28 and 56) of either PBS or GalXC-GYS2-1542 at 2 or 6 mg / kg, or a single dose (day 0) of GalXC-GYS2-1542 at 3 mg / kg.Figure 10 is a graph depicting a time course of glycogen synthase protein (GS) levels in NHPs at day 84 following 3 doses (days 0, 28 and 56) of either PBS or GalXC-GYS2-1542 at 2 or 6 mg / kg, or a single dose (day 0) of GalXC-GYS2-1542 at 3 mg / kg.

[0026] Figure 11 is a graph depicting a time course of hepatic glycogen levels in NHPs at day 84 following 3 doses (days 0, 28 and 56) of either PBS or GalXC-GYS2-1542 at 2 or 6 mg / kg, or a single dose (day 0) of GalXC-GYS2-1542 at 3 mg / kg.

[0027] Figure 12 is a graph depicting hepatic triglyceride (TG) levels in NHPs at day 84, following 3 doses (days 0, 28 and 56) of either PBS or GalXC-GYS2-1542 at 2 or 6 mg / kg, or a single dose (day 0) of GalXC-GYS2-1542 at 3 mg / kg.

[0028] DETAILED DESCRIPTION

[0029] The present invention features an anti-GKS2 siRNA molecule, having the SEQ ID NOs: 1 and 2, capable of selectively and potently inhibiting GYS2 expression in human subjects. Furthermore, the present application discloses methods of treating metabolic disorders (e.g., Type 2 Diabetes) using nucleic acid constructs which decrease GYS2 expression, including short interfering RNA (siRNA), antisense oligonucleotides (ASOs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) nucleic acid constructs. This invention is based in part on the observation that in two murine models of Type 2 Diabetes, treatment with an anti-Gys2 siRNA effectively decreases fasted blood glucose and improves insulin sensitivity without inducing deleterious hypoglycemia. Additionally, it has been observed that treatment with an siRNA directed to GYS2 reduces hepatic GYS2 mRNA, GYS2 protein, and glycogen in healthymonkeys without hypoglycemia or hepatic triglyceride accumulation and without inducing any observed adverse effects on liver health or hematological parameters.

[0030] Definitions

[0031] As used herein, “administer,” “administering,” “administration” and the like refers to providing a substance (e.g., a nucleic acid construct) to a subject.

[0032] As used herein, the term “antisense oligonucleotide”, or “ASO”, encompasses a nucleic acid-based molecule which has a sequence complementary to all or part of a target mRNA, and is thereby capable of forming a duplex with a mRNA.

[0033] As used herein, “CRISPR (guide) RNA”, or “gRNA”, refers to a nucleic acid construct including a CRISPR RNA (crRNA) that is complementary to a target DNA region of interest (e.g., an RNA oligonucleotide directed to GYS2 having a region of complementarity to GenBank accession number NG 016167.1), and a transactivating crRNA (tracrRNA) that hybridizes with the crRNA and which provides a stem-loop that binds a Cas nuclease.

[0034] As used herein, “CRISPR system” refers to transcripts and other elements involved in the expression of, or directing the activity of, CRISPR-associated genes (Cas genes). A “CRISPR system” can include a CRISPR (guide) RNA as defined, and a Cas endonuclease, an enzyme which cleaves DNA at the specific location to which it is directed by the CRISPR (guide) RNA.

[0035] As used herein, “directed to GYS2" refers to a nucleic acid construct having a region of complementarity to an mRNA encoding GYS2 or the genomic sequence of GYS2. Nucleic acid constructs of the present invention have regions of complementarity to the GYS2 mRNA transcript set forth in GenBank accession number NM 021957.4 (SEQ ID NO: 7) or the genomic sequence set forth in GenBank accession number NG 016167.1. When a cell expressing GYS2 is contactedwith a nucleic acid construct directed to GYS2, such contacting results in a selective decrease in the amount or level of GYS2 RNA transcript (e.g., GYS2 mRNA) or protein encoded by the GYS2 gene and / or a decrease in the amount or level of activity of GYS2 in a cell, a population of cells, a sample, or a subject, when compared to an appropriate reference (e.g., a reference cell, population of cells, sample or subject). For example, the act of contacting a cell with a nucleic acid herein may result in a decrease in the amount or level of GYS2 mRNA, protein and / or activity when compared to a cell that is not treated with the nucleic acid construct. Similarly, and as used herein, “reducing expression” refers to an act that results in reduced expression of GYS2, and can include a selective decrease in a GYS2. A “selective decrease”, as used herein, refers to a decrease in the amount or levels of a specific RNA transcript or protein in a cell, while not significantly (e.g., by more than 50%, more than 40%, more than 30%, more than 20% or more than 10%) decreasing or modulating the amount or levels of any non-GYS2 RNA transcript or protein. Similarly, as used herein, “selective gene editing ” refers to the ability to modify the GYS2 genomic sequence in a cell in a specific way. Selective gene editing can include adding, removing or changing the genomic sequence in a cell while not editing the DNA of any non-GYS2 genes (e.g. less than 0.5% edit occurring at unintended sites in the genome).

[0036] As used herein, “pharmaceutically acceptable carrier” refers to a non-therapeutic agent that may be included in a formulation of a composition, for example, to provide or contribute to a desired consistency or stabilizing effect.

[0037] As used herein, the term “GYS2” or “glycogen synthase 2” refers to the liver glycogen synthase gene. This gene encodes a protein, liver glycogen synthase, which enzymatically mediates the transfer of a glucose molecule from UDP-glucose to a terminal branch of the glycogen molecule. Homologs of GYS2 are conserved across a range of species, including human, mouse,rat, non-human primate species, and others (see, e.g., NCBI HomoloGene: 56580). In humans, GYS2 encodes multiple transcripts. Nucleic acid constructs of the present disclosure are directed to GYS2, meaning they have a region of complementarity to an mRNA encoding GYS2 or the genomic sequence of GYS2 (e.g., CRISPR (guide) strand directed to GYS2 having a region of complementarity to GenBank accession number NG 016167.1 or an siRNA or ASO directed to GYS2 having a region of complementarity to GenBank accession number NM 021957.4 (SEQ ID NO: 7)).

[0038] As used herein, “Metabolic Syndrome” (sometimes referred to as metabolic liver disease) refers to a disorder characterized by a cluster of related medical conditions and pathologies including any or all, but not limited to, the following medical conditions: Type 2 Diabetes, heart disease, stroke, abdominal obesity, elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, liver fibrosis, and low levels of high-density lipoprotein (HDL) levels.

[0039] As used herein, a “nucleic acid construct”, is an artificially engineered DNA or RNA molecule. Examples of nucleic acid constructs include, but are not limited to, antisense oligonucleotides (ASOs), aptamers, small interfering RNA (siRNA), CRISPR systems.

[0040] As used herein, “oligonucleotide” refers to a short nucleic acid (e.g., less than about 100 nucleotides in length). An oligonucleotide may be single-stranded (ss) or double-stranded (ds). An oligonucleotide may comprise deoxyribonucleosides, ribonucleosides, (including modified nucleosides) or a combination of both.

[0041] As used herein, “RNA oligonucleotide” refers to a short strand of RNA which may or may not have synthetic modifications to its sugar, phosphate, or nitrogenous bases. Synthetic modifications to an RNA oligonucleotide may comprise phosphorothioates, 2’ -Fluoro or 2’-O-methyl modifications or other modifications disclosed herein.As used herein, “short interfering RNA”, or “siRNA”, refers to a double-stranded oligonucleotide having an antisense strand, which is complementary to a target region of mRNA, and a sense strand that is complementary to the antisense strand. While not intending to be bound by any particular theory of the mechanism of siRNA activity, an siRNA works through the RNA interference pathway (RNAi) to effect gene silencing. An siRNA can engage the RNAi pathway upstream or downstream of the RNA-induced silencing complex (RISC) to induce gene silencing through nucleolytic degradation of the target gene through the Argonaut 2 endonuclease (Ago2, Slicer), or by inhibiting gene translation.

[0042] As used herein, “subject” means any mammal, including mice, rabbits, non-human primates (NHP), and humans. In one embodiment, the subject is a human or NHP. Moreover, “individual” or “patient” may be used interchangeably with “subject.”

[0043] As used herein, “treat” or “treating” refers to the act of providing care to a subject in need thereof, for example, by administering a therapeutic agent (e.g., a nucleic acid construct herein) to the subject, for purposes of improving the health and / or well-being of the subject with respect to an existing condition (e.g., a disease, disorder), or to prevent or decrease the likelihood of the occurrence of a condition. In some embodiments, treatment involves reducing the frequency or severity of at least one sign, symptom or contributing factor of a condition (e.g., disease, disorder) experienced by a subject.

[0044] Nucleic Acid Construct Inhibitors of GYS2 Expression

[0045] Nucleic acid constructs of the invention can inhibit expression of GYS2 in a number of ways. They can promote silencing of mRNA encoding GYS2 (e.g., an siRNA nucleic acid construct), block translation of GYS2 mRNA into a protein (e.g., an ASO nucleic acid construct),or modify the genomic DNA of GYS2 to prevent its expression (e.g., using a CRISPR system). Generally, each of these constructs includes a region of complementarity to either the GYS2 mRNA or GYS2 genomic DNA as described herein. Any of the nucleic acid construct types described herein or elsewhere are contemplated for use as a framework to incorporate a GYS2 targeting sequence for the purposes of inhibiting GYS2 expression.

[0046] Complementarity to GYS2 mRNA

[0047] In some embodiments, a nucleic acid construct herein (e.g., an siRNA or ASO) is directed to a target sequence (including a portion thereof) comprising a GYS2 mRNA. In some embodiments, the nucleic acid construct herein (e.g., an siRNA or ASO) have regions of complementarity to GYS2 mRNA (e.g., within a target sequence of GYS2 mRNA) for purposes of targeting the GYS2 mRNA in cells and inhibiting and / or reducing GYS2 expression. In some embodiments, the nucleic acid constructs herein comprise a GYS2 targeting sequence having a region of complementarity that binds or anneals to a GYS2 target sequence by complementary (Watson-Crick) base pairing. The targeting sequence, or region of complementarity, is generally of a suitable length and base content to enable binding or annealing of the nucleic acid construct (or oligonucleotide thereof) to a GYS2 mRNA for purposes of selectively inhibiting and / or reducing GYS2 expression.

[0048] In some embodiments, a nucleic acid construct herein (e.g., an siRNA or ASO) comprises a targeting sequence or a region of complementarity that is fully complementary to a GYS2 target sequence. In some embodiments, the targeting sequence or region of complementarity is partially complementary to a GYS2 target sequence. In some embodiments, the nucleic acid construct comprises a targeting sequence or region of complementarity that is fully complementary to aGYS2 target sequence. In some embodiments, the nucleic acid construct comprises a targeting sequence or region of complementarity that is partially complementary to a GYS2 target sequence.

[0049] In some embodiments, a nucleic acid construct herein (e.g., an siRNA or ASO) comprises a targeting sequence or region of complementarity having one or more base pair (bp) mismatches with the corresponding GYS2 target sequence. In some embodiments, the targeting sequence or region of complementarity may have up to about 1, up to about 2, up to about 3, up to about 4, up to about 5, mismatches with the corresponding GYS2 target sequence provided (1) that the ability of the targeting sequence or region of complementarity to bind or anneal to the GYS2 mRNA under appropriate hybridization conditions and / or the ability of the nucleic acid construct to inhibit GYS2 expression is maintained and (2) the targeting sequence remains selective for reducing GYS2 expression.

[0050] siRNA inhibitors of GYS2

[0051] In some embodiments, the nucleic acid constructs herein are siRNAs which inhibit GYS2 expression by engaging with the RNA interference (RNAi) pathway upstream or downstream of Dicer involvement. siRNAs of various lengths and structures have been developed to engage the RNAi pathway in different ways. For example, siRNA oligonucleotides have been developed with each oligonucleotide having sizes of about 19-25 nucleotides with at least one 3' overhang of 1 to 5 nucleotides see, e.g., US Patent No. 8,372,968 incorporated herein by reference). Longer oligonucleotides have also been developed that are processed by Dicer to generate active siRNA products (see, e.g., US Patent No. 8,883,996 incorporated herein by reference). Further work in the field produced extended dsRNAs, where at least one end of at least one oligonucleotide is extended beyond a duplex targeting region, including structures where one of the oligonucleotidesincludes a thermodynamically stabilizing tetraloop structure (see, e.g., US Patent Nos. 8,513,207 and 8,927,705, as well as Inti. Patent Application Publication No. WO 2010 / 033225, each incorporated herein by reference). Such structures may include single-oligonucleotide extensions (on one or both sides of the molecule) as well as double-oligonucleotide extensions.

[0052] In some embodiments, the nucleic acid constructs herein engage with the RNAi pathway downstream of the involvement of Dicer (e.g., Dicer cleavage). In some embodiments, siRNA described herein are Dicer substrates. In some embodiments, upon endogenous Dicer processing, double-stranded siRNAs of 19-23 nucleotides in length capable of reducing GYS2 expression are produced. In some embodiments, the siRNA comprises a first RNA oligonucleotide (also called a sense strand) comprising a portion of a target RNA sequence that is complementary to a 21-nucleotide RNA oligonucleotide (also called an antisense strand) in which both oligonucleotides anneal to form a 19-bp duplex and 2 nucleotide overhangs at either or both 3' ends. In some embodiments, the siRNA has an overhang (e.g., of 1, 2, or 3 nucleotides in length) in the 3’ end of the antisense strand. Longer siRNA designs are also available, including siRNAs having a first oligonucleotide of 23 nucleotides and a second oligonucleotide of 21 nucleotides, where there is a blunt end on the right side of the molecule (3' end of passenger oligonucleotide / 5' end of guide oligonucleotide) and a two nucleotide 3 '-guide oligonucleotide overhang on the left side of the molecule (5' end of the passenger oligonucleotide / 3' end of the guide oligonucleotide). In such molecules, there is a 21 bp duplex region. See, e.g., US Patent Nos. 9,012,138; 9,012,621 and 9,193,753 each incorporated herein by reference.

[0053] In some embodiments, siRNAs of this disclosure comprise a sense strand and an antisense strand that are both in the range of about 17 to 36 (e.g., 17 to 36, 20 to 25 or 21-23) nucleotides in length. In some embodiments, the siRNAs of this disclosure comprise an antisense strand of 19-30 nucleotides in length and a sense strand of 19-50 nucleotides in length, wherein the sense and antisense strands are separate oligonucleotides which form an asymmetric duplex region having an overhang of 1-4 nucleotides at the 3’ terminus of the antisense strand. In some embodiments, a nucleic acid construct comprises sense and antisense strands, such that there is a 3 '-overhang on the antisense strand.

[0054] In some embodiments, the antisense strand is 15-50 nucleotides in length. In some embodiments, the antisense strand is 15-25 nucleotides in length. In some embodiments, the antisense strand is 22 nucleotides in length. In some embodiments, the antisense strand differs by 1, 2, or 3 nucleotides from the target sequence.

[0055] In some embodiments, the sense strand and the antisense strand are separate oligonucleotides and are not covalently linked. In some embodiments, the sense strand and the antisense strand are covalently linked. In some embodiments, the sense strand and the antisense strand form a duplex region, wherein the sense strand and the antisense strand, or a portion thereof, bind with one another in a complementary fashion (e.g., by Watson-Crick base pairing).

[0056] In some embodiments, a first region (Rl) of the sense strand and the antisense strand form a first duplex (DI). In some embodiments, DI is at least about 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, at least 20 or at least 21) nucleotides in length. In some embodiments, DI is in the range of about 12 to 30 nucleotides in length (e.g., 12 to 30, 12 to 27, 15 to 22, 18 to 22, 18 to 25, 18 to 27, 18 to 30 or 21 to 30 nucleotides in length). In some embodiments, DI is at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 20, at least 25, or at least 30 nucleotides in length). In some embodiments, DI is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length. In some embodiments, DI is 20 nucleotides in length. In some embodiments, DI comprising sense strand and antisensestrand does not span the entire length of the sense strand and / or antisense strand. In some embodiments, DI comprising the sense strand and antisense strand spans the entire length of either the sense strand or antisense strand or both.

[0057] In some embodiments, the sense strand has a second region (R2), wherein R2 comprises a first subregion (S 1), a loop (L), such as a tetraloop (tetraL) or triloop (triL), and a second subregion (S2), wherein L is located between SI and S2, and wherein SI and S2 form a second duplex (D2). D2 may have various lengths. In some embodiments, D2 is about 1-6 bp in length. In some embodiments, D2 is 2-6, 3-6, 4-6, 5-6, 1-5, 2-5, 3-5 or 4-5 bp in length. In some embodiments, D2 is 1, 2, 3, 4, 5 or 6 bp in length. In some embodiments, D2 is 6 bp in length.

[0058] In some embodiments, an siRNA herein comprises a sense strand comprising a stem-loop (L) structure at the 3' end of the sense strand. In some embodiments, the stem-loop is formed by intraoligonucleotide base pairing. In some embodiments, a sense strand comprises a stem-loop structure at its 5' end. In some embodiments, the stem of the stem-loop comprises a duplex of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 2 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 3 nucleotides in length. In some embodiments, the stem of the stem-loop comprises a duplex of 4 nucleotides in length.

[0059] In some embodiments, a stem-loop provides the nucleic acid construct protection against degradation (e.g., enzymatic degradation), facilitates or improves targeting and / or delivery to a target cell, tissue, or organ (e.g., the liver), or both. For example, in some embodiments, the loop of a stem-loop is comprised of nucleotides comprising one or more modifications that facilitate, improve, or increase targeting to a target mRNA (e.g., a GYS2 mRNA), inhibition of target gene expression (e.g., GYS2 expression), and / or delivery, uptake, and / or penetrance into a target cell,tissue, or organ (e.g., the liver), or a combination thereof. In some embodiments, the stem-loop itself or modification(s) to the stem-loop do not affect or do not substantially affect the inherent gene expression inhibition activity of the nucleic acid construct, but facilitates, improves, or increases stability (e.g., provides protection against degradation) and / or delivery, uptake, and / or penetrance of the nucleic acid construct to a target cell, tissue, or organ (e.g., the liver). In certain embodiments, an siRNA herein comprises a sense strand further comprising (e.g., at its 3' end) a stem-loop set forth as: S1-L-S2, in which SI is complementary to S2, and in which L forms a single-oligonucleotide loop of linked nucleotides between SI and S2 of up to about 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length). In some embodiments, the loop (L) is 3 nucleotides in length (referred to herein as a “triloop”). In some embodiments, the loop (L) is 4 nucleotides in length (referred to herein as a “tetraloop”. In some embodiments, the loop (L) is 5 nucleotides in length. In some embodiments, the loop (L) is 6 nucleotides in length. In some embodiments, the loop (L) is 7 nucleotides in length. In some embodiments, the loop (L) is 8 nucleotides in length. In some embodiments, the loop (L) is 9 nucleotides in length. In some embodiments, the loop (L) is 10 nucleotides in length.

[0060] In some embodiments, the tetraloop comprises the sequence 5’-GAAA-3’. In some embodiments, the stem loop comprises the sequence 5’-GCAGCCGAAAGGCUGC-3’ (SEQ ID NO: 6).

[0061] In some embodiments, a loop (L) of a stem-loop having the structure S1-L-S2, as described above, is a tetraloop (tetraL) as describe in US Patent No. 10,131,912 incorporated herein by reference. In some embodiments, the tetraloop comprises ribonucleotides, deoxyribonucleotides, modified nucleotides, ligands (e.g., delivery ligands), and combinations thereof.It should be appreciated that, in some embodiments, sequences presented in the Sequence Listing may be referred to in describing the structure of an RNA oligonucleotide or other nucleic acid construct. In such embodiments, the actual oligonucleotide or other nucleic acid construct may have one or more alternative nucleotides (e.g., an RNA counterpart of a DNA nucleotide or a DNA counterpart of an RNA nucleotide) and / or one or more modified nucleotides and / or one or more modified internucleotide linkages and / or one or more other modification when compared with the specified sequence while retaining essentially same or similar complementary properties as the specified sequence.

[0062] In some embodiments, the siRNAs of this disclosure have one 5' end that is thermodynamically less stable when compared to the other 5' end. In some embodiments, an asymmetric nucleic acid construct is provided that includes a 3 '-overhang at the 3' end of an antisense strand. In some embodiments, the 3 '-overhang on the antisense strand is about 1-8 nucleotides in length (e.g., 1, 2, 3, 4, 5, 6, 7 or 8 nucleotides in length). In some embodiments, the nucleic acid construct has an overhang comprising two (2) nucleotides on the 3' end of the antisense strand. In some embodiments, an overhang is a 3 '-overhang comprising a length of between 1 and 6 nucleotides, optionally 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 6, 2 to 5, 2 to 4, 2 to 3, 3 to 6, 3 to 5, 3 to 4, 4 to 6, 4 to 5, 5 to 6 nucleotides, or 1, 2, 3, 4, 5 or 6 nucleotides.

[0063] In some embodiments, two (2) terminal nucleotides on the 3' end of an antisense strand are modified. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand are complementary with the target RNA (e.g., GYS2 mRNA). In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand are not complementary with the target RNA. In some embodiments, the two (2) terminal nucleotides on the 3' end of the antisense strand of a nucleic acid construct herein are unpaired. In some embodiments, the two (2) terminalnucleotides on the 3' end of the antisense strand of a nucleic acid construct herein comprise an unpaired GG. In some embodiments, the two (2) terminal nucleotides on the 3' end of an antisense strand of a nucleic acid construct herein are not complementary to the target RNA. In some embodiments, two (2) terminal nucleotides on each 3' end of a nucleic acid construct are GG. In some embodiments, one or both of the two (2) terminal GG nucleotides on each 3' end of a nucleic acid construct herein is not complementary with the target RNA.

[0064] In some embodiments, there is one or more (e.g., 1, 2, 3, 4 or 5) mismatch(s) between a sense and antisense strand of an siRNA. If there is more than one mismatch between a sense and antisense strand, they may be positioned consecutively (e.g., 2, 3 or more in a row), or interspersed throughout the region of complementarity. In some embodiments, the 3' end of the sense strand comprises one or more mismatches. In some embodiments, two (2) mismatches are incorporated at the 3' end of the sense strand. In some embodiments, base mismatches, or destabilization of segments at the 3' end of the sense strand of a nucleic acid construct herein improves or increases the potency of the construct.

[0065] Antisense Oligonucleotide Inhibitors of GYS2

[0066] In some embodiments, the present invention comprises an antisense oligonucleotide (ASO). ASOs are single stranded oligonucleotides which can be DNA or RNA. ASOs inhibit target gene expression by binding a target mRNA sequence within a cell to cause RNA cleavage or RNA blockage. The binding of a target mRNA sequence by an ASO can alter gene expression in a number of ways. One such mechanism is through the formation of RNA-DNA heteroduplexes between the ASO and the complementary mRNA sequence. Binding of the ASO to the mRNA sequence triggers the recruitment of the enzyme RNase H, which cleaves the RNA strand within the hybridized molecule, leading to the degradation of the targeted mRNA. This degradationinterrupts gene translation and results in reduced protein expression from the targeted gene. The binding of a target mRNA sequence by an ASO can also block ribosome binding and thereby inhibit target gene translation resulting in reduced protein expression. ASOs can comprise chemical modifications including, but not limited to, those described below (e.g., phosphorothioates, 2’ -Fluoro or 2’-O-methyl modifications).

[0067] ASO’s of the present disclosure are directed to GYS2 and comprise a region of complementarity to GYS2 mRNA. ASO’s of the present disclosure bind GYS2 mRNA and inhibit gene expression, resulting in reduced GYS2 protein expression in cells. Methods for designing ASOs that are selective for an individual gene (e.g., GYS2) are well understood in the art.

[0068] CRISPR System Inhibitors of GYS2

[0069] In some embodiments, the present invention comprises a CRISPR system comprising a CRISPR (guide) RNA (gRNA) that includes a CRISPR RNA (crRNA) that is complementary to a region of target DNA, and a transactivating crRNA (tracrRNA). In some embodiments the CRISPR system comprises a CRISPR (guide) RNA fused to a scaffold sequence from a transactivating CRISPR RNA (tracrRNA), connected by a short loop, forming a hairpin-like structure with key regions including the repeat, anti-repeat, and stem loop domains sequences encoding a Cas gene. In some embodiments the CRISPR (guide) RNA directs a CRISPR-associated (Cas) nuclease to a specific DNA sequence to be cut. In some embodiments, the guide RNA functionality includes the nicking of a target RNA sequence by a gRNA: Cas protein complex. In some embodiments, the guide RNA functionality includes the cleaving of a target RNA sequence by a gRNA:Cas protein complex. In some embodiments, the guide RNA functionality is any otherknown function of a guide RNA in a CRISPR-Cas system with a Cas protein, including an artificial CRISPR-Cas system with an engineered Cas protein.

[0070] In some embodiments, the Cas protein is a type II CRISPR system protein. In some embodiments, the Cas protein is a Cas9 protein. In some embodiments, the Cas9 protein is S. pneumoniae, S. pyogenes, or S. therm ophilus Cas9, and may include mutated Cas9 derived from these organisms. In some embodiments, the Cas protein is a type I CRISPR system protein. In some embodiments, the Cas protein is a Cas3 protein. In some embodiments, the Cas protein is a type III CRISPR system protein. In some embodiments, the Cas protein is a CaslO protein. In some embodiments, the Cas protein is a type V CRISPR system protein. In some embodiments, the Cas protein is a Casl2 protein. In some embodiments, the Cas protein is a type VI CRISPR system protein. In some embodiments, the Cas protein is a Cas 13 protein.

[0071] In certain embodiments, the compositions of the invention feature a nucleic acid construct including a CRISPR (guide) RNA which comprises a crRNA sequence is that is complementary to a target DNA region of interest (e.g. GYS2 DNA, GenBank accession number NG_016167.1) and directs the Cas nuclease there for editing to result in genomic modifications that disrupt expression of GYS2 in cells. Methods for designing CRISPR (guide) strands are well understood in the art.

[0072] RNA Oligonucleotide Modifications

[0073] In some embodiments, a nucleic acid construct described herein in comprises at least one RNA oligonucleotide (for example, within an siRNA, in certain ASO constructs and in certain CRISPR (guide) RNAs). RNA oligonucleotides may be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance from nuclease degradation,immunogenicity, base-pairing properties, RNA distribution and cellular uptake and other features relevant to therapeutic or research use.

[0074] In some embodiments, the modification is a modified sugar. In some embodiments, the modification is a 5 ’-terminal phosphate group. In some embodiments, the modification is a modified internucleotide linkage. In some embodiments, the modification is a modified base. In some embodiments, an RNA oligonucleotide described herein can comprise any one of the modifications described herein or any combination thereof. For example, in some embodiments, an RNA oligonucleotide described herein comprises at least one modified sugar, a 5 ’-terminal phosphate group, at least one modified internucleotide linkage, and at least one modified base.

[0075] The number of modifications on an RNA oligonucleotide and the position of those nucleotide modifications may influence the properties of an oligonucleotide. For example, oligonucleotides may be delivered in vivo by conjugating them to or encompassing them in a lipid nanoparticle (LNP) or similar carrier. However, when an RNA oligonucleotide is not protected by an LNP or similar carrier, it may be advantageous for at least some of the nucleotides to be modified. Accordingly, in some embodiments, all or substantially all the nucleotides of RNA oligonucleotide are modified. In some embodiments, more than half of the nucleotides are modified. In some embodiments, less than half of the nucleotides are modified. In some embodiments, the sugar moiety of all nucleotides comprising the RNA oligonucleotide is modified at the 2’ position. The modifications may be reversible or irreversible. In some embodiments, an RNA oligonucleotide as disclosed herein has a number and type of modified nucleotides sufficient to cause the desired characteristics (e.g., protection from enzymatic degradation, capacity to target a desired cell after in vivo administration, and / or thermodynamic stability).Sugar Modifications

[0076] In some embodiments, an RNA oligonucleotide described herein comprises a modified sugar. In some embodiments, a modified sugar (also referred herein to a sugar analog) includes a modified deoxyribose or ribose moiety in which, for example, one or more modifications occur at the 2', 3', 4' and / or 5' carbon position of the sugar. In some embodiments, a modified sugar may also include non-natural alternative carbon structures such as those present in locked nucleic acids (“LNA”; see, e.g., Koshkin et al. (1998) TETRAHEDON 54:3607-30), unlocked nucleic acids (“UNA”; see, e.g., Snead etal. (2013) MOL. THER-NUCL. ACIDS 2:el03) and bridged nucleic acids (“BNA”; see, e.g., Imanishi & Obika (2002) CHEM COMMUN. (CAMB) 21:1653-59).

[0077] In some embodiments, a nucleotide modification in a sugar comprises a 2'-modification. In some embodiments, a 2'-modification may be 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-fluoro (2'-F), 2'-aminoethyl (EA), 2'-O-methyl (2'-OMe), 2'-O-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA) or 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA). In some embodiments, the modification is 2'-F, 2'-OMe or 2'-M0E. In some embodiments, a modification in a sugar comprises a modification of the sugar ring, which may comprise modification of one or more carbons of the sugar ring. For example, a modification of a sugar of a nucleotide may comprise a 2'-oxygen of a sugar is linked to a 1 '-carbon or 4'-carbon of the sugar, or a 2'-oxygen is linked to the 1 '-carbon or 4'-carbon via an ethylene or methylene bridge. In some embodiments, a modified nucleotide has an acyclic sugar that lacks a 2'-carbon to 3'-carbon bond. In some embodiments, a modified nucleotide has a thiol group, e.g., in the 4' position of the sugar.

[0078] In some embodiments, an RNA oligonucleotide described herein comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, atleast 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, or more). In some embodiments, the sense strand of the nucleic acid construct comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, or more). In some embodiments, the antisense strand of the nucleic acid construct comprises at least about 1 modified nucleotide (e.g., at least 1, at least 5, at least 10, at least 15, at least 20, or more).

[0079] In some embodiments, all the nucleotides of the sense strand of the oligonucleotide are modified. In some embodiments, all the nucleotides of the antisense strand of the oligonucleotide are modified. In some embodiments, all the nucleotides of the nucleic acid construct (z.e., both the sense strand and the antisense strand) are modified. In some embodiments, the modified nucleotide comprises a 2'-modification (e.g., a 2'-F or 2'-OMe, 2'-M0E, and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid).

[0080] In some embodiments, the disclosure provides RNA oligonucleotides having different modification patterns. In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand having a modification pattern as set forth in the Examples and Sequence Listing and an antisense strand having a modification pattern as set forth in the Examples and Sequence Listing.

[0081] In some embodiments an RNA oligonucleotide disclosed herein comprises an antisense strand having nucleotides that are modified with 2'-F. In some embodiments, an RNA oligonucleotide disclosed herein comprises an antisense strand comprising nucleotides that are modified with 2'-F and 2'-OMe. In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2'-F. In someembodiments, an RNA oligonucleotide disclosed herein comprises a sense strand comprising nucleotides that are modified with 2'-F and 2'-0Me.

[0082] In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand with about 10-15%, 10%, 11%, 12%, 13%, 14% or 15% of the nucleotides of the sense strand comprising a 2’-fluoro modification. In some embodiments, about 11% of the nucleotides of the sense strand comprise a 2-fluoro modification. In some embodiments, an RNA oligonucleotide disclosed herein comprises an antisense strand with about 25-35%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34% or 35% of the nucleotides of the antisense strand comprising a 2’-fluoro modification. In some embodiments, about 32% of the nucleotides of the antisense strand comprise a 2’ -fluoro modification. In some embodiments, the oligonucleotide has about 15-25%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, or 25% of its nucleotides comprising a 2’ -fluoro modification. In some embodiments, about 19% of the nucleotides in the oligonucleotide comprise a 2’ -fluoro modification.

[0083] In some embodiments, one or more of positions 8, 9, 10 or 11 of the sense strand is modified with a 2'-F group. In some embodiments, one or more of positions 2, 3, 4, 5, 7, 10 and 14 of the antisense strand is modified with a 2'-F group. In some embodiments, the sugar moiety at each of nucleotides at positions 1-7, 12-27 and 31-36 in the sense strand is modified with a 2'-OMe. In some embodiments, the sugar moiety at each of nucleotides at positions 1, 6, 8, 9, 11-13 and 15-22 in the antisense strand is modified with a 2'-OMe.

[0084] In some embodiments, an RNA oligonucleotide disclosed herein comprises an antisense strand having the sugar moiety of each of the nucleotides at positions 2, 3, 4, 5, 7, 10, and 14 of the antisense strand modified with 2'-F and the sugar moiety of each of the remaining nucleotides of the antisense strand modified with a modification selected from the group consisting of 2'-O-propargyl, 2'-O-propylamin, 2'-amino, 2'-ethyl, 2'-aminoethyl (EA), 2'-0-methyl (2'-0Me), 2'-0-methoxyethyl (2'-M0E), 2'-O-[2-(methylamino)-2-oxoethyl] (2'-0-NMA), and 2'-deoxy-2'-fluoro-P-d-arabinonucleic acid (2'-FANA).

[0085] In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand having the sugar moiety at positions 8-11 modified with 2'-F. In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand having the sugar moiety at positions 1-7, 12-27 and 31-36 modified with 2’0Me.

[0086] 5 ’-Terminal Phosphate

[0087] In some embodiments, an RNA oligonucleotide disclosed herein comprises a sense strand and an antisense strand, wherein the antisense strand comprises a 5 ’-terminal phosphate. In some embodiments, 5 '-terminal phosphate groups of an siRNA enhance the interaction with Ago2. However, oligonucleotides comprising a 5 '-phosphate group may be susceptible to degradation via phosphatases or other enzymes, which can limit their performance and / or bioavailability in vivo. In some embodiments, an RNA oligonucleotide herein includes analogs of 5' phosphates that are resistant to such degradation. In some embodiments, the phosphate analog is oxymethyl phosphonate, vinyl phosphonate or malonyl phosphonate, or a combination thereof. In certain embodiments, the 5' terminus of an RNA oligonucleotide is attached to chemical moiety that mimics the electrostatic and steric properties of a natural 5 '-phosphate group (phosphate mimic).

[0088] In some embodiments, an RNA oligonucleotide herein has a phosphate analog at a d'carbon position of the sugar (referred to as a “4'-phosphate analog”). See, e.g., Inti. Patent Application Publication No. WO 2018 / 045317. In some embodiments, an RNA oligonucleotide herein comprises a 4'-phosphate analog at a 5 '-terminal nucleotide. In some embodiments, aphosphate analog is an oxymethyl phosphonate, in which the oxygen atom of the oxymethyl group is bound to the sugar moiety (e.g., at its 4'-carbon) or analog thereof. In other embodiments, a d'phosphate analog is a thiomethylphosphonate or an aminomethylphosphonate, in which the sulfur atom of the thiomethyl group or the nitrogen atom of the amino methyl group is bound to the d'carbon of the sugar moiety or analog thereof. In certain embodiments, a 4'-phosphate analog is an oxymethyl phosphonate. In some embodiments, an oxymethyl phosphonate is represented by the formula -O-CH2-PO(OH)2,-O-CH2-PO(OR)2, or -0-CH2-P0(0H)(R), in which R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, ClfcOCIfcCIfcSi (C 143)3 or a protecting group. In certain embodiments, the alkyl group is CH2CH3. More typically, R is independently selected from H, CH3 or CH2CH3. In some embodiments, R is CH3. In some embodiments, the 4’ -phosphate analog is 4’ -oxymethyl phosphonate.

[0089] In some embodiments, an RNA oligonucleotide disclosed herein comprises an antisense strand comprising a 4'-phosphate analog at the 5 '-terminal nucleotide, wherein 5 ’-terminal nucleotide comprises the following structure (Chem 1):

[0090]

[0091] Chem 1

[0092] 4’-O-monomethylphosphonate-2’-O-methyluridine phosphorothioate [MePhosphonate-40-mUs],Modified Internucleotide Linkage

[0093] In some embodiments, an RNA oligonucleotide disclosed herein comprises a modified internucleotide linkage. In some embodiments, phosphate modifications or substitutions result in an RNA oligonucleotide that comprises at least about 1 (e.g., at least 1, at least 2, at least 3 or at least 5) modified internucleotide linkage. In some embodiments, any one of the oligonucleotides disclosed herein comprises about 1 to about 10 (e.g., 1 to 10, 2 to 8, 4 to 6, 3 to 10, 5 to 10, 1 to 5, 1 to 3 or 1 to 2) modified intemucleotide linkages. In some embodiments, any one of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified internucleotide linkages.

[0094] A modified internucleotide linkage may be a phosphorodithioate linkage, a phosphorothioate linkage, a phosphotriester linkage, a thionoalkylphosphonate linkage, a thionalkylphosphotriester linkage, a phosphoramidite linkage, a phosphonate linkage or a boranophosphate linkage. In some embodiments, at least one modified internucleotide linkage of any one of the oligonucleotides as disclosed herein is a phosphorothioate linkage.

[0095] In some embodiments, an RNA oligonucleotide provided herein has a phosphorothioate linkage between one or more of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 3 and 4 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand. In some embodiments, an RNA oligonucleotide described herein has a phosphorothioate linkage between each of positions 1 and 2 of the sense strand, positions 1 and 2 of the antisense strand, positions 2 and 3 of the antisense strand, positions 20 and 21 of the antisense strand, and positions 21 and 22 of the antisense strand.N-acetylgalactosanune (GalNAc) Ligands

[0096] GalNAc is a high affinity carbohydrate ligand for the asialoglycoprotein receptor (ASGPR), which is primarily expressed on the surface of hepatocyte cells and has a major role in binding, internalizing and subsequent clearing circulating glycoproteins that contain terminal galactose or GalNAc residues (asialoglycoproteins). Conjugation (either indirect or direct) of GalNAc moieties to nucleic acid constructs of the instant disclosure can be used to target these nucleic acid constructs to the ASGPR expressed on cells. In some embodiments, a nucleic acid construct of the instant disclosure (e.g., an siRNA or ASO) is conjugated to at least one or more GalNAc moieties, wherein the GalNAc moieties target the oligonucleotide to an ASGPR expressed on human liver cells (e.g., human hepatocytes). In some embodiments, the GalNAc moiety target the oligonucleotide to the liver.

[0097] In some embodiments, a nucleic acid construct of the instant disclosure (e.g., an siRNA or ASO) is conjugated directly or indirectly to a monovalent GalNAc moiety. In some embodiments, the nucleic acid construct is conjugated directly or indirectly to more than one monovalent GalNAc (z.e., is conjugated to 2, 3 or 4 monovalent GalNAc moieties and is typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, a nucleic acid construct is conjugated to one or more bivalent GalNAc, trivalent GalNAc or tetravalent GalNAc moieties. In some embodiments, a bivalent, trivalent or tetravalent GalNAc moiety is conjugated to a nucleic acid construct via a branched linker. In some embodiments, a monovalent GalNAc moiety is conjugated to a first nucleotide and a bivalent, trivalent, or tetravalent GalNAc moiety is conjugated to a second nucleotide via a branched linker.In some embodiments, one (1) or more (e.g., 1, 2, 3, 4, 5 or 6) nucleotides of a nucleic acid construct described herein (e.g., an siRNA or ASO) are each conjugated to a GalNAc moiety. In some embodiments, two (2) to four (4) nucleotides of a tetraloop are each conjugated to a separate GalNAc moiety. In some embodiments, one (1) to three (3) nucleotides of a triloop are each conjugated to a separate GalNAc moiety. In some embodiments, targeting ligands are conjugated to two (2) to four (4) nucleotides at either ends of a sense or antisense strand (e.g., ligands are conjugated to a two (2) to four (4) nucleotide overhang or extension on the 5' or 3' terminus of a sense or antisense strand) such that the GalNAc moieties resemble bristles of a toothbrush and the nucleic acid construct resembles a toothbrush. In some embodiments, GalNAc moieties are conjugated to a nucleotide of the sense strand. For example, three (3) or four (4) GalNAc moieties can be conjugated to nucleotides in the tetraloop of the sense strand where each GalNAc moiety is conjugated to one (1) nucleotide.

[0098] In some embodiments, a nucleic acid construct described herein (e.g., an siRNA) comprises a tetraloop, wherein the tetraloop (tetraL) is any combination of adenine (A) and guanine (G) nucleotides. In some embodiments, the tetraloop (tetraL) comprises a monovalent GalNAc moiety attached to any one or more guanine (G) nucleotides of the tetraloop via any linker described herein, as depicted below in Chem 2 (X=heteroatom):

[0099]

[0100] Chem 2

[0101] In some embodiments, the tetraloop has a monovalent GalNAc attached to any one or more adenine nucleotides of the tetraloop via any linker described herein, as depicted below in Chem 3 (X=heteroatom):

[0102]

[0103] Chem 3

[0104] In some embodiments, a nucleic acid construct herein comprises a monovalent GalNAc moiety attached to a guanine (G) nucleotide referred to as [ademG-GalNAc] or 2'-aminodiethoxymethanol-Guanine-GalNAc, as depicted below in Chem 4:

[0105]

[0106] Chem 4

[0107] In some embodiments, a nucleic acid construct herein comprises a monovalent GalNAc moiety attached to an adenine nucleotide, referred to as [ademA-GalNAc] or 2'-aminodiethoxymethanol-Adenine-GalNAc, as depicted below in Chem 5:

[0108]

[0109] Chem 5An example of such conjugation is shown below for a loop comprising from 5' to 3' the nucleotide sequence GAAA (L = linker, X = heteroatom). Such a loop may be present, for example, at positions 27-30 of a sense strand provided herein. In the chemical formula,

[0110]

[0111] is used to describe an attachment point to the nucleic acid construct (Chem 6).

[0112]

[0113] Chem 6

[0114] Appropriate methods or chemistry (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotidecomprising a nucleic acid construct herein using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the nucleic acid constructs described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. W02016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is stable. An example is shown below for a loop comprising from 5' to 3' the nucleotides GAAA, in which GalNAc moi eties are attached to nucleotides of the loop using an acetal linker (Chem 7 and Chem 8). Such a loop may be present,

[0115] for example, at positions 27-30 of the any one of the sense strands. In the chemical formula,

[0116]

[0117] is an attachment point to the nucleic acid construct.

[0118]

[0119] Chem 7

[0120]

[0121] Chem 8As mentioned, various appropriate methods or chemistry synthetic techniques (e.g., click chemistry) can be used to link a targeting ligand to a nucleotide. In some embodiments, a targeting ligand is conjugated to a nucleotide using a click linker. In some embodiments, an acetal-based linker is used to conjugate a targeting ligand to a nucleotide of any one of the nucleic acid constructs described herein. Acetal-based linkers are disclosed, for example, in Inti. Patent Application Publication No. WO 2016 / 100401. In some embodiments, the linker is a labile linker. However, in other embodiments, the linker is a stable linker.

[0122] Exemplary Nucleic Acid Constructs for Reducing GYS2 Expression

[0123] In some embodiments, a nucleic acid construct provided herein is an siRNA and includes a first RNA oligonucleotide having a sequence of SEQ ID NO: 4 and a second RNA oligonucleotide including a sequence of SEQ ID NO: 5.

[0124] In some embodiments, the first RNA oligonucleotide having a sequence of SEQ ID NO: 4 of this construct includes a 2'-F modified nucleotide at positions 8-11, a 2'-0Me modified nucleotide at positions 1-7, 12-27, and 31-36, a GalNAc-conjugated nucleotide at position 28, 29 and 30; and a phosphorothioate linkage between positions 1 and 2.

[0125] In some embodiments the second RNA oligonucleotide having a sequence of SEQ ID NO: 5 of this construct includes a 2'-F modified nucleotide at positions 2, 3, 4, 5, 7, 10 and 14, a 2'-OMe at positions 1, 6, 8, 9, 11-13, and 15-22, a phosphorothioate linkage between positions 1 and 2, positions 2 and 3, positions 3 and 4, positions 20 and 21, and positions 21 and 22, and a 5’-terminal nucleotide at position 1 comprising a 4’-phosphate analog, optionally wherein the 5’-terminal nucleotide includes 4’-O-monomethylphosphonate-2’-O-methyluridine [MePhosphonate-4O-mU]; where positions 1-20 of the antisense strand form a duplex region withpositions 1-20 of the first oligonucleotide, where positions 21-36 of the first oligonucleotide form a stem-loop, where positions 27-30 form the loop of the stem-loop, optionally where positions 27-30 form a tetraloop, and positions 21 and 22 of the antisense strand form an overhang.

[0126] In some embodiments, the disclosure provides a nucleic acid construct that is an siRNA directed to GYS2, wherein the nucleic acid construct includes a chemical modification pattern of the first strand (e.g., a sense strand having the sequence of SEQ ID NO: 1) and a second strand (e.g., an antisense strand having the sequence of SEQ ID NO: 2) as indicated below:

[0127] Sense Strand: 5 ’ -mX-X-m X-mX-m X-m X-m X-m X-fX-fX-fX-fX-m X-m X-mX-m X-m X-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX-mX- [ademX-GalNAc] -[ademX-GalNAc] - [ademX-GalNAc]-mX-mX-mX-mX-mX-mX- 3’;

[0128] hybridized to:

[0129] Antisense Strand: S’-fMePhosphonate-dO-mXJ-X-fX-X-fX- X-fX-fX-mX-fX-mX-mX-fX-mX-mX-mX-fX-mX-mX-mX-mX-mX-mX-X-mX-X-mX-3 ’ ;

[0130] wherein mX= 2’-( -methyl modified nucleotide, fX =2’- fluoro modified nucleotide, -S- = phosphorothioate linkage, - = phosphodiester linkage, [MePhosphonate-4O-mX] = 4’-O-monomethylphosphonate-2’-O-methyluridine, and ademX-GalNAc = GalNAc attached to a nucleotide.

[0131] In some of the above embodiments, a nucleic acid construct directed to GYS2 provided by the disclosure includes a first strand having the nucleotide sequence as set forth in SEQ ID NO: 1 and a second strand having the nucleotide sequence as set forth in SEQ ID NO: 2.Formulations

[0132] Various formulations (e.g., pharmaceutical formulations) have been developed for nucleic acid constructs use. For example, nucleic acid constructs (e.g., an siRNA or ASO) can be delivered to a subject or a cellular environment using a formulation that minimizes degradation, facilitates delivery and / or uptake, or provides another beneficial property to the oligonucleotides in the formulation. In some embodiments, provided herein are compositions comprising nucleic acid constructs (e.g., siRNA, ASO or CRIPSR system) reduce the expression of GYS2. Such compositions can be suitably formulated such that when administered to a subject, either into the immediate environment of a target cell or systemically, a sufficient portion of the nucleic acid constructs to enter the cell to reduce GYS2 expression. Any variety of suitable nucleic acid construct formulations can be used to deliver oligonucleotides for the reduction of GYS2 as disclosed herein. In some embodiments, a nucleic acid construct is formulated in a sterile aqueous solution. In some embodiments, a nucleic acid construct is formulated in buffer solutions such as phosphate buffered saline solution.

[0133] Methods of Use

[0134] Reducing GYS2 Expression

[0135] In some embodiments, the disclosure provides methods for contacting or delivering to a cell or population of cells an effective amount of a nucleic acid construct provided herein (e.g., an siRNA, ASO or CRIPSR system) to reduce GYS2 expression. In some embodiments, a reduction of GYS2 expression is determined by measuring a reduction in the amount or level of GYS2 mRNA, GYS2 protein, or GYS2 activity in a cell. The methods include those described herein and known to one of ordinary skill in the art.Methods provided herein are useful in any appropriate cell type. In some embodiments, a cell is any cell that expresses GYS2 mRNA (e.g., hepatocytes). In some embodiments, the cell is a primary cell obtained from a subject. In some embodiments, the primary cell has undergone a limited number of passages such that the cell substantially maintains its natural phenotypic properties. In some embodiments, a cell to which the nucleic acid construct is delivered is ex vivo or in vitro (i.e., can be delivered to a cell in culture or to an organism in which the cell resides).

[0136] In some embodiments, the nucleic acid constructs herein are delivered to a cell or population of cells using a nucleic acid delivery method known in the art including, but not limited to, injection of a solution containing the nucleic acid constructs, bombardment by particles covered by the nucleic acid constructs, exposing the cell or population of cells to a solution containing the nucleic acid constructs, or electroporation of cell membranes in the presence of the nucleic acid constructs. Other methods known in the art for delivering nucleic acid constructs to cells may be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection such as calcium phosphate, and others.

[0137] In some embodiments, reduction of GYS2 expression is determined by an assay or technique that evaluates one or more molecules, properties, or characteristics of a cell or population of cells associated with GYS2 expression, or by an assay or technique that evaluates molecules that are directly indicative of GYS2 expression in a cell or population of cells (e.g., GYS2 mRNA or GYS2 protein). In some embodiments, the extent to which a nucleic acid construct provided herein reduces GYS2 expression is evaluated by comparing GYS2 expression in a cell or population of cells contacted with the oligonucleotide to an appropriate control (e.g., an appropriate cell or population of cells not contacted with the nucleic acid construct or contacted with a control nucleic acid construct). In some embodiments, a control amount or level of GYS2expression in a control cell or population of cells is predetermined, such that the control amount or level need not be measured in every instance the assay or technique is performed. The predetermined level or value can take a variety of forms. In some embodiments, a predetermined level or value can be single cut-off value, such as a median or mean.

[0138] In some embodiments, contacting or delivering a nucleic acid construct described herein to a cell or a population of cells results in a reduction in GYS2 expression in a cell or population of cells not contacted with the nucleic acid construct or contacted with a control nucleic acid construct. In some embodiments, the reduction in GYS2 expression is about 1% or lower, about 5% or lower, about 10% or lower, about 15% or lower, about 20% or lower, about 25% or lower, about 30% or lower, about 35% or lower, about 40% or lower, about 45% or lower, about 50% or lower, about 55% or lower, about 60% or lower, about 70% or lower, about 80% or lower, or about 90% or lower relative to a control amount or level of GYS2 expression. In some embodiments, the control amount or level of GYS2 expression is an amount or level of GYS2 mRNA and / or GYS2 protein in a cell or population of cells that has not been contacted with a nucleic acid construct herein. In some embodiments, GYS2 mRNA expression is measured using methods known in the art. In some embodiments, GYS2 mRNA expression is measured by qPCR. In some embodiments, GYS2 protein expression is measured using methods known in the art. In some embodiments GYS2 protein expression is measured by ELISA. In some embodiments, GYS2 protein expression is measured by western blot. In some embodiments, the effect of delivery of a nucleic acid construct herein to a cell or population of cells according to a method herein is assessed after any finite period or amount of time (e.g., minutes, hours, days, weeks, months).Treatment Methods

[0139] The disclosure provides nucleic acid constructs for use as a medicament, in particular for use in a method for the treatment of diseases, disorders, and conditions associated with, or modulated by, expression of GYS2. The disclosure also provides nucleic acid constructs for use, or adaptable for use, to treat a subject (e.g., a human having a disease, disorder or condition associated with GYS2 expression) that would benefit from reducing GYS2 expression. In some respects, the disclosure provides nucleic acid constructs for use, or adapted for use, to treat a subject having a disease, disorder or condition associated with expression of GYS2. The disclosure also provides nucleic acid constructs for use, or adaptable for use, in the manufacture of a medicament or pharmaceutical composition for treating a disease, disorder or condition associated with GYS2 expression. In some embodiments, the nucleic acid constructs for use, or adaptable for use, target GYS2 mRNA and reduce GYS2 expression (e.g., via the RNAi pathway). In some embodiments, the nucleic acid constructs for use, or adaptable for use, target GYS2 mRNA and reduce the amount or level of GYS2 mRNA, GYS2 protein and / or GYS2 activity.

[0140] In some embodiments, GYS2 expression, the amount or level of GYS2 mRNA, GYS2 protein, GYS2 activity, or a biomarker related to or affected by modulation of GYS2 expression, or any combination thereof, is reduced in a cell (e.g., a hepatocyte), a population or a group of cells (e.g., an organoid), an organ (e.g., liver), blood or a fraction thereof (e.g., plasma), a tissue (e.g., liver tissue), a sample (e.g., a liver biopsy sample), or any other appropriate biological material obtained or isolated from the subject. In some embodiments, GYS2 expression, the amount or level of GYS2 mRNA, GYS2 protein, GYS2 activity, or a biomarker related to or affected by modulation of GYS2 expression, or any combination thereof, is reduced in more than one type of cell (e.g., a hepatocyte and one or more other type(s) of cell), more than one groups ofcells, more than one organ (e.g., liver and one or more other organ(s)), more than one fraction of blood (e.g., plasma and one or more other blood fraction(s)), more than one type of tissue (e.g., liver tissue and one or more other type(s) of tissue), or more than one type of sample (e.g., a liver biopsy sample and one or more other type(s) of biopsy sample).

[0141] In addition, in some embodiments of the methods herein, a subject having a disease, disorder, or condition associated with GYS2 expression or is predisposed to the same is selected for treatment with a nucleic acid construct provided herein (e.g., an siRNA, ASO or CRISPR system). In some embodiments, the method comprises selecting an individual having a marker (e.g., a biomarker) for a disease, disorder, or condition associated with GYS2 expression or predisposed to the same.

[0142] The disclosure also provides methods of treating a subject having, suspected of having, or at risk of developing a disease, disorder or condition associated with GYS2 expression (e.g., Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes as described below) with a nucleic acid construct provided herein. In some aspects, the disclosure provides methods of treating or attenuating the onset or progression of a disease, disorder or condition associated with GYS2 expression using the nucleic acid constructs herein. In some embodiments of the methods herein, the subject is treated by administering a therapeutically effective amount of any one or more of the nucleic acid constructs provided herein. In some embodiments, treatment comprises reducing GYS2 expression. In some embodiments, the subject is treated therapeutically. In some embodiments, the subject is treated prophylactically.

[0143] Examples of a disease, disorder or condition associated with GYS2 expression include, but are not limited to, Metabolic Syndrome, a condition associated with metabolic syndrome and Type 2 Diabetes. Metabolic Syndrome is diagnosed based on the presence of three or more of thefollowing associated conditions: elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, liver fibrosis, and low levels of high-density lipoprotein (HDL) levels. Patients with Metabolic Syndrome are at higher risk for Type 2 Diabetes, but T2D may also occur independently and is diagnosed and characterized by elevated blood glucose levels. In some embodiments, other conditions associated with metabolic syndrome include hyperuricemia, fatty liver (which may progress to nonalcoholic fatty liver disease), polycystic ovarian syndrome (in women), erectile dysfunction (in men), and acanthosis nigricans. In addition, although metabolic dysfunction-associated steatohepatitis (MASH) is not part of the cluster of disorders which define Metabolic Syndrome, it is also a frequent hepatic complication.

[0144] In some embodiments, a subject is administered any one of the compositions herein (e.g., a composition comprising an nucleic acid construct described herein) either enterally (e.g., orally, by gastric feeding tube, by duodenal feeding tube, via gastrostomy or rectally), parenterally (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intraosseous infusion, intramuscular injection, intracerebral injection, intracerebroventricular injection, intrathecal), topically (e.g., epicutaneous, inhalational, via eye drops, or through a mucous membrane), or by direct injection into a target organ (e.g., the liver of a subject). Typically, oligonucleotides herein are administered intravenously or subcutaneously.

[0145] Kits

[0146] In some embodiments, the disclosure provides a kit comprising a nucleic acid construct herein (e.g., an siRNA, ASO or CRISPR system), and instructions for use. In some embodiments, the kit comprises a nucleic acid construct herein, and a package insert containing instructions for use of the kit and / or any component thereof. In some embodiments, the kit comprises, in a suitablecontainer, a nucleic acid construct herein, one or more controls, and various buffers, reagents, enzymes and other standard ingredients well known in the art. In some embodiments, the container comprises at least one vial, well, test tube, flask, bottle, syringe, or other container means, into which the oligonucleotide is placed, and in some instances, suitably aliquoted. In some embodiments where an additional component is provided, the kit contains additional containers into which this component is placed. The kits can also include a means for containing the oligonucleotide and any other reagent in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained. Containers and / or kits can include labeling with instructions for use and / or warnings.

[0147] In some embodiments, a kit comprises a nucleic acid construct herein, and a pharmaceutically acceptable carrier, or a pharmaceutical composition comprising the oligonucleotide and instructions for treating or delaying progression of a disease, disorder or condition associated with GYS2 expression in a subject in need thereof.

[0148] In some embodiments, a kit comprises a CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. A CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0149] LIST OF FURTHER EMBODIMENTS OF THE INVENTION

[0150] The invention is further described by the following non-limiting embodiments:

[0151] 1. A composition comprising a nucleic acid construct having a first RNA oligonucleotide and a second RNA oligonucleotide, wherein the first RNA oligonucleotide comprises the sequence setforth in SEQ ID NO: 1, and the second RNA oligonucleotide comprises the sequence set forth in SEQ ID NO: 2.

[0152] 2. The composition of embodiment 1, wherein the first RNA oligonucleotide consists of the sequence set forth in SEQ ID NO: 1.

[0153] 3. The composition of embodiment 1 or 2, wherein the second RNA oligonucleotide consists of the sequence set forth in SEQ ID NO: 2.

[0154] 4. A pharmaceutical composition comprising the composition according to any one of embodiments 1-3, and a pharmaceutically acceptable carrier.

[0155] 5. A method of treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof, the method comprising administering to the subject the composition of any one of embodiments 1-3 or the pharmaceutical composition of embodiment 4.

[0156] 6. A method of treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof, the method comprising administering to the subject a nucleic acid construct directed to GYS2.

[0157] 7. The method of embodiment 5 or 6, wherein the method is a method of treating Metabolic Syndrome.8. The method of any one of embodiment 7, wherein the Metabolic Syndrome is diagnosed by the presence of at least three of five following conditions in the subject: elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, liver fibrosis, and low levels of high-density lipoprotein (HDL) levels.

[0158] 9. The method of embodiment 5 or 6, wherein the method is a method of treating Type 2 Diabetes.

[0159] 10. The method of embodiment 9, wherein the Type 2 Diabetes occurs in a subject with Metabolic Syndrome.

[0160] 11. The method of embodiment 9, wherein the Type 2 Diabetes occurs in a subject in the absence of Metabolic Syndrome.

[0161] 12. The method of any one of embodiments 6 to 11, wherein said nucleic acid construct is directed to a nucleic acid having the sequence set forth in SEQ ID NO: 7.

[0162] 13. The method of any one of embodiments 6 to 12, wherein the nucleic acid construct directed to GYS2 is an siRNA.

[0163] 14. The method of any one of embodiments 6 to 11, wherein the nucleic acid construct directed to GYS2 is an ASO.15. The method of any one of embodiments 6 to 11 , wherein the nucleic acid construct directed to GYS2 comprises a CRISPR (guide) RNA which comprises a region of complementarity to the genomic sequence of GYS2 (GenBank accession number NG 016167.1).

[0164] 16. The method of embodiment 15, wherein the method further comprises administering a Cas protein or a nucleic acid encoding a Cas protein.

[0165] 17. The method of embodiment 16, wherein the Cas protein or nucleic acid encoding a Cas protein and the CRISPR (guide) RNA are administered in a single composition.

[0166] 18. The method of embodiment 16 or 17, wherein the Cas protein is a Type II Cas9 protein.

[0167] 19. The method of embodiment 16 or 17, wherein the Cas protein is a Type I Cas3 protein.

[0168] 20. The method of embodiment 16 or 17 wherein the Cas protein is a Type III CaslO protein.

[0169] 21. The method of embodiment 16 or 17 wherein the Cas protein is a Type V Casl2 protein.

[0170] 22. The method of embodiment 16 or 17 wherein the Cas protein is a Type VI Casl3 protein.

[0171] EXAMPLESExample 1. The Effects of Inhibition of Gys2 on Blood Glucose and Insulin Levels in a db / db Mouse Model of Obesity

[0172] A study was conducted to assess the effects of reduction of Gys2 mRNA on blood glucose and insulin levels in both healthy mice (wild-type or wild-type BKS) and a mouse model of obesity and diabetes db / db). Additionally, Gys2 mRNA and liver glycogen content were also quantified.

[0173] Experimental Design

[0174] Twenty-seven male wildtype BKS and twenty-seven male db / db mice aged 5 weeks were purchased from Jackson Laboratories. Animals were maintained on a standard mouse chow diet (NIH #3 IM). After one week of acclimation, lean and obese mice were randomly assigned to study groups. Groups were housed 3 mice per cage, as shown in

[0175] Table

[0176] Table 1: Experimental Design

[0177]

[0178] Abbreviations: PBS = phosphate buffered saline; SC = subcutaneous

[0179] On study days 0, 7, 14, 21, 28, 35, 42, and 49, mice received a subcutaneous (SC) dose of either PBS or GalXC-GYS2 (a mouse active siRNA targeting Gys2) at 5mg / kg, for a total of eight doses.

[0180] Methods

[0181] Fasted Blood Glucose Measurements

[0182] Fasted blood glucose was measured on study days 0, 14, and 42 after the mice were fasted for 6 hours. A small volume (~2 ul) of whole blood obtained from a tail vein puncture using a point-of-care glucometer, the Contour Next EZ (Ascensia), was used to determine blood glucose. Additionally, animals were fasted for 6 hours prior to necropsy on day 56, and terminal serum samples were used to determine fasted glucose for this date.

[0183] Glucose Tolerance Test

[0184] On study day 28, a glucose tolerance test (GTT) was performed on all study animals. After 6 hours of fasting, the mice were administered Ig / kg D-glucose via subcutaneous injection, and blood glucose was determined at 15, 30-, 60-, 90-, and 120-minutes post glucose administration as described above. Data were analysed by comparing the area under the curve (AUC) of blood glucose over time, where the curve baseline is the fasted blood glucose level.

[0185] RT-qPCRAt terminal dissection, approximately 50 mg of liver was flash-frozen on dry ice and stored at -80 degrees C for RT-qPCR analysis. An additional 50 mg of liver was placed in a tissue cassette and fixed in 10% neutral buffered formalin for 48 hours prior to paraffin embedding.

[0186] To determine levels of Gys2 mRNA by RT-qPCR, approximately 10 mg liver tissue was homogenized in 0.75 mL phenol / guanidine-based QIAzol® Lysis Reagent (Qiagen ®, Valencia, CA) using a Tissuelyser II (Qiagen®). The homogenate was extracted with lbromo3 chloropropane (Sigma-Aldrich, St. Louis, MO). RNA was extracted from the aqueous phase using the MagMax Technology (ThermoFisher Scientific) according to the manufacturer’s instructions. RNA was quantified using spectrometry at 260 and 280 nm. A highcapacity cDNA reverse transcription kit (ThermoFisher Scientific, catalog no:4368814) was used to prepare cDNA. RT-qPCR assays from ThermoFisher Scientific and Integrated DNA Technologies (IDT; Coralville, IA) were used with reagents from Bio-Rad according to the manufacturer’s instructions (catalog no: 172-5281, Bio-Rad, Hercules, CA) to measure mRNA expression (see Table 2 for primer sets) with normalization to hypoxanthineguanine phosphoribosyl transferase (Hprf) mRNA levels. RTqPCR experiments were run in triplicate for each sample. Reduction in mRNA expression in the GalXC-GYS2 treated groups were calculated as the percent expression relative to the time-matched PBS chow group using log 10 transformed AACT values. Data were analyzed with GraphPad Prism (GraphPad version 9.0, La Jolla, CA) using an unpaired ttest to determine significance. Statistical differences between the PBS chow group and the HFD PBS or the HFD GalXC-GYS2 treated group were evaluated by two-tailed t-test. Graphs of mean ± standard error of the mean (SEM) were generated using GraphPad Prism.

[0187]

[0188] Table 2: RT-qPCR Primers

[0189]

[0190] Western Blot

[0191] To determine levels of glycogen synthase protein by Western Blot, approximately 10 mg of liver tissue was homogenized in TPER (ThermoFisher Scientific, catalog no: 78510) with added protease inhibitors (ThermoFisher Scientific, catalog no: A32959). Total protein was quantified using the Pierce 660 nm reagent per manufacturer’s instructions (ThermoFisher Scientific, catalog no: 23208). Tissue homogenate was diluted to 2 ug of total protein per uL with additional TPER and mixed with LDS sample buffer (ThermoFisher Scientific, catalog no: NP0007) per manufacturer’s instructions. Samples were heated to 70 degrees C for 10 minutes, then allowed to cool to room temperature, and 15 ul (equivalent to 30ug total protein) was loaded onto NuPAGENovex 4-12% Bis-Tris Midi protein gels (ThermoFisher Scientific, catalog no: WG1403BOX).

[0192] Proteins were separated on these gels with NuPAGE Sample reducing reagent (ThermoFisher Scientific, catalog no: NP0004) and NuPAGE MES SDS Running Buffer (ThermoFisher Scientific, catalog no: NP0002) per manufacturer’s instructions. Licor Molecular Weight Marker (Licor, catalog no: 928-40000) was used as a molecular weight standard. After gel separation, proteins were transferred to nitrocellulose membranes using iBlot Nictrocellulose transfer stacks (Life Technologies, catalog no: IB3010-01) per manufacturer’s instructions and blocked in Odyssey blocking buffer (Licor, catalog no: 927-40000) before incubating with antibodies listed in Table 3. Proteins were visualized and quantified using the Odyssey imaging system (Licor) per manufacturer’s instructions.

[0193] Table 3: Western Blot Antibodies

[0194]

[0195] Liver Glycogen Measurements

[0196] To determine liver glycogen content, approximately 25 mg of liver tissue was homogenized in 1 ml of PBS per 25 mg of tissue. Tissue homogenate was heated to 95 degrees C for 5 minutes and allowed to cool to room temperature. Supernatant containing glycogen was isolated by centrifugation at 13,000xg for 5 minutes. Supernatant was assayed for glycogen content using the Glycogen Assay Kit (Sigma Aldrich catalog no: MAK016) and SpectraMax i3x Plate reader(Molecular Devices) per manufacturer’s instructions. Glycogen content was normalized to initial tissue weights.

[0197] Serum Insulin Measurements

[0198] At terminal dissection approximately 1 ml of whole blood was collected by cardiac puncture and transferred to a serum collection tube. Blood was allowed to clot for 30 minutes at room temperature before serum separation was performed by spinning samples at 2000 x g. Serum was removed and stored at -80 degrees C for analysis.

[0199] Serum insulin was quantified using the Mouse / Rat Insulin Kit (MSD catalog no: K152BZC-1) and MSD 600 instrument (Meso Scale Discovery) per manufacturer’s instructions. Homeostasis Model Assessment of Insulin Resistance (HOMA-IR) was calculated with the following formula:

[0200] HOMA-IR = (insulin in mU / L * glucose in mg / dL) / 405

[0201] Results

[0202] Wild-type BKS animals dosed with GalXC-GYS2 had a significant reduction in fasted blood glucose as compared to PBS-dosed animals beginning at study day 14 and persisting throughout the remainder of the study. BKS animals dosed with GalXC-GYS2 demonstrated impaired glucose clearance as determined by AUC of glucose measurements taken during the glucose tolerance test on study day 28. Hypoglycemia (blood glucose <65 mg / dl) was not observed. This is illustrated in Figure 1. db / db animals dosed with GalXC-GYS2 or PBS control demonstrated extremely high blood glucose (>300 mg / dL), beginning at week 2 post-treatment, as expected with this model. Due to inaccuracy of the glucometer at these high glucose readinglevels, it was not possible to determine if GalXC-GYS2 treatment had an effect on blood glucose during the course of the experiment, nor during the glucose tolerance test at study day 28. At study termination, serum glucose was determined and was observed to be significantly reduced in GalXC-GYS2 treated db / db animals compared to PBS-treated control animals. Hypoglycemia (blood glucose <65 mg / dl) was not observed.

[0203] As shown in Figure 2, wild-type BKS animals dosed with GalXC-GYS2 had a significant reduction in mRNA expression of Gys2 compared to PBS dosed animals (reduction of 91.46%; p < 0.0001). db / db animals had lower Gys2 mRNA expression compared to wild-type animals (69.91% reduction), but treatment with GalXC-GYS2 significantly reduced this expression further, showing a 98.02% (reduction p < 0.0001) compared to wild-type control animals.

[0204] Both wild-type BKS and db / db animals dosed with GalXC-GYS2 had levels of hepatic glycogen and glycogen synthase (GS) protein that were undetectable. This is illustrated by Figure 3A and Figure 3B.

[0205] Wild-type BKS animals dosed with GalXC-GYS2 showed a decrease in serum insulin (p=0.0008) and a reduction in HOMA-IR at study termination on day 56 (p=0.0042). GalXC-GYS2 treated db / db animals show no significant change in fasted insulin or HOMA-IR (Figures 4A-D). db / db animals showed an increase in fasted insulin and HOMA-IR compared to wild-type controls.

[0206] The db / db animals did effectively model a Type 2 Diabetes phenotype based on elevated fasted blood glucose and increased serum insulin and HOMA-IR, which together suggest insulin insensitivity. However, extremely high fasting blood glucose in this model impaired our ability to measure fasted blood glucose at interim timepoints and to assess glucose clearance via GTT. Despite this, it was determined that reduction of Gys2 mRNA and protein using GalXC-GYS2resulted in reduction of liver glycogen and lower fasting blood glucose levels in this model. Similar data were observed in the wild-type BKS mice. No group of treated animals showed hypoglycaemia or liver triglyceride accumulation.

[0207] In summary, eight (8) weekly doses of GalXC-GYS2 significantly reduced Gys2 mRNA levels and reduced both glycogen synthase protein and hepatic glycogen to undetectable levels in wild-type and db / db animals. Wild-type animals dosed with GalXC-GYS2 demonstrated reduced fasted blood glucose, insulin, and HOMA-IR, and showed reduced glucose clearance in a glucose tolerance test, db / db animals, which model T2D, show reduced fasting blood glucose. No deleterious hypoglycemia was observed during the study in any group. In conclusion, GalXC-GYS2 effectively decreases fasted blood glucose in healthy animals and in an animal model of Type 2 Diabetes.

[0208] Example 2. The Effects of Inhibition of Gys2 on Blood Glucose and Insulin Levels in a DIO Mouse Model

[0209] A study was conducted to assess the effects of reduction of Gys2 mRNA in both healthy mice and a mouse model of obesity and diabetes (diet-induced obese mice, or DIO) on blood glucose and insulin levels. Additionally, Gys2 mRNA and liver glycogen content were also quantified.

[0210] Experimental Design

[0211] As illustrated by the experimental design shown in Table 4, twenty-nine male C57BL / 6NTac mice, 12 weeks of age, were purchased from Taconic. Twenty-four of these mice had been maintained on high-fat diet (HFD, 60% kcal, Research Diets #D 12492) from 6 weeks ofage and 5 control animals were fed a diet of standard mouse chow (NIH #3 IM). For the remainder of the study, mice were maintained on these respective diets. HFD-fed mice were randomly assigned to study groups (3 mice per cage).

[0212] Table 4: Experimental Design

[0213]

[0214] Abbreviations: HFD = D12492 60% high fat diet; PBS = phosphate buffered saline; SC = subcutaneous

[0215] On study days 0, 7, 14, 21, 28, 35, 42, and 49 mice received an SC dose of either PBS or GalXC-GYS2 (a mouse active siRNA targeting Gys2) at 5 mg / kg, for a total of 8 doses.

[0216] Methods

[0217] For detailed methods on determining fasted blood glucose, glucose tolerance testing, RT- qPCR, Western Blot, liver glycogen measurements and serum insulin measurements, see Example 1

[0218] Results

[0219] Animals dosed with GalXC-GYS2 had a significant reduction in fasted blood glucose compared to PBS-dosed DIO animals beginning at study day 14 and persisting throughout theremainder of the study as shown in Figure 5. Animals dosed with GalXC-GYS2 did not have impaired glucose clearance as determined by AUC of glucose measurements taken during the glucose tolerance test on study day 28. DIO animals showed a significant increase in glucose clearance as determined by AUC compared to PBS-dosed chow fed animals.

[0220] As shown in Figure 6, DIO animals dosed with GalXC-GYS2 had a significant reduction in mRNA expression of Gys2 as compared to PBS dosed DIO animals (97.22%; p < 0.0001; 2). In response to the HFD, a decrease in Gys2 mRNA was observed compared to PBS-dosed chow diet animals.

[0221] DIO animals dosed with GalXC-GYS2 did not have significant liver triglyceride content compared to PBS-dosed DIO animals. As expected, DIO animals showed a significant increase in liver triglycerides compared to PBS-dosed, chow fed animals. DIO animals dosed with GalXC-GYS2 had undetectable levels of hepatic glycogen as illustrated by Figure 7.

[0222] DIO animals dosed with GalXC-GYS2 showed no significant change in serum insulin 15 minutes after a 1 g / kg glucose challenge on study day 42, however they do demonstrate a reduction in HOMA-IR at this time point (p=0.0257). At study termination on day 56, GalXC-GYS2 treated animals show a significant reduction in fasted insulin (p < 0.0001) and HOMA-IR (p < 0.0001). DIO animals showed a significant increase in fasted insulin and HOMA-IR compared to PBS-dosed chow fed controls. This data is represented by Figure 8A and Figure 8B.

[0223] In this study, the DIO animals did effectively model an early-stage Type 2 Diabetes phenotype, based on elevated fasted blood glucose, impaired glucose clearance in the glucose tolerance test, and increased serum insulin and HOMA-IR, which together suggest insulin insensitivity. In summary, eight (8) weekly doses of GalXC-GYS2 significantly reduced Gys2 mRNA levels (97%) and reduced both glycogen synthase protein and hepatic glycogen toundetectable levels. DIO animals dosed with GalXC-GYS2 demonstrated reduced fasted blood glucose, insulin, and HOMA-IR, and showed no defect in glucose clearance in a glucose tolerance test. No deleterious hypoglycemia was observed during the study. In conclusion, GalXC-GYS2 effectively decreases fasted blood glucose and improves insulin sensitivity in an animal model of Type 2 Diabetes.

[0224] Example 3: The Effect of Multiple Administrations of GalXC-GYS2-1542 in Lean Cynomolgus Primates

[0225] A set of N-acetylgalactosamine small interfering RNAs (GalNAc siRNAs) were designed to reduce GYS2 mRNA expression via an RNAi mechanism of action in hepatocytes (see, US Patent No. 11,572,562 incorporated herein by reference). This study was designed to evaluate the efficacy of one of these compounds, GalXC-GYS2-1542, in reducing mRNA and protein expression of GYS2, and to understand the potential impact of hepatocyte specific GYS2 knockdown on hepatic lipids, hepatic glycogen content, and glycemia in lean, healthy non-human primates.

[0226] Experimental Design

[0227] As illustrated by the experimental design in Table 5, lean, healthy cynomolgus monkeys (non-human primates or NHPs) were randomly assigned to 4 groups of 4 animals.

[0228] Table 5: Experimental Design

[0229]

[0230]

[0231] Abbreviations: PBS = phosphate buffered saline; SC = subcutaneous

[0232] Animals were administered either 3 monthly doses of GalXC-GYS2-1542 test article at 2 or 6 mg / kg, or PBS (control) by subcutaneous (SC) injection on study days 0, 28, and 56, or administered a single 3 mg / kg SC injection GalXC-GYS2-1542 on study day 0 (as shown in Table 5).

[0233] Methods

[0234] Predose liver biopsies were collected from animals in all groups. Liver biopsies were collected from the animals in all groups on study days 0, 28, 56, and 84. Biopsies were stored for RT-qPCR, protein, triglyceride, and glycogen analysis. Effects on GYS2 mRNA expression,glycogen synthase protein, hepatic glycogen and triglyceride content was quantified in all liver biopsy samples.

[0235] Blood and serum samples were collected on study days 0, 28, 56, and 84. Blood was analysed for glucose, HbAlc, and haematological parameters including neutrophils, white and red blood cells, and haemoglobin. Serum samples were used to evaluate the effects of GalXC-GYS2- 1542 on liver function alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) enzyme levels, cholesterol, triglycerides, glycose, insulin, lactate, and ketones.

[0236] Liver Glycogen Measurement

[0237] Approximately 10 mg of frozen liver tissue from study days 0, 28, and 56 was homogenized in 0.35 ml of PBS and 100 ul of lysate was removed for analysis by Western Blot. Remaining tissue homogenate was heated to 95 degrees C for 5 minutes and allowed to cool to room temperature. Supernatant containing glycogen was isolated by centrifugation at 13,000 x g for 5 minutes. Supernatant was assayed for glycogen content using the Glycogen-Glo Assay Kit (Promega catalog no: J5052) per manufacturer’s instructions. Glycogen content was normalized to lysate protein content as determined by Pierce 660 nm assay.

[0238] RT-qPCR

[0239] To determine GYS2 levels by RT-qPCR, liver biopsies stored in RNAlater from study days 0, 28, 56, and 84 (approximately 8-20 mg) were used for RNA isolation. Tissues samples were homogenized in 0.70 mL phenol / guanidine-based QIAzol® Lysis Reagent (Qiagen®, Valencia, CA) using a Tissuelyser II (Qiagen®). The homogenate was extracted with lbromo3 chloropropane (Sigma-Aldrich, St. Louis, MO). RNA was extracted from aqueous phaseusing the MagMax Technology (ThermoFisher Scientific) according to the manufacturer’s instructions. RNA was quantified using spectrometry at 260 and 280 nm. A highcapacity cDNA reverse transcription kit (catalog no:4368814, ThermoFisher Scientific) was used to prepare cDNA. RT-qPCR assays from ThermoFisher Scientific and Integrated DNA

[0240] Technologies (IDT; Coralville, IA) were used with reagents from Bio-Rad (catalog no: 172-5281 or 172-5271, Bio-Rad, Hercules, CA) to measure GYS2 mRNA expression according to the manufacturer’s instructions (see Table 6 for primer sequences) with normalization to peptidylprolyl isomerase B (PPIB) mRNA levels. RTqPCR experiments were run in triplicate and the average % expression calculated for each sample. Reduction in mRNA expression in the GalXC-GYS2-1542 treated groups was calculated as the percent expression relative to the time- matched PBS control group and pre-dose samples. Data were analyzed with GraphPad Prism (GraphPad version 10.0, La Jolla, CA) using an unpaired t-test to determine significance. Graphs of mean ± standard error of the mean (SEM) were generated using GraphPad Prism (GraphPad).

[0241]

[0242] (2A(average CT Target Gene — average CT Housekeeping Gene))

[0243] X 100 = % Expression NR1H3

[0244]

[0245] Table 6 : RT-qPCR primer sequences

[0246]

[0247] Western Blot

[0248] To measure glycogen synthase protein by Western Blot, samples from study days 0, 28, and 56, 100 ul of liver homogenate in PBS was mixed with lOOul of TPER (ThermoFisher Scientific, catalog no: 78510) with added protease inhibitors (ThermoFisher Scientific, catalog no: A32959). For day 84 samples approximately 10 mg of liver tissue was homogenized in TPER (ThermoFisher Scientific, catalog no: 78510) with added protease inhibitors (ThermoFisher Scientific, catalog no: A32959). Total protein was quantified using the Pierce 660 nm reagent per manufacturer’s instructions (ThermoFisher Scientific, catalog no: 23208). Tissue homogenate was diluted to 2 ug of total protein per uL with additional TPER and mixed with LDS sample buffer (ThermoFisher Scientific, catalog no: NP0007) per manufacturer’s instructions. Samples were heated to 70 degrees C for 10 minutes, then allowed to cool to room temperature, and 15ul (equivalent to 30 ug total protein) was loaded onto NuPAGE Novex 4-12% Bis-Tris Midi protein gels (ThermoFisher Scientific, catalog no: WG1403BOX). Proteins were separated on these gels with NuPAGE Sample reducing reagent (ThermoFisher Scientific, catalog no: NP0004) and NuPAGE MES SDS Running Buffer (ThermoFisher Scientific, catalog no: NP0002) per manufacturer’s instructions. Licor Molecular Weight Marker (Licor, catalog no: 928-40000) was used as a molecular weight standard. After gel separation, proteins were transferred to nitrocellulose membranes using iBlot Nictrocellulose transfer stacks (Life Technologies, catalog no: IB3010-01) per manufacturer’s instructions and blocked in Odyssey blocking buffer (Licor, catalog no: 927-40000) before incubating with antibodies listed in Table 7. Proteins were visualized and quantified using the Odyssey imaging system (Licor) per manufacturer’s instructions.Table 7: Western Blot Antibodies

[0249]

[0250] Liver Triglyceride Measurements

[0251] To measure liver triglyceride content, samples from study day 84, approximately 25 mg of liver tissue, were homogenized in 1ml of PBS per 25 mg of tissue. Tissue homogenate was assayed immediately for triglyceride content using the Triglyceride-Glo Assay Kit (Promega catalog no: J3 161) according to manufacturer’s instructions. Remaining tissue homogenate was heated to 95 degrees C for 5 minutes and allowed to cool to room temperature. Supernatant containing glycogen was isolated by centrifugation at 13,000 x g for 5 minutes. Supernatant was assayed for glycogen content using the Glycogen-Glo Assay Kit (Promega catalog no: J5052) per manufacturer’s instructions. Glycogen and triglyceride content were normalized to lysate protein content as determined by Pierce 660 nm assay.Circulating Blood Parameters

[0252] See Table 8 for a list of circulating parameters evaluated and the assay format used for analysis. Approximately 150ul of serum from each animal was aliquoted on dry ice for additional analysis (ALP, AST, ALT, cholesterol, glucose, and triglycerides).

[0253] Table 8: Circulating Parameters Evaluated

[0254]

[0255] aAll assays were run according to the manufacturer’s instructions

[0256] Results

[0257] As shown in Figure 9, significant reduction of liver GYS2 mRNA was achieved after a single 2, 3, or 6 mg / kg dose of GalXC-GYS2-1542 (55.75%, 41.80%, and 68.38%, respectively on study day 28). GYS2 mRNA reduction was sustained through day 84 in the 2 and 6 mg / kg multiple dose groups (63.23% and 75.8%, respectively), while recovery was observed at day 84 in the 3 mg / kg single dose group at day 84 (10.11% reduction). Maximum reduction in GYS2 mRNA expression was observed on study day 84 and was 63.23% (p < 0.0001) and 75.8% (p < 0.0001) for the 2 and 6 mg / kg groups, respectively, relative to the time matched PBS group.

[0258] Figure 10 illustrates that glycogen synthase protein (GS) was reduced at day 28 and sustained through day 84 in all groups. Reduction of glycogen synthase protein was observed following a single 2, 3, or 6 mg / kg dose of GalXC-GYS2-1542 (reduction of 69.1%, 71.1%, and 85.5%, respectively on study day 28). GS protein was further reduced through day 84 in the 2 and6 mg / kg multiple dose groups (90.0% and 92.3%, respectively) and was maintained at day 84 in the 3 mg / kg single dose group (64.3% reduction). Maximum reduction in GS protein was observed on study day 84 and was 90.0% (p < 0.0001) and 92.3% (p < 0.0001) for the 2 and 6 mg / kg groups, respectively, relative to the time matched PBS group. No effects on liver function (liver enzymes), liver TG, circulating lipids, glucose levels, HbAlc, serum insulin, or haematological parameters were detected in blood and plasma samples. Administration of GalXC-GYS2-1542 did not result in any significant body weight change.

[0259] Reduction of hepatic glycogen was observed following a single 2, 3, or 6 mg / kg dose of GalXC-GYS2-1542 (62.9%, 79.6%, and 76.4%, respectively, on study day 28). Glycogen reduction was further reduced at day 84 in the 2 mg / kg multiple dose group (80.4%) and maintained in the 6 mg / kg multiple dose group (74.4%) and the 3 mg / kg single dose group (84.3%) as illustrated by Figure 11. Additionally, as shown in Figure 12, administration of GalCX-GYS2-1542 did not result in any changes in hepatic triglyceride content in healthy monkeys at study day 84.

[0260] Administration of GalXC-GYS2-1542 did not result in any dose-dependent changes in neutrophil levels, white blood cells (WBCs), lymphocytes (LYMPH), eosinophils (EOS), basophils (BASO), red blood cells (RBCs), or hemoglobin subunit g (HBG) concentrations) in healthy monkeys.

[0261] In this study, treatment with GalXC-GYS2-1542 successfully reduces hepatic GYS2 mRNA, GYS2 protein, and glycogen in the livers of healthy animals. Results from this study indicate that treatment with GalXC-GYS2-1542 and subsequent reduction in hepatic glycogen do not cause fasting hypoglycemia. GalXC-GYS2-1542 successfully reduces hepatic GYS2 mRNA, protein, and glycogen in healthy monkeys without hypoglycemia or hepatic triglycerideaccumulation and without inducing any observed adverse effects on liver health or hematological parameters.

[0262] Reference is made to U.S. Patent Applications Nos. 8,372,968; 8,883,996; 8,513,207; 8,927,705; 9,012,138; 9,012,621; 10,131,912; 11,572,562. Reference is also made to Inti. Patent Application Publication No. WO 2010 / 033225.

[0263] The above applications, and all documents cited in those applications or during their prosecution ("application cited documents") and all documents cited or referenced in those application cited documents, and all documents cited or referenced herein ("herein cited documents") and all documents cited or referenced in the herein cited documents, are hereby incorporated by reference and may be used in the practice of the present invention, together with any manufacturer's instructions, manuals, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein. More specifically, all references are incorporated by reference to the extent that each individual document is individually and specifically indicated to be incorporated by reference.

[0264] Table 9: Sequences

[0265]

[0266]

[0267]

[0268]

Claims

CLAIMS1. A composition comprising a nucleic acid construct having a first RNA oligonucleotide and a second RNA oligonucleotide, wherein the first RNA oligonucleotide comprises the sequence set forth in SEQ ID NO: 1, and the second RNA oligonucleotide comprises the sequence set forth in SEQ ID NO: 2.

2. The composition of claim 1, wherein the first RNA oligonucleotide consists of the sequence set forth in SEQ ID NO: 1.

3. The composition of claim 1 or 2, wherein the second RNA oligonucleotide consists of the sequence set forth in SEQ ID NO: 2.

4. A pharmaceutical composition comprising the composition according to any one of claims 1-3, and a pharmaceutically acceptable carrier.

5. The composition of any one of claims 1-3 or the pharmaceutical composition of claim 4 for use in treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof.

6. A nucleic acid construct directed to GYS2 for use in treating Metabolic Syndrome, a condition associated with Metabolic Syndrome, or Type 2 Diabetes in a subject in need thereof.

7. The composition, pharmaceutical composition or nucleic acid according to claim 5 or 6 for use in treating Metabolic Syndrome.

8. The composition, pharmaceutical composition or nucleic acid for use according to claim 7, wherein the Metabolic Syndrome is diagnosed by the presence of at least three of five following conditions in a subject: elevated blood pressure, elevated fasting plasma glucose, high serum triglycerides, liver fibrosis, and low levels of high-density lipoprotein (HDL) levels.

9. The composition, pharmaceutical composition or nucleic acid according to claim 5 or 6 for use intreating Type 2 Diabetes.

10. The composition, pharmaceutical composition or nucleic acid for use in according to claim 9, wherein the Type 2 Diabetes occurs in a subject with Metabolic Syndrome.

11. The composition, pharmaceutical composition or nucleic acid for use according to claim 9, wherein the Type 2 Diabetes occurs in a subject in the absence of Metabolic Syndrome.

12. The composition, pharmaceutical composition or nucleic acid for use according to any one of claims 6 to 11, wherein said nucleic acid construct is directed to a nucleic acid having the sequence set forth in SEQ ID NO: 7.

13. The composition, pharmaceutical composition or nucleic acid for use according to any one of claims 6 to 12, wherein the nucleic acid construct directed to GYS2 is an siRNA.

14. The composition, pharmaceutical composition or nucleic acid for use according to any one of claims 6 to 11, wherein the nucleic acid construct directed to GYS2 is an antisense oligonucleotide.

15. The composition, pharmaceutical composition or nucleic acid for use according to any one of claims 6 to 11, wherein the nucleic acid construct directed to GYS2 comprises a CRISPR (guide) RNA which comprises a region of complementarity to the genomic sequence of GYS2 (GenBank accession number NG 016167.1).

16. The composition, pharmaceutical composition or nucleic acid for use according to claim 15, wherein the composition, pharmaceutical composition or nucleic acid is provided with a Cas protein or a nucleic acid encoding a Cas protein.

17. The composition, pharmaceutical composition or nucleic acid for use according to claim 16, wherein the composition, pharmaceutical composition or nucleic acid and the Cas protein or nucleic acid encoding a Cas protein and the CRISPR (guide) RNA form a single composition.

18. The composition, pharmaceutical composition or nucleic acid for use according to claim 16 or 17, wherein the Cas protein is a Type II Cas9 protein.

19. The composition, pharmaceutical composition or nucleic acid for use according to claim 16 or 17, wherein the Cas protein is a Type I Cas3 protein.

20. The composition, pharmaceutical composition or nucleic acid for use according to claim 16 or 17, wherein the Cas protein is a Type III Cas 10 protein.

21. The composition, pharmaceutical composition or nucleic acid for use according to claim 16 or 17, wherein the Cas protein is a Type V Casl2 protein.

22. The composition, pharmaceutical composition or nucleic acid for use according to claim 16 or 17, wherein the Cas protein is a Type VI Cas 13 protein.

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