Compositions and methods for inhibiting gys2 expression

Abstract

The present disclosure relates to oligonucleotides, compositions, and methods useful for reducing GYS2 expression, particularly in hepatocytes. The disclosed oligonucleotides for reducing GYS2 expression can be double-stranded or single-stranded, and can be modified to improve properties such as stronger resistance to nucleases and lower immunogenicity. The disclosed oligonucleotides for reducing GYS2 expression can also include targeting ligands to target specific cells or organs, such as hepatocytes of the liver, and can be useful for treating glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and related conditions.

Description

[0001] Related applications

[0002] This application claims the benefits of U.S. Provisional Application No. 62 / 637574, filed March 2, 2018, entitled “COMPOSITIONS AND METHODS FOR INHIBITING GYS2 EXPRESSION”, pursuant to 35 USC §119(e), the full contents of which are incorporated herein by reference. Invention Field

[0003] This application relates to oligonucleotides and their uses, particularly to the use in the treatment of glycogen storage diseases and related conditions.

[0004] References to sequence lists

[0005] This application is submitted together with the sequence listing in electronic format. The sequence listing is provided as a file entitled D0800.70014WO00-SEQ.txt (132 kilobytes in size) created on February 15, 2019. The information of the sequence listing in electronic format is incorporated herein by reference in its entirety. Background of the Invention

[0007] Glycogen is a complex sugar that the body uses to store glucose. When the body needs more glucose to function, it typically breaks down stored glycogen for use in cellular processes. Several enzymes are involved in the processes of storing glucose as glycogen (glycogen synthesis) and breaking down glycogen into glucose (glycogenolysis). When one or more of these enzymes are inhibited, glycogen storage diseases may result, in which a lack of glycogen storage, accumulation of glycogen in affected cells (e.g., liver and / or muscle cells), or the formation of abnormally structured glycogen may be observed. When glycogen storage or decomposition disorders occur, those affected may suffer from a number of symptoms, including but not limited to: hepatomegaly, increased hepatotoxicity (e.g., higher levels of AST, ALT, and / or ALP), liver fibrosis, fatty acid deposition in the liver, hepatomegaly, hepatocellular adenoma, and / or hepatocellular carcinoma. A non-limiting group of exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

[0008] Invention Summary

[0009] Various aspects of this disclosure relate to oligonucleotides and related methods for treating glycogen storage disorders (e.g., diseases or conditions affecting glycogen breakdown or storage, such as GSD1a, GSDIII, GSDIV, GSDVI, or GSDIX) in subjects. In some embodiments, effective RNAi oligonucleotides have been developed for selectively inhibiting GYS2 expression in subjects. In some embodiments, RNAi oligonucleotides can be used to reduce overall GYS2 activity in hepatocytes, thereby reducing or preventing hepatomegaly, hepatotoxicity (e.g., levels of AST, ALT, and / or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and / or hepatocellular carcinoma. In some embodiments, key regions of GYS2 mRNA (referred to as hotspots) have been identified herein, which are particularly suitable for targeting using such oligonucleotide-based methods (see, for example, Example 1).

[0010] One aspect of this disclosure provides oligonucleotides for reducing GYS2 expression. In some embodiments, the oligonucleotide comprises an antisense strand containing a sequence as shown in any one of SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand consists of a sequence as shown in any one of SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the antisense strand comprises or consists of a sequence as shown in any one of SEQ ID NO:417-466, 575-580, 586-598, or 620-627. In some embodiments, the oligonucleotide further comprises a sense strand containing a sequence as shown in any one of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of or is composed of a sequence as shown in any one of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand comprises or is composed of a sequence as shown in any one of SEQ ID NO:385-416, 569-574, 581-585, or 612-619.

[0011] One aspect of this disclosure provides an oligonucleotide for reducing the expression of GYS2, wherein the oligonucleotide comprises an antisense strand of 15 to 30 nucleotides in length. In some embodiments, the antisense strand has a region complementary to a target sequence of GYS2 as shown in any of SEQ ID NO:599-608. In some embodiments, the complementary region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, or at least 22 consecutive nucleotides in length. In some embodiments, the complementary region is completely complementary to the target sequence of GYS2. In some embodiments, the region complementary to GYS2 is at least 19 consecutive nucleotides in length.

[0012] In some embodiments, the antisense strand is 19 to 27 nucleotides long. In some embodiments, the antisense strand is 21 to 27 nucleotides long. In some embodiments, the oligonucleotide further comprises a sense strand of 15 to 40 nucleotides long, wherein the sense strand and the antisense strand form a double-stranded region. In some embodiments, the sense strand is 19 to 40 nucleotides long. In some embodiments, the antisense strand is 27 nucleotides long, and the sense strand is 25 nucleotides long. In some embodiments, the double-stranded region is at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 nucleotides long. In some embodiments, the antisense strand and the sense strand form a double-stranded region of 25 nucleotides long.

[0013] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand, each ranging in length from 21 to 23 nucleotides. In some embodiments, the oligonucleotide comprises a duplex structure ranging in length from 19 to 21 nucleotides. In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of one or more nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, the sense strand, or both. In some embodiments, the oligonucleotide further comprises a 3'-overhang sequence of two nucleotides in length on the antisense strand. In some embodiments, the oligonucleotide comprises a 3'-overhang sequence of two nucleotides in length, wherein the 3'-overhang sequence is present on the antisense strand, and wherein the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length, such that the sense strand and the antisense strand form a duplex of 21 nucleotides in length.

[0014] In some embodiments, the sense strand comprises a sequence as shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the sense strand consists of a sequence as shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the antisense strand comprises a sequence as shown in any of SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some implementations, the antisense strand consists of sequences such as those shown in SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598 or 620-627.

[0015] In some embodiments, the sense strand includes a stem-loop at its 3' end as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2.

[0016] Another aspect of this disclosure provides oligonucleotides for reducing GYS2 expression, the oligonucleotides comprising an antisense strand and a sense strand, wherein the antisense strand is 21 to 27 nucleotides in length and has a region complementary to GYS2, wherein the sense strand comprises a stem-loop at its 3' end as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 3 to 5 nucleotides in length between S1 and S2, and wherein the antisense strand and the sense strand form a duplex structure of at least 19 nucleotides in length, but are not covalently linked (see example...). Figure 3 In some embodiments, the complementary region is fully complementary to at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 consecutive nucleotides of the GYS2 mRNA. In some embodiments, L is a tetracyclic ring. In some embodiments, L is 4 nucleotides in length. In some embodiments, L contains a sequence indicated as GAAA.

[0017] In some embodiments, the oligonucleotide comprises at least one modified nucleotide. In some embodiments, the modified nucleotide comprises a 2'-modification. In some embodiments, the 2'-modification is selected from: 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinose. In some embodiments, all nucleotides of the oligonucleotide are modified.

[0018] In some embodiments, the oligonucleotide comprises at least one modified internucleotide bond. In some embodiments, the at least one modified internucleotide bond is a thiophosphate bond. In some embodiments, the 4'-carbon of the sugar in the 5'-nucleotide of the antisense strand comprises a phosphate ester analog. In some embodiments, the phosphate ester analog is an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate.

[0019] In some embodiments, at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands. In some embodiments, each targeting ligand comprises a carbohydrate, amino sugar, cholesterol, polypeptide, or lipid. In some embodiments, each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety. In some embodiments, the GalNAc moiety is a monovalent, divalent, trivalent, or tetravalent GalNAc moiety. In some embodiments, up to four nucleotides of the stem-loop L are each conjugated to a monovalent GalNAc moiety. In some embodiments, the targeting ligand comprises an aptamer.

[0020] Another aspect of this disclosure provides compositions comprising the oligonucleotides and excipients of this disclosure. Another aspect of this disclosure provides methods comprising administering the compositions of this disclosure to a subject. In some embodiments, said methods result in a reduction or prevention of hepatomegaly, hepatic nodule formation, hepatotoxicity (e.g., levels of AST, ALT, and / or ALP), liver fibrosis, hepatocyte proliferation, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and / or hepatocellular carcinoma. Another aspect of this disclosure provides methods for treating glycogen storage disease or one or more symptoms of glycogen storage disease. A group of non-limiting exemplary glycogen storage diseases may include: GSDI (e.g., GSDIa), GSDIII, GSDIV, GSDVI, and GSDIX.

[0021] Another aspect of this disclosure provides an oligonucleotide for reducing the expression of GYS2, the oligonucleotide comprising a sense strand of 15 to 40 nucleotides in length and an antisense strand of 15 to 30 nucleotides in length, wherein the sense strand and the antisense strand form a double-stranded region, wherein the sense strand comprises a sequence as shown in any one of SEQ ID NO: 1-192, 385-416, 467-517, 569-574, 581-585 or 612-619, and causes the antisense strand to comprise a complementary sequence selected from SEQ ID NO: 193-384, 417-466, 518-568, 575-580, 586-598 or 620-627.

[0022] In some embodiments, the oligonucleotide comprises a pair of sense and antisense strands selected from rows of the table shown in Table 4. Brief description of the attached diagram

[0024] The accompanying drawings, which are incorporated in and form part of this specification, illustrate certain embodiments and, together with the written description, serve to provide non-limiting examples of certain aspects of the compositions and methods disclosed herein.

[0025] Figure 1A and 1B This is a graph showing the percentage of GYS2 mRNA remaining after screening 264 GYS2 conjugates in HEK-293 cells. The nucleotide positions corresponding to the 3' ends of the sense strand of each siRNA are indicated on the x-axis in NM_021957.3.

[0026] Figure 2A and 2B This is a set of graphs showing the percentage of remaining mRNA in HEK-293 cells after screening for 71 GYS2 oligonucleotides at two or three different concentrations (0.1 nM and 1.0 nM, or 0.03 nM, 0.1 nM and 1.0 nM).

[0027] Figure 3 This is a schematic diagram showing a non-restrictive example of a double-stranded oligonucleotide with a nicked tetracyclic structure that has been conjugated to four GalNAc moieties (rhombuses).

[0028] Figure 4 This is a graph showing the screening results of GYS2 oligonucleotides with different base sequences in HEK-293 cells using one or two different modification patterns. The X-axis lists the 3' end of the sense strand targeted by the evaluated oligonucleotide. The negative control sequence (NC1), untransfected cells, and cells transfected with the mimic are shown on the left as controls.

[0029] Figure 5 This figure shows the screening results of GYS2 oligonucleotides with different base sequences in a nicked tetracyclic structure in monkey hepatocytes. The same modification pattern was used, and the oligonucleotides were tested at three different concentrations (0.1 μM, 0.3 μM, and 1.0 μM). Untransfected cells are shown on the left as a control.

[0030] Figure 6A and 6B This is an indicator of the IC50 in HEK-293 cells for GYS2 oligonucleotides selected from dose-response curve screening. 50 A series of graphs showing the results.

[0031] Figure 7This is a graph showing the in vivo activity evaluation of GalNAc-conjugated GYS2 oligonucleotides with a nicked tetracyclic structure. Eight different oligonucleotide sequences were tested. The oligonucleotides were administered subcutaneously at 0.5 mg / kg to mice expressing human GYS2. Data show the amount of residual GYS2 mRNA on day 4 post-administration, normalized to the PBS control. Invention Details

[0033] According to some aspects, this disclosure provides oligonucleotides targeting GYS2 mRNA that effectively reduce GYS2 expression in cells, particularly liver cells (e.g., hepatocytes), to treat glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of glycogen storage diseases. Therefore, in a related aspect, this disclosure provides methods for treating glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of glycogen storage diseases, comprising selectively reducing GYS2 gene expression in the liver. In some embodiments, the GYS2-targeting oligonucleotides provided herein are designed to be delivered to selected cells (e.g., hepatocytes of the liver) of a target tissue to treat glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) or one or more symptoms of glycogen storage diseases in a subject.

[0034] Further aspects of this disclosure, including a description of the defined terms, are provided below.

[0035] I. Definition

[0036] Administration: As used herein, the term “administering” or “administration” means the delivery of a substance (e.g., oligonucleotide) to a subject in a pharmacologically useful manner (e.g., to treat a condition in the subject).

[0037] Approximate: As used herein, the term “approximate” or “about” when applied to one or more target values ​​means a value similar to the reference value. In some embodiments, unless otherwise stated or otherwise apparent from the context, the term “approximate” or “about” means a range of values ​​falling within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less of the reference value in any direction (unless such a number would exceed 100% of the possible value).

[0038] Desialyl glycoprotein receptor (ASGPR): As used herein, the term "desialyl glycoprotein receptor" or "ASGPR" refers to a dichotomous C-type lectin formed by a major 48 kDa (ASGPR-1) and a minor 40 kDa (ASGPR-2) subunit. ASGPRs are primarily expressed on the surface of hepatocyte sinusoids and play a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins (desialyl glycoproteins) containing terminal galactose or N-acetylgalactosamine residues.

[0039] Complementarity: As used herein, the term "complementarity" refers to a structural relationship between nucleotides (e.g., two nucleotides on opposite nucleic acids or on opposite regions of a single nucleic acid strand) that allows the nucleotides to form base pairs with each other. For example, a purine nucleotide complementary to a pyrimidine nucleotide of an opposite nucleic acid can be base-paired together by forming hydrogen bonds with each other. In some embodiments, complementary nucleotides may be base-paired in a Watson-Crick manner or in any other manner that allows the formation of a stable duplex. In some embodiments, the two nucleic acids may have nucleotide sequences that are complementary to each other to form complementary regions, as described herein.

[0040] Deoxyribonucleotide: As used herein, the term "deoxyribonucleotide" refers to a nucleotide that has a hydrogen atom at the 2' position of its pentose sugar compared to a ribonucleotide. Modified deoxyribonucleotides are deoxyribonucleotides that have modifications or substitutions (including modifications or substitutions in sugars, phosphate groups, or bases) that have one or more atoms other than at the 2' position.

[0041] Double-stranded oligonucleotide: As used herein, the term "double-stranded oligonucleotide" refers to an oligonucleotide that is substantially in a double-stranded form. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently separated nucleic acid chains of a double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed between antiparallel sequences of nucleotides in covalently linked nucleic acid chains of a double-stranded oligonucleotide. In some embodiments, complementary base pairings are formed from one or more double-stranded regions of a double-stranded oligonucleotide from a single nucleic acid chain that is folded (e.g., via a hairpin) to provide complementary antiparallel sequences of nucleotides that are base-paired together. In some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are fully double-stranded with each other. However, in some embodiments, the double-stranded oligonucleotide comprises two covalently separated nucleic acid chains that are partially double-stranded, for example, having overhangs at one or both ends. In some embodiments, the double-stranded oligonucleotide comprises antiparallel sequences of nucleotides that are partially complementary, and therefore may have one or more mismatches, which may include internal mismatches or terminal mismatches.

[0042] Double strand: As used in this article, the term “double strand” in relation to nucleic acids (e.g., oligonucleotides) refers to a structure formed by the pairing of complementary bases in two antiparallel sequences of nucleotides.

[0043] Excipients: As used herein, the term “excipient” refers to a non-therapeutic agent that may be included in a composition, for example, to provide or facilitate a desired consistency or stabilizing effect.

[0044] Glycogen storage disease: As used herein, the terms “glycogen storage disease,” “GSD,” or “glycogen storage diseases” refer to metabolic disorders caused by a deficiency of enzymes that affect glycogen synthesis, glycogenolysis, and / or glucose breakdown (glycolysis). Various types of glycogen storage diseases have been characterized, including GSD 0, GSD I (also known as GSD 1 or von Gilke's disease; e.g., GSDIa), GSD II (also known as Pompey's disease or acid maltase deficiency), GSD III (also known as GSD3, Currie's disease, or Fobes' disease), GSD IV (GSD 4 or Anderson's disease), GSD V (also known as McCardell's disease), GSD VI (also known as GSD 6 or Hess' disease), GSD VII (also known as GSD 7 or Tauri's disease), GSD VIII, and GSD IX (also known as GSD9). In some implementations, individuals with glycogen storage disease exhibit one or more of a number of symptoms, including, but not limited to: hepatomegaly, increased hepatotoxicity (e.g., higher levels of AST, ALT, and / or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and / or hepatocellular carcinoma.

[0045] GYS2: As used herein, the term “GYS2” or “glycogen synthase 2” refers to the liver glycogen synthase gene. This gene encodes the protein liver glycogen synthase, which catalyzes the rate-limiting pathway in glycogen synthesis (i.e., the transfer of glucose molecules from UDP-glucose to the terminal branch of the glycogen molecule). GYS2 is expressed in liver cells, such as hepatocytes. Homologues of GYS2 are conserved across a range of species, including humans, mice, rats, non-human primates, and more (see, for example, NCBI HomoloGene:56580). In humans, GYS2 encodes multiple transcripts, namely, those shown in GenBank accessions NM_021957.3 (SEQ ID NO:609), XM_006719063.3, and XM_017019245.1, each encoding different isoforms, namely GenBank accessions NP_068776.2, XP_006719126.1 (isoform X1), and XP_016874734.1 (isoform X2). An exemplary monkey (macaque) transcript sequence is shown in GenBank accession XM_001098578.2 (SEQ ID NO:610). An exemplary mouse transcript is shown in GenBank accession NM_145572.2 (SEQ ID NO:611).

[0046] Hepatocytes: As used herein, the term “hepatocyte” (or hepatocytes) refers to the cells of the parenchymal tissue of the liver. These cells constitute approximately 70–85% of the liver mass and produce serum albumin, fibrinogen, and the prothrombin group of coagulation factors (except factors 3 and 4). Markers of hepatocyte lineage cells may include, but are not limited to, transthyretin (Ttr), glutamine synthetase (Glul), hepatocyte nuclear factor 1a (Hnf1a), and hepatocyte nuclear factor 4a (Hnf4a). Markers of mature hepatocytes may include, but are not limited to, cytochrome P450 (Cyp3a11), fumarate acetoacetate hydrolase (Fah), glucose-6-phosphate (G6p), albumin (Alb), and OC2-2F8. See, for example, Huch et al., (2013), Nature, 494(7436):247–250, the content of which relates to hepatocyte markers is incorporated herein by reference.

[0047] Loop: As used herein, the term “loop” refers to an unpaired region of a nucleic acid (e.g., an oligonucleotide) flanked by two antiparallel regions of the nucleic acid that are sufficiently complementary to each other, such that under appropriate hybridization conditions (e.g., in phosphate buffer, in cells), the two antiparallel regions flanked by the unpaired region hybridize to form a double strand (referred to as a “stem”).

[0048] Modified internucleotide bonds: As used herein, the term "modified internucleotide bond" refers to an internucleotide bond that has one or more chemical modifications compared to a reference internucleotide bond containing a phosphodiester bond. In some embodiments, the modified nucleotide is a non-naturally occurring bond. Typically, modified internucleotide bonds endow nucleic acids in which the modified internucleotide bonds are present with one or more desired properties. For example, modified nucleotides can improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, and reduce immunogenicity, etc.

[0049] Modified nucleotides: As used herein, the term "modified nucleotide" refers to a nucleotide having one or more chemical modifications compared to a corresponding reference nucleotide selected from the following: adenine ribonucleotide, guanine ribonucleotide, cytosine ribonucleotide, uracil ribonucleotide, adenine deoxyribonucleotide, guanine deoxyribonucleotide, cytosine deoxyribonucleotide, and thymidine deoxyribonucleotide. In some embodiments, the modified nucleotide is a non-naturally occurring nucleotide. In some embodiments, the modified nucleotide has one or more chemical modifications in its sugar, nucleotide, and / or phosphate ester groups. In some embodiments, the modified nucleotide has one or more chemical moieties conjugated to the corresponding reference nucleotide. Generally, the modified nucleotide imparts one or more desired properties to the nucleic acid in which the modified nucleotide is present. For example, the modified nucleotide may improve thermal stability, resistance to degradation, nuclease resistance, solubility, bioavailability, biological activity, and reduce immunogenicity. In some embodiments, the modified nucleotide contains a 2'-O-methyl or 2'-F substitution at the 2' position of the ribose ring.

[0050] A nicked tetra-loop structure: A nicked tetra-loop structure is a structure of RNAi oligonucleotides characterized by the presence of separate sense (passenger) and antisense (guide) strands, wherein the sense strand has a region complementary to the antisense strand, such that the two strands form a bistrand, and wherein at least one of the strands, typically the sense strand, extends from the bistrand, wherein the extension contains a tetra-loop and two self-complementary sequences forming a stem region adjacent to the tetra-loop, wherein the tetra-loop is configured to stabilize the adjacent stem region formed by the self-complementary sequences of at least one strand.

[0051] Oligonucleotide: As used herein, the term "oligonucleotide" refers to a short nucleic acid, such as a short nucleic acid less than 100 nucleotides in length. Oligonucleotides may comprise ribonucleotides, deoxyribonucleotides, and / or modified nucleotides, including, for example, modified ribonucleotides. Oligonucleotides may be single-stranded or double-stranded. Oligonucleotides may or may not have a double-stranded region. As a set of non-limiting examples, oligonucleotides may be, but are not limited to, small interfering RNA (siRNA), microRNA (miRNA), short hairpin RNA (shRNA), cleatase substrate interfering RNA (dsiRNA), antisense oligonucleotides, short siRNA, or single-stranded siRNA. In some embodiments, the double-stranded oligonucleotide is an RNAi oligonucleotide.

[0052] Overhang: As used herein, the term "overhang" refers to a terminal non-base-pairing nucleotide arising from a chain or region extending beyond the end of the complementary chain, said chain or region forming a duplex with said complementary chain. In some embodiments, the overhang comprises one or more unpaired nucleotides extending from a duplex region at the 5' or 3' end of the double-stranded oligonucleotide. In some embodiments, the overhang is a 3' or 5' overhang on the antisense or sense strand of the double-stranded oligonucleotide.

[0053] Phosphate ester analogs: As used herein, the term "phosphate ester analog" refers to a chemical moiety that mimics the electrostatic and / or steric properties of a phosphate ester group. In some embodiments, the phosphate ester analog replaces the 5'-phosphate ester, which is often readily removable by enzymatic reactions, located at the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the 5'-phosphate ester analog contains a phosphatase-resistant bond. Examples of phosphate ester analogs include 5'-phosphonates, such as 5'-methylenephosphonate (5'-MP) and 5'-(E)-vinylphosphonate (5'-VP). In some embodiments, the oligonucleotide has a phosphate ester analog at the 4'-carbon position of the sugar at the 5'-terminal nucleotide (referred to as a "4'-phosphate ester analog"). An example of a 4'-phosphate ester analog is an oxymethylphosphonate or an analog thereof in which the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon). See, for example, International Patent Application PCT / US2017 / 049909, filed September 1, 2017; U.S. Provisional Application No. 62 / 383,207, filed September 2, 2016; and U.S. Provisional Application No. 62 / 393,401, filed September 12, 2016, the contents of which relate to phosphate ester analogs are incorporated herein by reference. Other modifications have been developed for the 5' end of oligonucleotides (see, for example, WO 2011 / 133871; U.S. Patent No. 8,927,513; and Prakash et al. (2015), Nucleic Acids Res., 43(6):2993-3011, the contents of which relate to phosphate ester analogs are incorporated herein by reference).

[0054] Reduced expression: As used herein, the term "reduced expression" of a gene refers to a decrease in the amount of RNA transcripts or proteins encoded by the gene and / or a decrease in the amount of the gene's activity in cells or subjects compared to appropriate reference cells or subjects. For example, treating cells with a double-stranded oligonucleotide (e.g., a double-stranded oligonucleotide having an antisense strand complementary to the GYS2 mRNA sequence) can result in a reduction in the amount of RNA transcripts, proteins, and / or enzymatic activities (e.g., encoded by the GYS2 gene) compared to cells not treated with the double-stranded oligonucleotide. Similarly, as used herein, "reduced expression" refers to actions that result in a decrease in the expression of a gene (e.g., GYS2).

[0055] Complementary Region: As used herein, the term "complementary region" refers to a sequence of nucleotides in a nucleic acid (e.g., a double-stranded oligonucleotide) that is sufficiently complementary to an antiparallel sequence of the nucleotide (e.g., a target nucleotide sequence within mRNA) to allow hybridization between the two sequences of the nucleotide under appropriate hybridization conditions (e.g., in phosphate-buffered saline, in cells, etc.). A complementary region may be completely complementary to a nucleotide sequence (e.g., a target nucleotide sequence present within mRNA or a portion thereof). For example, a complementary region that is completely complementary to a nucleotide sequence present in mRNA has a continuous nucleotide sequence that is complementary to the corresponding sequence in mRNA without any mismatches or gaps. Alternatively, a complementary region may be partially complementary to a nucleotide sequence (e.g., a nucleotide sequence present in mRNA or a portion thereof). For example, a complementary region that is partially complementary to a nucleotide sequence present in mRNA has a continuous nucleotide sequence that is complementary to the corresponding sequence in mRNA but contains one or more mismatches or gaps (e.g., 1, 2, 3, or more mismatches or gaps) compared to the corresponding sequence in mRNA, provided that the complementary region remains capable of hybridizing with mRNA under appropriate hybridization conditions.

[0056] Ribonucleotide: As used herein, the term "ribonucleotide" refers to a nucleotide having ribose as its pentose sugar and a hydroxyl group at its 2' position. Modified ribonucleotides are ribonucleotides with modifications or substitutions (including modifications or substitutions of ribose, phosphate groups, or bases) having one or more atoms other than at the 2' position.

[0057] RNAi oligonucleotides: As used herein, the term “RNAi oligonucleotide” means (a) a double-stranded oligonucleotide having a sense strand (passenger) and an antisense strand (guide), wherein the antisense strand or a portion thereof is used by the Argonaute 2 (Ago2) endonuclease to cleave target mRNA; or (b) a single-stranded oligonucleotide having a single antisense strand, wherein the antisense strand (or a portion thereof) is used by the Ago2 endonuclease to cleave target mRNA.

[0058] Chain: As used herein, the term "chain" refers to a single, continuous sequence of nucleotides linked together by internucleotide bonds (e.g., phosphodiester bonds, thiophosphate bonds). In some embodiments, the chain has two free ends, such as a 5'-end and a 3'-end.

[0059] Subject: As used herein, the term "subject" means any mammal, including mice, rabbits, and humans. In one embodiment, the subject is a human or a non-human primate. The terms "individual" or "patient" may be used interchangeably with "subject".

[0060] Synthetic: As used herein, the term “synthetic” refers to nucleic acids or other molecules that are artificially synthesized (e.g., using a machine (e.g., a solid-state nucleic acid synthesizer)) or are not otherwise derived from the natural source (e.g., a cell or organism) that normally produces the molecule.

[0061] Targeting Ligand: As used herein, the term "targeting ligand" refers to a molecule (e.g., a carbohydrate, amino sugar, cholesterol, peptide, or lipid) that selectively binds to a homologous molecule (e.g., a receptor) of a target tissue or cell and can be conjugated to another substance for the purpose of targeting other substances to the target tissue or cell. For example, in some embodiments, a targeting ligand may be conjugated to an oligonucleotide for the purpose of targeting a specific target tissue or cell. In some embodiments, the targeting ligand selectively binds to a cell surface receptor. Thus, in some embodiments, when conjugated to an oligonucleotide, the targeting ligand facilitates the delivery of the oligonucleotide to a specific cell by selectively binding to a receptor expressed on the cell surface and by the cellular internalization of the complex comprising the oligonucleotide, the targeting ligand, and the receptor. In some embodiments, the targeting ligand is conjugated to the oligonucleotide via a linker that is cleaved after or during cell internalization, thereby releasing the oligonucleotide from the targeting ligand within the cell.

[0062] Tetra-loop: As used herein, the term "tetra-loop" refers to a loop that increases the stability of adjacent double strands formed by hybridization of nucleotide side sequences. The increase in stability is detectable as the melting temperature (Tm) of the adjacent stem duplex increases above the average expected Tm of the adjacent stem duplex from a loop of a reasonably long length composed of a set of randomly selected nucleotide sequences. For example, a tetra-loop can impart a melting temperature of at least 50°C, at least 55°C, at least 56°C, at least 58°C, at least 60°C, at least 65°C, or at least 75°C in 10 mM NaHPO4 to a hairpin containing a duplex of at least two base pairs. In some embodiments, the tetra-loop can stabilize base pairs in adjacent stem duplexes through stacking interactions. Furthermore, the interactions between nucleotides in the tetracycle include, but are not limited to, non-Watson-Crick base pairing, stacking interactions, hydrogen bonding, and contact interactions (Cheong et al., Nature 1990 Aug. 16; 346(6285):680-2; Heus and Pardi, Science 1991 Jul. 12; 253(5016):191-4). In some embodiments, the tetracycle comprises or consists of 3 to 6 nucleotides, and is typically 4 to 5 nucleotides. In some embodiments, the tetracycle comprises or consists of 3, 4, 5, or 6 nucleotides that may or may not be modified (e.g., may or may not be conjugated to the target moiety). In one embodiment, the tetracycle consists of four nucleotides. Any nucleotide can be used in the tetracycle, and the standard IUPAC-IUB notation for such nucleotides can be used, as described in Cornish-Bowden (1985) Nucl. Acids Res. 13:3021-3030. For example, the letter “N” can be used to indicate that any base can be at that position, the letter “R” can be used to indicate that A (adenine) or G (guanine) can be at that position, and “B” can be used to indicate that C (cytosine), G (guanine), or T (thymine) can be at that position. Examples of tetracyclic rings include the tetracyclic UNCG family (e.g., UUCG), the tetracyclic GNRA family (e.g., GAAA), and the CUUG tetracyclic ring. (Woese et al., Proc Natl Acad Sci USA. 1990 November; 87(21): 8467-71; Antao et al., Nucleic Acids Res. 1991 Nov. 11; 19(21): 5901-5). Examples of tetracyclic DNA include the tetracyclic d(GNNA) family (e.g., d(GTTA)), the tetracyclic d(GNRA) family, the tetracyclic d(GNAB) family, the tetracyclic d(CNNG) family, and the tetracyclic d(TNCG) family (e.g., d(TTCG)).See, for example: Nakano et al. Biochemistry, 41(48), 14281-14292, 2002. SHINJI et al. Nippon Kagakkai Koen Yokoshu VOL. 78th; NO. 2; PAGE. 731 (2000), the contents of which are incorporated herein by reference. In some embodiments, the four rings are contained within a slit four-ring structure.

[0063] Treatment: As used herein, the term "treatment" refers to the act of providing care to a subject in need, for the purpose of improving the health and / or well-being of a subject with respect to an existing condition (e.g., disease, symptom) or to prevent or reduce the likelihood of the occurrence of the condition, such as by administering a therapeutic agent (e.g., oligonucleotide) to the subject. 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, symptom) experienced by the subject.

[0064] II. Oligonucleotide-based inhibitors

[0065] i.GYS2-targeted oligonucleotides

[0066] This article identifies potent oligonucleotides by examining GYS2 mRNA, including mRNA from different species (human and macaque (see, for example, Example 1)) and by in vitro and in vivo assays. Such oligonucleotides can achieve therapeutic benefits for subjects with glycogen storage diseases (e.g., GSD1a, GSD3, GSD4, GSD5, and GSD6) or one or more symptoms of glycogen storage diseases by reducing GYS2 activity and thus by reducing or preventing hepatomegaly, hepatotoxicity (proven, such as levels of AST, ALT, and / or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and / or hepatocellular carcinoma. For example, this document provides effective RNAi oligonucleotides having a sense strand comprising or consisting of sequences shown as any one of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585 or 612-619, and an antisense strand comprising or consisting of complementary sequences selected from SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598 or 620-627, as also arranged in the tables provided in Table 4 (e.g., a sense strand comprising a sequence shown as SEQ ID NO:1 and an antisense strand comprising a sequence shown as SEQ ID NO:193).

[0067] The sequence can be placed in a variety of different oligonucleotide structures (or formats) as described herein.

[0068] In some embodiments, certain regions of GYS2 mRNA have been identified as hotspots for targeting because they are more suitable for oligonucleotide-based repression than other regions. In some embodiments, the hotspot regions of GYS2 consist of sequences as shown in any of SEQ ID NO: 599-608. For the purpose of inhibiting GYS2 mRNA expression, these regions of GYS2 mRNA can be targeted using oligonucleotides as discussed herein.

[0069] Therefore, in some embodiments, for the purpose of targeting mRNA in cells and inhibiting its expression, the oligonucleotides provided herein are designed to have regions complementary to GYS2 mRNA (e.g., within hotspots of GYS2 mRNA). For the purpose of inhibiting its expression, the complementary regions typically have suitable length and base content such that the oligonucleotide (or its chain) can anneal to GYS2 mRNA.

[0070] In some embodiments, the oligonucleotides disclosed herein include complementary regions (e.g., on the antisense strand of a double-stranded oligonucleotide) that are at least partially complementary to sequences shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 (which include sequences localized to hotspot regions of GYS2 mRNA). In some embodiments, the oligonucleotides disclosed herein include complementary regions (e.g., on the antisense strand of a double-stranded oligonucleotide) that are fully complementary to sequences shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the complementary region of the oligonucleotide complementary to a consecutive nucleotide of the sequence shown in SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans the entire length of the antisense strand. In some embodiments, the complementary region of the oligonucleotide complementary to a consecutive nucleotide of the sequence shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619 spans a portion of the entire length of the antisense strand (all nucleotides except the two at the 3' end of the antisense strand). In some embodiments, the oligonucleotides disclosed herein include complementary regions (e.g., on the antisense strand of a double-stranded oligonucleotide) that are at least partially (e.g., completely) complementary to successive extensions of nucleotides 1-19 spanning sequences such as those shown in SEQ ID NO: 1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

[0071] In some embodiments, the complementary region is at least 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the oligonucleotides provided herein have a region complementary to GYS2 mRNA with a length ranging from 12 to 30 nucleotides (e.g., 12 to 30, 12 to 22, 15 to 25, 17 to 21, 18 to 27, 19 to 27, or 15 to 30). In some embodiments, the oligonucleotides provided herein have a region complementary to GYS2 mRNA with a length of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides.

[0072] In some embodiments, the region complementary to GYS2 mRNA may have one or more mismatches compared to the corresponding sequence of GYS2 mRNA. The complementary region on the oligonucleotide may have up to one, two, three, four, or more mismatches, provided it maintains its ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. Alternatively, the complementary region on the oligonucleotide may have no more than one, two, three, or four mismatches, provided it maintains its ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions. In some embodiments, if more than one mismatch exists in the complementary region, they may be sequentially located (e.g., two, three, four, or more consecutively) or scattered throughout the complementary region, provided the oligonucleotide maintains its ability to form complementary base pairs with GYS2 mRNA under appropriate hybridization conditions.

[0073] Still, in some embodiments, the double-stranded oligonucleotides provided herein comprise or consist of the following: a sense strand having a sequence shown as in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585 or 612-619 and an antisense strand having a complementary sequence selected from SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598 or 620-627, as arranged in the tables provided in Table 4 (e.g., a sense strand comprising a sequence shown as in SEQ ID NO:1 and an antisense strand comprising a sequence shown as in SEQ ID NO:193).

[0074] ii. Oligonucleotide structure

[0075] Various oligonucleotide structures, including RNAi, miRNA, etc., are available for use in the methods of this disclosure to target GYS2 mRNA. Any structure described herein or elsewhere can be used as a framework to incorporate or target sequences described herein (e.g., hotspot sequences of GYS2, such as those exemplified in SEQ ID NO:599-608, or containing sequences or sense or antisense strands as shown in SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585 or 612-619, or as shown in SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598 or 620-627, respectively). Double-stranded oligonucleotides used to target GYS2 expression (e.g., via the RNAi pathway) typically have sense and antisense strands that form a duplex with each other. In some embodiments, the sense and antisense strands are not covalently linked. However, in some implementations, the meaningful and antisense chains are covalently linked.

[0076] In some embodiments, the sequence described herein may be incorporated into an oligonucleotide comprising both sense and antisense strands in the range of 17-40 nucleotides in length, or a target may be used with an oligonucleotide comprising both sense and antisense strands in the range of 17-40 nucleotides in length. In some embodiments, an oligonucleotide incorporating such a sequence is provided having a tetracyclic structure within the 3' extension of its sense strand and two terminal overhanging nucleotides at the 3' end of its antisense strand. In some embodiments, the two terminal overhanging nucleotides are GG. Typically, one or both of the two terminal GG nucleotides of the antisense strand are not complementary to the target.

[0077] In some embodiments, oligonucleotides incorporating such sequences are provided, having sense and antisense strands, each ranging in length from 21 to 23 nucleotides. In some embodiments, 3' overhangs are provided on the sense strand, antisense strand, or sense and antisense strands, each being 1 or 2 nucleotides in length. In some embodiments, the oligonucleotide has a 23-nucleotide leader strand and a 21-nucleotide guest strand, wherein the 3' end of the guest strand and the 5' end of the leader strand form blunt ends, and wherein the leader strand has a two-nucleotide 3' overhang.

[0078] In some embodiments, the double-stranded oligonucleotides used to reduce GYS2 expression participate in RNA interference (RNAi). For example, RNAi oligonucleotides have been developed in which each strand has a size of 19-25 nucleotides and has at least one 3' overhang of 1 to 5 nucleotides (see, for example, U.S. Patent No. 8,372,968). Longer oligonucleotides have also been developed, which are processed by cleavage enzymes to generate active RNAi products (see, for example, U.S. Patent No. 8,883,996). Further work yields extended double-stranded oligonucleotides in which at least one end of at least one strand extends beyond the double-stranded target region, including structures in which one of the strands comprises a thermodynamically stable tetracyclic structure (see, for example, U.S. Patent Nos. 8,513,207 and 8,927,705, and WO 2010033225, the disclosure of which is incorporated herein by reference). Such structures may include single-strand extensions (on one or both sides of the molecule) and double-strand extensions.

[0079] In some embodiments, the oligonucleotide may be in the range of 21 to 23 nucleotides in length. In some embodiments, the oligonucleotide may have a 3' overhang (e.g., 1, 2, or 3 nucleotides in length) at the 3' end of the sense strand and / or antisense strand. In some embodiments, the oligonucleotide (e.g., siRNA) may comprise a 21-nucleotide leader strand antisense to the target RNA and a complementary guest strand, wherein the two strands are annealed to form a 19-bp duplex and a 2-nucleotide overhang at either or both 3' ends. See, for example, US9012138, US9012621, and US9193753, the contents of which are incorporated herein by reference for their respective disclosures.

[0080] In some embodiments, the oligonucleotide of the present invention has a 36-nucleotide sense strand comprising a region extending beyond the antisense duplex, wherein the extended region has a stem-quadruplex structure, wherein the stem is a six-base-pair duplex and wherein the quadruplex has four nucleotides. In some embodiments, the stem-quadruplex is as follows: S1-L-S2, wherein S1 and S2 are complementary to form a duplex, and wherein L forms a quadruplex between S1 and S2.

[0081] In some of those implementations, three or four of the tetracyclic nucleotides are each conjugated to a monovalent GalNac ligand.

[0082] In some embodiments, the oligonucleotide of the present invention comprises a sense strand of 25 nucleotides and an antisense strand of 27 nucleotides, which, when acted upon by cleatase, produces an antisense strand incorporated into the mature RISC.

[0083] Other oligonucleotide designs for use with the compositions and methods disclosed herein include: 16-meric siRNAs (see, e.g., Nucleic Acids in Chemistry and Biology. Blackburn (ed.), Royal Society of Chemistry, 2006), shRNAs (e.g., with stems of 19 bp or less; see, e.g., Moore et al., Methods Mol. Biol. 2010; 629:141-158), blunt-ended siRNAs (e.g., 19 bp in length; see, e.g., Kraynack and Baker, RNA Vol. 12, p163-176 (2006)), asymmetric siRNAs (aiRNA; see, e.g., Sun et al., Nat. Biotechnol. 26, 1379–1382 (2008)), and asymmetric shorter double-stranded siRNAs (see, e.g., Chang et al., Mol. Biol ... Ther. 2009 Apr; 17(4):725-32), bifurcated siRNAs (see, e.g., Hohjoh, FEBS Letters, Vol 557, issues 1-3; Jan 2004, p 193-198), single-stranded siRNAs (Elsner; Nature Biotechnology 30, 1063 (2012)), dumbbell-shaped circular siRNAs (see, e.g., Abe et al. J Am Chem Soc 129:15108-15109 (2007)) and small internally segmented interfering RNAs (sisiRNA; see, e.g., Bramsen et al., Nucleic Acids Res. 2007 Sep; 35(17):5886–5897). The relevant publications in each of the foregoing references are incorporated herein by reference in their entirety. Other non-limiting examples of oligonucleotide structures that can be used to reduce or inhibit GYS2 expression in some implementations are microRNAs (miRNAs), short hairpin RNAs (shRNAs), and short siRNAs (see, for example, Hamilton et al., Embo J., 2002, 21(17):4671-4679; see also U.S. Application No. 20090099115).

[0084] a. antisense chain

[0085] In some embodiments, the oligonucleotides disclosed herein for targeting GYS2 comprise an antisense strand comprising or consisting of a sequence as shown in any of SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598, or 620-627. In some embodiments, the oligonucleotide comprises an antisense strand comprising or consisting of at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides as shown in any of SEQ ID NO:193-384, 417-466, 518-568, 575-580, 586-598, or 620-627.

[0086] In some embodiments, the double-stranded oligonucleotide may have an antisense strand of up to 40 nucleotides in length (e.g., up to 40, up to 35, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide may have an antisense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 22, at least 25, at least 27, at least 30, at least 35, or at least 38 nucleotides in length). In some embodiments, the oligonucleotide may have an antisense strand of 12 to 40 nucleotides in length (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 22, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40). In some embodiments, the oligonucleotide may have an antisense strand of 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length.

[0087] In some implementations, the antisense strand of the oligonucleotide may be referred to as the "guide strand." For example, if the antisense strand can bind to the RNA-induced silencing complex (RISC) and bind to the Argonaut protein, or bind to one or more similar factors and guide the silencing of a target gene, it may be referred to as the guide strand. In some implementations, the sense strand complementary to the guide strand may be referred to as the "passenger strand."

[0088] b. Chain of meaning

[0089] In some embodiments, the oligonucleotides disclosed herein for targeting GYS2 comprise or consist of a sense strand sequence as shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619. In some embodiments, the oligonucleotide has a sense strand comprising or consists of at least 12 (e.g., at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23) consecutive nucleotides of the sequence shown in any of SEQ ID NO:1-192, 385-416, 467-517, 569-574, 581-585, or 612-619.

[0090] In some embodiments, the oligonucleotide may have a sense strand (or guest strand) of up to 40 nucleotides in length (e.g., up to 40, up to 36, up to 30, up to 27, up to 25, up to 21, up to 19, up to 17, or up to 12 nucleotides in length). In some embodiments, the oligonucleotide may have a sense strand of at least 12 nucleotides in length (e.g., at least 12, at least 15, at least 19, at least 21, at least 25, at least 27, at least 30, at least 36, or at least 38 nucleotides in length). In some embodiments, the oligonucleotide may have a sense strand of length ranging from 12 to 40 nucleotides (e.g., 12 to 40, 12 to 36, 12 to 32, 12 to 28, 15 to 40, 15 to 36, 15 to 32, 15 to 28, 17 to 21, 17 to 25, 19 to 27, 19 to 30, 20 to 40, 22 to 40, 25 to 40, or 32 to 40). In some embodiments, the oligonucleotide may have a sense strand of length 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides.

[0091] In some embodiments, the sense strand includes a stem-loop structure at its 3' end. In some embodiments, the sense strand includes a stem-loop structure at its 5' end. In some embodiments, the stem is a double strand of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 base pairs in length. In some embodiments, the stem-loop provides the molecule with better protection against degradation (e.g., enzymatic degradation) and facilitates targeting features for delivery to target cells. For example, in some embodiments, the loop provides an added nucleotide that can be modified without substantially affecting the gene expression repressive activity of the oligonucleotide. In some embodiments, oligonucleotides are provided herein in which the sense strand includes (e.g., at its 3' end) a stem-loop as follows: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of up to 10 nucleotides in length (e.g., 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length) between S1 and S2.

[0092] In some implementations, the stem-loop loop (L) is a tetracycle (e.g., within a cleaved tetracycle structure). The tetracycle may contain ribonucleotides, deoxyribonucleotides, modified nucleotides, and combinations thereof. Typically, a tetracycle has 4 to 5 nucleotides.

[0093] c. Length of the double chain

[0094] In some embodiments, the length of the double strand formed between the sense and antisense strands is at least 12 nucleotides (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 some embodiments, the length of the double strand formed between the sense and antisense strands is in the range of 12-30 nucleotides (e.g., 12-30, 12-27, 12-22, 15-25, 18-30, 18-22, 18-25, 18-27, 18-30, 19-30, or 21-30 nucleotides). In some embodiments, the length of the double strand formed between the sense and antisense strands is 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides. In some embodiments, the double strand formed between the sense and antisense strands does not span the entire length of the sense and / or antisense strands. In some embodiments, the double strand between the sense and antisense strands spans the entire length of either the sense or antisense strand. In some embodiments, the double strand between the sense and antisense strands spans the entire length of both the sense and antisense strands.

[0095] d. Oligonucleotide terminus

[0096] In some embodiments, the oligonucleotides provided herein comprise a sense strand and an antisense strand, such that a 3'-protrusion is present on the sense strand or the antisense strand, or both. In some embodiments, the oligonucleotides provided herein have a 5' end that is thermodynamically less stable than another 5' end. In some embodiments, an asymmetric oligonucleotide is provided comprising a blunt end at the 3' end of the sense strand and a protruding end at the 3' end of the antisense strand. In some embodiments, the 3' protrusion on the antisense strand is 1-8 nucleotides long (e.g., 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides).

[0097] Typically, the oligonucleotide of RNAi has a two-nucleotide overhang at the 3' end of the antisense (guide) strand. However, other overhangs are possible. In some embodiments, the overhang is a 3' overhang containing a length 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. However, in some embodiments, the overhang is a 5' overhang, which comprises a length 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.

[0098] In some embodiments, one or more (e.g., 2, 3, 4) terminal nucleotides at the 3' or 5' end of the sense and / or antisense strand are modified. For example, in some embodiments, one or two terminal nucleotides at the 3' end of the antisense strand are modified. In some embodiments, the last nucleotide at the 3' end of the antisense strand is modified, for example, by including a 2'-modification, such as 2'-O-methoxyethyl. In some embodiments, the last or two terminal nucleotides at the 3' end of the antisense strand are complementary to the target. In some embodiments, the last or two nucleotides at the 3' end of the antisense strand are not complementary to the target. In some embodiments, the 5' and / or 3' ends of the sense or antisense strand have an inverse cap nucleotide.

[0099] e. Mismatch

[0100] In some embodiments, one or more (e.g., 1, 2, 3, or 4) mismatches exist between the sense and antisense strands. If more than one mismatch exists between the sense and antisense strands, they can be sequentially located (e.g., two, three, or more consecutively) or scattered throughout the complementary region. In some embodiments, the 3' end of the sense strand contains one or more mismatches. In one embodiment, two mismatches are incorporated into the 3' end of the sense strand. In some embodiments, base mismatches or destabilization of the 3'-terminal segment of the oligonucleotide's sense strand enhance the efficiency of bistrand synthesis in RNAi, possibly by facilitating cleatosyl processing.

[0101] iii. Single-stranded oligonucleotides

[0102] In some embodiments, the oligonucleotides used to reduce GYS2 expression, as described herein, are single-stranded. Such structures may include, but are not limited to, single-stranded RNAi oligonucleotides. Recent efforts have demonstrated the activity of single-stranded RNAi oligonucleotides (see, for example, Matsui et al. (May 2016), Molecular Therapy, Vol. 24(5), 946-955). However, in some embodiments, the oligonucleotides provided herein are antisense oligonucleotides (ASOs). Antisense oligonucleotides are single-stranded oligonucleotides having a nucleobase sequence that, when written in the 5' to 3' orientation, contains the reverse complement of a target segment of a specific nucleic acid and is suitably modified (e.g., as a gapmer) to induce RNaseH-mediated cleavage of its target RNA in the cell or (e.g., as a mixmer) to inhibit the translation of the target mRNA in the cell. The antisense oligonucleotides used in this disclosure may be modified in any suitable manner known in the art, including, for example, as shown in U.S. Patent No. 9,567,587, the disclosure of which regarding modifications of the antisense oligonucleotides (including, for example, changes in length, the sugar moiety of the nucleobase (pyrimidine, purine), and the heterocyclic moiety of the nucleobase) is incorporated herein by reference. Furthermore, antisense molecules have been used for decades to reduce the expression of specific target genes (see, for example, Bennett et al.; Pharmacology of Antisense Drugs, Annual Review of Pharmacology and Toxicology, Vol. 57:81-105).

[0103] iv. Oligonucleotide modification

[0104] Oligonucleotides can be modified in various ways to improve or control specificity, stability, delivery, bioavailability, resistance to nuclease degradation, immunogenicity, base-pairing properties, RNA distribution and cellular uptake, and other characteristics relevant to therapeutic or research use. See, for example, Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881; Bramsen and Kjems (Frontiers in Genetics, 3(2012): 1-22). Therefore, in some embodiments, the oligonucleotides of this disclosure may include one or more suitable modifications. In some embodiments, the modified nucleotide has modifications in its bases (or nucleobases), sugars (e.g., ribose, deoxyribose), or phosphate ester groups.

[0105] The number of modifications on an oligonucleotide and the position of those modifications can affect the properties of the oligonucleotide. For example, oligonucleotides can be delivered in vivo by conjugating them to lipid nanoparticles (LNPs) or similar carriers, or by encapsulating them in lipid nanoparticles (LNPs) or similar carriers. However, when the oligonucleotide is not protected by an LNP or similar carrier (e.g., “naked delivery”), it can be advantageous for at least some of its nucleotides to be modified. Thus, in some embodiments of any oligonucleotide provided herein, all or substantially all of the nucleotides of the 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. Typically, in the case of naked delivery, each nucleotide is modified at the 2'-position of the sugar group of that nucleotide. These modifications can be reversible or irreversible. Typically, 2'-position modifications are 2'-fluorine, 2'-O-methyl, etc. In some embodiments, oligonucleotides such as those disclosed herein have a number and type of modified nucleotides sufficient to induce desired characteristics (e.g., protection against enzymatic degradation, ability to target desired cells after in vivo administration, and / or thermodynamic stability).

[0106] a. Sugar modification

[0107] In some embodiments, the modified sugar (also referred to herein as a sugar analogue) comprises a modified deoxyribose or ribose moiety, for example, wherein one or more modifications occur at the 2', 3', 4' and / or 5' carbon positions of the sugar. In some embodiments, the modified sugar may also comprise non-natural alternative carbon structures, such as those present in: locked nucleic acids (“LNA”) (see, e.g., Koshkin et al. (1998), Tetrahedron 54, 3607-3630), unlocked nucleic acids (“UNA”) (see, e.g., Snead et al. (2013), Molecular Therapy–Nucleic Acids, 2, e103), and bridged nucleic acids (“BNA”) (see, e.g., Imanishi and Obika (2002), The Royal Society of Chemistry, Chem. Commun., 1653-1659). Koshkin et al., Snead et al., and Imanishi and Obika’s public disclosures concerning sugar modifications are incorporated herein by reference.

[0108] In some embodiments, the nucleotide modification in the sugar includes a 2'-modification. In some embodiments, the 2'-modification can be 2'-aminoethyl, 2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl, or 2'-deoxy-2'-fluoro-β-d-arabinose. Typically, the modification is 2'-fluoro, 2'-O-methyl, or 2'-O-methoxyethyl. However, a variety of 2'-position modifications have been developed for use in oligonucleotides and can be used in the oligonucleotides disclosed herein. See, for example, Bramsen et al., Nucleic Acids Res., 2009, 37, 2867-2881. In some embodiments, the modification in the sugar includes a modification of the sugar ring, which may include a modification of one or more carbons of the sugar ring. For example, the sugar modification of the nucleotide may include a bond between the 2'-carbon and the 1'-carbon or 4'-carbon of the sugar. For example, the bond may include an ethylene or methylene bridge. In some embodiments, the modified nucleotide has an acyclic sugar lacking 2'-to-3'-carbon bonds. In some embodiments, the modified nucleotide has a thiol group, for example, at the 4' position of the sugar.

[0109] In some embodiments, the terminal 3'-terminal group (e.g., 3'-hydroxyl) is a phosphate ester group or other group that can be used, for example, to connect adapters, adaptors or markers, or to directly link oligonucleotides to another nucleic acid.

[0110] b.5' terminal phosphate

[0111] In some cases, the 5'-terminal phosphate group of an oligonucleotide can enhance its interaction with Argonaut 2. However, oligonucleotides containing a 5'-phosphate group are readily degraded by phosphatases or other enzymes, which can limit their bioavailability in vivo. In some embodiments, the oligonucleotide comprises an analog of a 5'-phosphate group that is resistant to such degradation. In some embodiments, the phosphate analog may be an oxymethylphosphonate, a vinylphosphonate, or a malonylphosphonate. In some embodiments, the 5' end of the oligonucleotide chain is attached to a chemical moiety that mimics the electrostatic and steric properties of the native 5'-phosphate group (“phosphate analog”) (see, for example, Prakash et al. (2015), Nucleic Acids Res., Nucleic Acids Res. 2015 Mar 31; 43(6):2993–3011, the contents of which relate to phosphate analogs are incorporated herein by reference). Numerous phosphate ester analogs that can be attached to the 5' end have been developed (see, for example, U.S. Patent No. 8,927,513, the contents of which relate to phosphate ester analogs are incorporated herein by reference). Other modifications have been developed for the 5' end of oligonucleotides (see, for example, WO 2011 / 133871, the contents of which relate to phosphate ester analogs are incorporated herein by reference). In some embodiments, a hydroxyl group is attached to the 5' end of the oligonucleotide.

[0112] In some embodiments, the oligonucleotide has a phosphate ester analog (referred to as a "4'-phosphate ester analog") at the 4'-carbon position of the sugar. See, for example, International Patent Application PCT / US2017 / 049909, filed September 1, 2017; U.S. Provisional Application No. 62 / 383,207, filed September 2, 2016, entitled "4'-Phosphate Analogs and Oligonucleotides Comprising the Same"; and U.S. Provisional Application No. 62 / 393,401, filed September 12, 2016, each relating to phosphate ester analogs, the contents of which are incorporated herein by reference. In some embodiments, the oligonucleotide provided herein contains a 4'-phosphate ester analog at the 5'-terminal nucleotide. In some embodiments, the phosphate ester analog is an oxymethylphosphonate or an analog thereof in which the oxygen atom of the oxymethyl group is bonded to the sugar moiety (e.g., at its 4'-carbon). In other embodiments, the 4'-phosphate analog is a thiomethylphosphonate or aminomethylphosphonate or its analogue, wherein the sulfur atom of the thiomethyl group or the nitrogen atom of the aminomethyl group is bonded to the 4'-carbon of the sugar moiety. In some embodiments, the 4'-phosphate analog is an oxymethylphosphonate. In some embodiments, the oxymethylphosphonate is represented by the formula -O-CH2-PO(OH)2 or -O-CH2-PO(OR)2, wherein R is independently selected from H, CH3, an alkyl group, CH2CH2CN, CH2OCOC(CH3)3, CH2OCH2CH2Si(CH3)3, or a protecting group. In some embodiments, the alkyl group is CH2CH3. More generally, R is independently selected from H, CH3, or CH2CH3.

[0113] c. Modified nucleotide interbonds

[0114] In some embodiments, the oligonucleotide may comprise modified nucleotide internucleotides. In some embodiments, phosphate modification or substitution can produce an oligonucleotide comprising at least one (e.g., at least 1, at least 2, at least 3, at least 4, or at least 5) modified nucleotide internucleotides. In some embodiments, any of the oligonucleotides disclosed herein comprises 1 to 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 nucleotide internucleotides. In some embodiments, any of the oligonucleotides disclosed herein comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 modified nucleotide internucleotides.

[0115] The modified internucleotide bond can be a dithiophosphate bond, a thiophosphate bond, a phosphate trimer bond, a thiocarbonyl phosphonate bond, a thiocarbonyl phosphonate bond, a phosphoramide bond, a phosphonate bond, or a borate phosphate bond. In some embodiments, at least one modified internucleotide bond of any of the oligonucleotides disclosed herein is a thiophosphate bond.

[0116] d. Base modification

[0117] In some embodiments, the oligonucleotides provided herein have one or more modified nucleobases. In some embodiments, the modified nucleobase (also referred to herein as a base analog) is linked at the 1' position of the nucleotide sugar moiety. In some embodiments, the modified nucleobase is a nitrogenous base. In some embodiments, the modified nucleobase does not contain a nitrogen atom. See, for example, U.S. Patent Application Publication No. 20080274462. In some embodiments, the modified nucleotide contains a universal base. However, in some embodiments, the modified nucleotide does not contain a nucleobase (base-free).

[0118] In some embodiments, the universal base is a heterocyclic portion located at the 1' position of the nucleotide sugar moiety in the modified nucleotide or at an equivalent position in a nucleotide sugar moiety substitution. When present in a duplex, it can be positioned in relatively more than one type of base without substantially altering the duplex structure. In some embodiments, a single-stranded nucleic acid containing a universal base forming a duplex with a target nucleic acid has a lower Tm than a duplex formed with a complementary nucleic acid, compared to a reference single-stranded nucleic acid (e.g., an oligonucleotide) that is fully complementary to the target nucleic acid. However, in some embodiments, a single-stranded nucleic acid containing a universal base forming a duplex with a target nucleic acid has a higher Tm than a duplex formed with a nucleic acid containing a mismatched base, compared to a reference single-stranded nucleic acid in which the universal base has been replaced to generate a single mismatch. m .

[0119] Non-limiting examples of universally binding nucleotides include inosine, 1-β-D-ribofuranosyl-5-nitroindole, and / or 1-β-D-ribofuranosyl-3-nitropyrrole (Quay et al., U.S. Patent Application Publication No. 20070254362; Van Aerschot et al., An acyclic 5-nitroindazole nucleoside analogue as ambiguous nucleoside. Nucleic Acids Res. 1995 Nov 11; 23(21):4363-70; Loakes et al., 3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR. Nucleic Acids Res. 1995 Jul 11; 23(13):2361-6; Loakes and Brown, 5-Nitroindole as an universal base analogue. Nucleic Acids Res. 1994 Oct 11; 22(20): 4039-43. The aforementioned disclosures concerning their respective base modifications are incorporated herein by reference.

[0120] e. Reversible modification

[0121] Although certain modifications can be made to protect oligonucleotides from the effects of the in vivo environment before they reach target cells, once the oligonucleotides reach the cytosol of the target cells, the target cells can reduce the efficacy or activity of the oligonucleotides. Reversible modifications can be made so that the molecule retains the desired properties outside the cell, and is then removed upon entering the cellular cytosol environment. Reversible modifications can be removed, for example, by the action of intracellular enzymes or by intracellular chemical conditions (e.g., by reduction by intracellular glutathione).

[0122] In some embodiments, the reversibly modified nucleotides contain a glutathione-sensitive moiety. Typically, nucleic acid molecules have been chemically modified with a cyclic disulfide moiety to mask the negative charge generated by the internucleotide diphosphate bond and to improve cellular uptake and nuclease resistance. See U.S. Publication No. 2011 / 0294869, originally assigned to Traversa Therapeutics, Inc. (“Traversa”), PCT Publication No. WO 2015 / 188197, assigned to Solstice Biologics, Ltd. (“Solstice”), Meade et al., Nature Biotechnology, 2014, 32:1256-1263 (“Meade”), and PCT Publication No. WO 2014 / 088920, assigned to Merck Sharp & Dohme Corp., each of which is incorporated herein by reference for its disclosure of such modifications. This reversible modification of the internucleotide diphosphate bond is designed to be cleaved intracellularly via a reducing environment in the cytosol (e.g., glutathione). Earlier examples include neutral phosphate triester modifications, which have been reported to be cleaved intracellularly (Dellinger et al. J. Am. Chem. Soc. 2003, 125: 940-950).

[0123] In some embodiments, this reversible modification allows for protection during in vivo administration (e.g., transport via lysosomes / endosome compartments in the blood and / or cells), where the oligonucleotide will be exposed to nucleases and other harsh environmental conditions (e.g., pH). The modification is reversed upon release into the cytosol of the cell (where glutathione levels are higher compared to the extracellular space), resulting in a cleaved oligonucleotide. Using a reversible glutathione-sensitive moiety allows for the introduction of spatially larger chemical groups into the target oligonucleotide compared to options available using irreversible chemical modifications. This is because these larger chemical groups will be removed in the cytosol and therefore should not interfere with the biological activity of the oligonucleotide within the cytosol of the cell. As a result, these larger chemical groups can be engineered to confer various advantages to the nucleotide or oligonucleotide, such as nuclease resistance, lipophilicity, charge, thermal stability, specificity, and reduced immunogenicity. In some embodiments, the glutathione-sensitive moiety can be structurally engineered to modify its release kinetics.

[0124] In some embodiments, the glutathione-sensitive moiety is linked to the sugar of the nucleotide. In some embodiments, the glutathione-sensitive moiety is linked to the 2'-carbon of the sugar of the modified nucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 5'-carbon of the sugar, particularly when the modified nucleotide is the 5'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety is located at the 3'-carbon of the sugar, particularly when the modified nucleotide is the 3'-terminal nucleotide of the oligonucleotide. In some embodiments, the glutathione-sensitive moiety comprises a sulfonyl group. See, for example, International Patent Application PCT / US2017 / 048239, published as International Patent Publication WO2018 / 039364 on March 1, 2018, entitled Compositions Comprising Reversibly Modified Oligonucleotides and Uses Thereof, filed on August 23, 2016, the contents of which are incorporated herein by reference.

[0125] v. Targeted ligands

[0126] In some embodiments, it is desirable to target the oligonucleotides of this disclosure to one or more cells or organs. This strategy can help avoid undesirable effects in other organs or prevent excessive loss of the oligonucleotides to cells, tissues, or organs that do not benefit from them. Therefore, in some embodiments, the oligonucleotides disclosed herein can be modified to facilitate targeting of specific tissues, cells, or organs, for example, to facilitate oligonucleotide delivery to the liver. In some embodiments, the oligonucleotides disclosed herein can be modified to facilitate oligonucleotide delivery to hepatocytes of the liver. In some embodiments, the oligonucleotides comprise nucleotides conjugated to one or more targeting ligands.

[0127] The targeting ligand may comprise carbohydrates, amino sugars, cholesterol, peptides, polypeptides, proteins or protein portions (e.g., antibodies or antibody fragments), or lipids. In some embodiments, the targeting ligand is an aptamer. For example, the targeting ligand may be an RGD peptide for targeting tumor vascular systems or glioma cells, a CREKA peptide for targeting tumor vascular systems or stomas, transferrin, lactoferrin, or an aptamer for targeting transferrin receptors expressed on CNS vascular systems, or an anti-EGFR antibody targeting EGFR on glioma cells. In some embodiments, the targeting ligand is one or more GalNAc portions.

[0128] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, 2 to 4 nucleotides of the oligonucleotide are each conjugated to a separate targeting ligand. In some embodiments, the targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to a protruding end or extension of 2 to 4 nucleotides at the 5' or 3' end of the sense or antisense strand), such that the targeting ligand resembles the bristles of a toothbrush, and the oligonucleotide resembles a toothbrush. For example, the oligonucleotide may comprise a stem-loop at the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the stem-loop may be individually conjugated to the targeting ligand, as described, for example, in International Patent Application Publication WO 2016 / 100401, published June 23, 2016 (the relevant contents of which are incorporated herein by reference).

[0129] In some implementations, it is desirable to target oligonucleotides that reduce GYS2 expression to hepatocytes in the liver of a subject. Any suitable hepatocyte-targeting motif can be used for this purpose.

[0130] GalNAc is a high-affinity ligand for the desialyl glycoprotein receptor (ASGPR), which is primarily expressed on the sinusoidal surface of hepatocytes and plays a major role in the binding, internalization, and subsequent clearance of circulating glycoproteins (desialyl glycoproteins) containing terminal galactose or N-acetylgalactosamine residues. Conjugation (indirectly or directly) of the GalNAc moiety to oligonucleotides of this disclosure can be used to target these oligonucleotides to ASGPRs expressed on these hepatocytes.

[0131] In some embodiments, the oligonucleotides of this disclosure are directly or indirectly conjugated to monovalent GalNAc. In some embodiments, the oligonucleotides are directly or indirectly conjugated to more than one monovalent GalNAc (i.e., conjugated to 2, 3, or 4 monovalent GalNAc moieties, and typically conjugated to 3 or 4 monovalent GalNAc moieties). In some embodiments, the oligonucleotides of this disclosure are conjugated to one or more divalent, trivalent, or tetravalent GalNAc moieties.

[0132] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, or 6) nucleotides of the oligonucleotide are each conjugated to the GalNAc moiety. In some embodiments, 2 to 4 nucleotides of the loop (L) of the stem-loop are each conjugated to a separate GalNAc. In some embodiments, a targeting ligand is conjugated to 2 to 4 nucleotides at either end of the sense or antisense strand (e.g., the ligand is conjugated to a 2 to 4 nucleotide overhang or extension at the 5' or 3' end of the sense or antisense strand), such that the GalNAc moiety resembles the bristles of a toothbrush, and the oligonucleotide resembles a toothbrush. For example, the oligonucleotide may comprise a stem-loop at the 5' or 3' end of the sense strand, and 1, 2, 3, or 4 nucleotides of the loop of the stem may be conjugated individually to the GalNAc moiety. In some embodiments, the GalNAc moiety is conjugated to a nucleotide of the sense strand. For example, the four GalNAc moieties can be conjugated to nucleotides in a tetracyclic sense chain, with each GalNAc moiety conjugated to one nucleotide.

[0133] Targeting ligands can be attached to nucleotides using suitable methods or chemical approaches (e.g., click chemistry). In some embodiments, click connectors are used to conjugate the targeting ligand to the nucleotide. In some embodiments, acetal-based connectors are used to conjugate the targeting ligand to any of the oligonucleotides described herein. Acetal-based connectors are disclosed, for example, in International Patent Application Publication No. WO2016100401 A1, published June 23, 2016, and the contents relating to such connectors are incorporated herein by reference. In some embodiments, the connector is an unstable connector. However, in other embodiments, the connector is fairly stable. In some embodiments, a double-stranded extension (up to 3, 4, 5, or 6 base pairs in length) is provided between the targeting ligand (e.g., the GalNAc moiety) and the double-stranded oligonucleotide.

[0134] III. Preparations

[0135] Various formulations have been developed to facilitate the use of oligonucleotides. For example, oligonucleotides can be delivered to a subject or cellular environment using formulations that minimize degradation, facilitate delivery and / or uptake, or provide another beneficial property to the oligonucleotide in the formulation. In some embodiments, compositions comprising oligonucleotides (e.g., single-stranded or double-stranded oligonucleotides) to reduce GYS2 expression are provided herein. Such compositions can be suitably formulated such that, when administered to a subject (in the direct environment of target cells or systemically), a sufficient fraction of the oligonucleotide enters the cells to reduce GYS2 expression. Any of a variety of suitable oligonucleotide formulations can be used to deliver oligonucleotides for reducing GYS2, as disclosed herein. In some embodiments, the oligonucleotide is formulated in a buffer solution, such as an aqueous solution of phosphate-buffered saline, liposomes, micelle structures, and capsids. In some embodiments, the naked oligonucleotide or its conjugate is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the naked oligonucleotide or its conjugate is formulated in an alkaline buffer solution (e.g., PBS).

[0136] Formulations of oligonucleotides containing cationic lipids can be used to facilitate the transfection of oligonucleotides into cells. For example, cationic lipids such as lipofectin, cationic glycerol derivatives, and polycationic molecules (e.g., polylysine) can be used. Suitable lipids include Oligofectamine, Lipofectamine (Life Technologies), NC388 (Ribozyme Pharmaceuticals, Inc., Boulder, Colo.), or FuGene 6 (Roche), all of which can be used according to the manufacturer's instructions.

[0137] Therefore, in some embodiments, the formulation comprises lipid nanoparticles. In some embodiments, the excipient comprises liposomes, lipids, lipid complexes, microspheres, microparticles, nanospheres, or nanoparticles, or may otherwise be formulated for administration to the cells, tissues, organs, or bodies of a subject in need (see, for example, Remington: The Science and Practice of Pharmacy, 22nd edition, Pharmaceutical Press, 2013).

[0138] In some embodiments, the formulations disclosed herein include excipients. In some embodiments, the excipients impart to the composition enhanced stability, enhanced absorption, enhanced solubility, and / or therapeutic enhancement of the active ingredient. In some embodiments, the excipients are buffers (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or mediators (e.g., buffer solutions, petrolatum, dimethyl sulfoxide, or mineral oil). In some embodiments, the oligonucleotides are lyophilized to extend their shelf life and then formulated into solutions prior to use (e.g., administration to a subject). Therefore, the excipients in compositions comprising any of the oligonucleotides described herein may be lyophilization protectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or collapse temperature modifiers (e.g., dextran, ficoll, or gelatin).

[0139] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Typically, the route of administration is intravenous or subcutaneous.

[0140] Pharmaceutical compositions suitable for injectable applications include sterile aqueous solutions (in the case of water solubility) or dispersions, as well as sterile powders for the ad hoc preparation of sterile injectable solutions or dispersions. For intravenous or subcutaneous administration, suitable carriers include physiological saline, antibacterial water, Cremophor EL.™ (BASF, Parsippany, NJ), or phosphate-buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In many cases, isotonic agents, such as sugars, and polyols such as mannitol, sorbitol, and sodium chloride, are preferably included in the composition. Sterile injectable solutions can be prepared by incorporating the desired amount of oligonucleotides with one or a combination of the desired ingredients listed above into a selected solvent, followed by filtration sterilization.

[0141] In some embodiments, the composition may contain at least about 0.1% or more of a therapeutic agent (e.g., an oligonucleotide for reducing GYS2 expression), although the percentage of one or more active ingredients may be between about 1% and about 80% or more of the total composition by weight or volume. Those skilled in the art of preparing such pharmaceutical formulations will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations, and therefore various dosages and treatment regimens may be desirable.

[0142] Even though many implementations involve liver-targeted delivery of any oligonucleotides disclosed herein, targeting other tissues is also considered.

[0143] IV. Instructions for Use

[0144] i. Reduce GYS2 expression in cells

[0145] In some embodiments, methods are provided for delivering an effective amount of any of the oligonucleotides disclosed herein to cells for the purpose of reducing GYS2 expression in cells. The methods provided herein can be used in any suitable cell type. In some embodiments, the cells are any cells expressing GYS2 (e.g., liver cells, such as hepatocytes or adipocytes). In some embodiments, the cells are primary cells that have been obtained from a subject and may have undergone a limited number of generations, such that the cells substantially maintain their native phenotypic characteristics. In some embodiments, the cells to which the oligonucleotides are delivered are ex vivo or in vitro (i.e., cells that can be delivered to a culture or an organism in which the cells reside). In specific embodiments, methods are provided for delivering an effective amount of any of the oligonucleotides disclosed herein to cells for the purpose of reducing GYS2 expression solely or primarily in hepatocytes.

[0146] In some embodiments, the oligonucleotides disclosed herein can be introduced using suitable nucleic acid delivery methods, including injecting a solution containing the oligonucleotide, bombarding a cell or organism with a particle coated with the oligonucleotide, exposing a cell or organism to a solution containing the oligonucleotide, or electroporating a cell membrane in the presence of the oligonucleotide. Other suitable methods for delivering oligonucleotides to cells can be used, such as lipid-mediated carrier transport, chemical-mediated transport, and cationic liposome transfection of calcium phosphate and others.

[0147] The consequences of inhibition can be confirmed by appropriate assays assessing one or more characteristics of cells or subjects or by biochemical techniques assessing molecules (e.g., RNA, protein) that indicate GYS2 expression. In some embodiments, the extent to which the oligonucleotides provided herein reduce GYS2 expression levels is assessed by comparing GYS2 expression levels (e.g., mRNA or protein levels) with appropriate controls (e.g., the level of GYS2 expression in cells or cell populations to which oligonucleotides have not been delivered or to which negative controls have been delivered). In some embodiments, the appropriate control level for GYS2 expression can be a predetermined level or value, such that it is not necessary to measure the control level every time. Predetermined levels or values ​​can take various forms. In some embodiments, the predetermined level or value can be a single cutoff value, such as a median or average.

[0148] In some embodiments, the administration of the oligonucleotide as described herein results in a reduction in the level of GYS2 expression in cells. In some embodiments, the reduction in GYS2 expression can be a reduction to 1% or less, 5% or less, 10% or less, 15% or less, 20% or less, 25% or less, 30% or less, 35% or less, 40% or less, 45% or less, 50% or less, 55% or less, 60% or less, 70% or less, 80% or less, or 90% or less compared to an appropriate control level of GYS2. An appropriate control level can be the level of GYS2 expression in cells or cell populations that have not been contacted with the oligonucleotide as described herein. In some embodiments, the effectiveness of delivering the oligonucleotide to cells according to the methods disclosed herein is evaluated after a limited time period. For example, the level of GYS2 in cells can be analyzed at least 8 hours, 12 hours, 18 hours, 24 hours after the oligonucleotide is introduced into the cells; or at least one, two, three, four, five, six, seven, or fourteen days after the introduction of the oligonucleotide.

[0149] In some embodiments, the oligonucleotide is delivered in the form of a transgene engineered to express the oligonucleotide in cells (e.g., its sense and antisense strands). In some embodiments, the oligonucleotide is delivered using a transgene engineered to express any oligonucleotide disclosed herein. The transgene can be delivered using a viral vector (e.g., adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, or herpes simplex virus) or a non-viral vector (e.g., plasmid or synthetic mRNA). In some embodiments, the transgene can be injected directly into a subject.

[0150] ii. Treatment methods

[0151] This disclosure relates to methods for reducing GYS2 expression in the treatment of glycogen storage diseases in subjects. In some embodiments, the methods may include administering an effective amount of any of the oligonucleotides disclosed herein to a subject in need. Such treatments can be used, for example, to reduce or prevent hepatomegaly, hepatotoxicity (e.g., reducing or decreasing levels of AST, ALT, and / or ALP), liver fibrosis, fatty acid deposition in the liver, hepatic hyperplasia, hepatocellular adenoma, and / or hepatocellular carcinoma. Such treatments can also be used, for example, to treat or prevent one or more symptoms associated with glycogen storage diseases selected from GSD1a, GSDIII, GSDIV, GSDVI, and GSDIX, or to treat or prevent one or more symptoms of such glycogen storage diseases. This disclosure provides preventive and therapeutic methods for treating subjects at risk of (or susceptible to) glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX) and / or symptoms or conditions associated with glycogen storage diseases (e.g., GSDIa, GSDIII, GSDIV, GSDVI, and GSDIX).

[0152] In some aspects, this disclosure provides methods for preventing diseases, conditions, symptoms, or illnesses as described herein in a subject by administering a therapeutic agent (e.g., an oligonucleotide or a vector or transgene encoding it) to the subject. In some embodiments, the subject to be treated is a subject who would benefit from a reduction in, for example, the amount of GYS2 protein in the liver.

[0153] The methods described herein generally involve administering an effective amount (i.e., an amount capable of producing the desired therapeutic outcome) of an oligonucleotide to a subject. A therapeutically acceptable amount may be an amount capable of treating a disease or condition. The appropriate dose for any subject will depend on several factors, including the subject's body size, body surface area, age, the specific composition to be administered, one or more active ingredients in the composition, the time and route of administration, overall health, and any other medications administered concurrently.

[0154] In some embodiments, any of the compositions disclosed herein are administered to a subject via the enteral (e.g., orally, via a gastric feeding tube, via a duodenal feeding tube, via a gastrostomy, or via the rectum), parenteral (e.g., subcutaneous injection, intravenous injection or infusion, intra-arterial injection or infusion, intramuscular injection), local (e.g., epidermal, inhalation, via eye drops, or via mucous membranes), or by direct injection into a target organ (e.g., the subject's liver). Typically, the oligonucleotides disclosed herein are administered intravenously or subcutaneously.

[0155] In some embodiments, the oligonucleotide is administered at a dose ranging from 0.1 mg / kg to 25 mg / kg (e.g., 1 mg / kg to 5 mg / kg). In some embodiments, the oligonucleotide is administered at a dose ranging from 0.1 mg / kg to 5 mg / kg or from 0.5 mg / kg to 5 mg / kg.

[0156] As a set of non-limiting examples, the oligonucleotides disclosed herein will be applied typically once a year, twice a year, quarterly (once every three months), every two months (once every two months), monthly, or weekly.

[0157] In some implementations, the subject to be treated is a human (e.g., a human patient) or a non-human primate or other mammal subject. Other exemplary subjects include domestic animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and animals such as mice, rats, guinea pigs, and hamsters. Example

[0158] Example 1: Development of GYS2 oligonucleotide inhibitors using human and mouse cell-based assays

[0159] Candidate oligonucleotides for inhibiting GYS2 expression were developed using human and mouse-based assays. First, a computer-based algorithm was used to generate candidate oligonucleotide sequences (25-27-mers) for GYS2 inhibition. Then, cell-based and PCR assays were used to evaluate the ability of the candidate oligonucleotides to reduce GYS2 expression.

[0160] Computer-based algorithms provided oligonucleotides complementary to human GYS2 mRNA (SEQ ID NO:609, Table 1), some of which were also complementary to macaque GYS2 mRNA (SEQ ID NO:610, Table 1).

[0161] Table 1. Sequences of GYS2 mRNA in humans and macaques

[0162] Species GenBank RefSeq# SEQ ID NO. people NM_021957.3 609 macaques XM_001098578.2 610

[0163] Of the oligonucleotides provided by the algorithm, 264 were selected as candidates for experimental evaluation in a HEK-293 cell-based assay. In this assay, HEK-293 human embryonic kidney cells stably expressing GYS2 (referred to as HEK-GYS2 cells) were transfected with the oligonucleotides. After transfection, the cells were maintained for a period of time, and then a assay based on… The level of residual GYS2 mRNA was determined using qPCR assays. Two qPCR assays, a 3' assay and a 5' assay, were used to determine mRNA levels as measured by HEX and FAM probes, respectively. Results from a HEK-293 cell-based assay using 264 oligonucleotides were shown. Figure 1A and 1B The 3' assay (circle) and 5' assay (diamond) each display the percentage of remaining mRNA. Oligonucleotides that result in the lowest percentage of remaining mRNA compared to the negative control are considered hits. Oligonucleotides with low complementarity to the human genome are used as negative controls.

[0164] Hotspots on human GYS2 mRNA were defined based on the activity and location of these oligonucleotides. Hotspots were identified as extensions in the human GYS2 mRNA sequence associated with at least two oligonucleotides that resulted in mRNA levels less than or equal to 35% compared to controls in any assay. Therefore, the following hotspots within the human GYS2 mRNA sequence were identified: 579-618, 691-738, 1089-1125, 1175-1211, 1431-1486, 2341-2383, 2497-2543, 2660-2698, 2808-2851, and 3014-3050.

[0165] The hotspot sequence is summarized in Table 2.

[0166] Table 2. Hotspot sequence

[0167]

[0168] Dose response analysis

[0169] Of the 264 oligonucleotides evaluated in the initial HEK-293 cell-based assay, 71 particularly active oligonucleotides were selected as hits based on their ability to knock down GYS2 levels and then subjected to secondary screening.

[0170] In this secondary screening, candidate oligonucleotides were tested using the same assays as in the primary screening, but at two or three different concentrations (1 nM, 0.1 nM, and 0.03 nM). Figure 2A and 2B The target mRNA level is typically normalized based on arginine / serine-rich splicing factor 9 (SFRS9) (a housekeeping gene that provides a stable expression reference between samples) to generate... Figure 2A and 2B The percentage of mRNA shown is compared to the negative control sequence (NC1) and mimic transfection. Figure 2A and 2BEach of the 71 oligonucleotides tested has the same modification pattern (referred to as M1), which contains a combination of ribonucleotides, deoxyribonucleotides, and 2'-O-methyl modified nucleotides. The sequences of the 71 oligonucleotides tested are provided in Table 3.

[0171] Table 3. Candidate oligonucleotide sequences based on HEK-293 cell assay

[0172]

[0173] Hs: human, and Rm: macaque; the sense and antisense SEQ ID NO. lists are provided in relative order for hybridization to produce a sense strand and a corresponding antisense strand for each oligonucleotide. For example, the sense strand of SEQ ID NO:1 is hybridized with the antisense strand of SEQ ID NO:193.

[0174] In this phase, 36 of the most effective sequences were selected from the tests for further analysis. The selected sequences were then converted into a nicked four-loop structure (36-mer transit chain and 22-mer leader chain). For a general four-loop structure, see [link to relevant documentation]. Figure 3 Then, these oligonucleotides were tested as before, and the ability of each oligonucleotide to reduce GYS2 mRNA expression in HepG2 cells was assessed at three concentrations.

[0175] Figure 4 Data on oligonucleotides made from different base sequences (each adapted to one or two different modification modes) with a nicked tetracyclic structure are displayed. The X-axis lists the 3' end of the sense strand targeted by the evaluated oligonucleotide. Target mRNA levels were normalized as described above to generate... Figure 4 The percentage of mRNA shown is displayed, and the oligonucleotides tested are shown compared to the negative control sequence (NC1) and mimic transfection.

[0176] In monkey hepatocytes, the concentrations of each compound were 0.1 μM, 0.3 μM, and 1.0 μM. Figure 5 Further testing of certain tetracyclic-modified oligonucleotides was conducted using the same modification pattern. Figure 5 The oligonucleotides tested were compared with untransfected cells. Some oligonucleotides were further tested in HEK-293 cells using full-dose response curves to determine the half-maximal inhibitory concentration (IC50) of each compound. 50 (See also) Figure 6A and 6B ).

[0177] In vivo mouse screening

[0178] Data from the aforementioned in vitro experiments were evaluated to identify the tetracyclic and modification patterns that would improve delivery properties while maintaining the activity used to reduce GYS2 expression in mouse hepatocytes. Based on this analysis, selected oligonucleotides were then conjugated to the GalNAc moieties. Four GalNAc moieties were conjugated to nucleotides with sense strands in the tetracyclic rings. Click adapters were used for conjugation. The GalNAcs used are shown below:

[0179]

[0180] N-acetyl-bD-galactosamine (CAS#:14131-60-3)

[0181] A total of 65 potent GalNAc-conjugated GYS2 oligonucleotides from 18 different base sequences and with varying modification patterns and nicked tetracyclic structures were tested. The selected GYS2 oligonucleotide sequences were active against human and monkey mRNA sequences but inactive against mouse Gys2. GYS2 oligonucleotides were administered subcutaneously to CD-1 mice transiently expressing human GYS2 mRNA via hydrodynamic injection of human GYS2 expression plasmids at 0.5–5 mg / kg. Mice were euthanized on day 4 post-administration. Liver samples were obtained and RNA was extracted to evaluate GYS2 mRNA levels by RT-qPCR. The percentage of GYS2 mRNA was determined based on these measurements, compared to PBS control mRNA.

[0182] Eight of the 65 conjugates tested were identified as the most potent base sequences, and each was tested using the same modification modality by subcutaneous injection of 0.5 mg / kg into CD-1 mice transiently expressing human GYS2 mRNA. Mice were euthanized on day 4 post-administration. Liver samples were obtained and RNA extracted for GYS2 mRNA level evaluation by RT-qPCR. The percentage of GYS2 mRNA was determined based on these measurements, compared to PBS control mRNA, and displayed as a percentage. Figure 7 middle.

[0183] Materials and methods

[0184] transfection

[0185] For the first screening, Lipofectamine RNAiMAX was used. TMTo facilitate effective transfection, a complex oligonucleotide was used. The oligonucleotide, RNAiMAX, and Opti-MEM were incubated together at room temperature for 20 minutes, and then 50 μL of this mixture was added to each well of the plate for transfection. The culture medium was aspirated from flasks containing actively passaged cells, and the cells were incubated at 37°C in the presence of trypsin for 3–5 minutes. After the cells no longer adhered to the flasks, cell growth medium (without penicillin and streptomycin) was added to neutralize the trypsin and suspend the cells. A 10 μL aliquot was taken and counted using a hemocytometer to quantify cells per milliliter. For cells, 10,000 to 25,000 cells / well were seeded in culture medium (e.g., 100 μL of medium). The diluted cell suspension was added to 96-well transfection plates already containing the oligonucleotide in Opti-MEM. The transfection plates were then incubated at 37°C for 24 hours. After 24 hours of incubation, the culture medium was aspirated from each well. Lyse cells using lysis buffer from the Promega RNA Isolation Kit. Add lysis buffer to each well. Then transfer the lysed cells to a Corbett XtractorGENE (QIAxtractor) for RNA isolation or store at -80°C.

[0186] For subsequent screening and experiments, such as secondary screening, Lipofectamine RNAiMAx was used to complex the oligonucleotides for reverse transfection. The complex was prepared by mixing RNAiMAX and siRNA in OptiMEM medium for 15 minutes. The transfection mixture was transferred to a multi-well plate, and cell suspension was added to the wells. After 24 hours of incubation, the cells were washed once with PBS and then lysed using lysis buffer from the Promega SV96 kit. RNA was purified using an SV96 plate in a vacuum manifold. Four μL of purified RNA was then heated at 65°C for 5 minutes and cooled to 4°C. The RNA was then used for reverse transcription in a 10 μL reaction using a high-capacity reverse transcription kit (Life Technologies). The cDNA was then diluted to 50 μL with nuclease-free water and used for quantitative PCR using multiplexed 5'-endonuclease assay and SSoFastqPCR master mix (Bio-Rad laboratories).

[0187] cDNA synthesis

[0188] Using Corbett X-tractor Gene TM(QIAxtractor) RNA was isolated from mammalian cells in tissue cultures. A modified SuperScript II protocol was used to synthesize cDNA from the isolated RNA. The isolated RNA (approximately 5 ng / μL) was heated to 65°C for 5 minutes and incubated with dNP, random hexamer, oligodT, and water. The mixture was cooled for 15 seconds. A solution of water, 5X first-strand buffer, DTT, and Superase In... TM An enzyme mixture consisting of an RNA inhibitor and SuperScript II RTase was added to the mixture. Using a thermal cycler, the contents were heated to 42°C for 1 hour, then to 70°C for 15 minutes, and then cooled to 4°C. The resulting cDNA was then subjected to a process based on… qPCR. The qPCR reaction is multiplexed, with each reaction containing two 5' endonuclease assays.

[0189] qPCR assay

[0190] Initially used based on The qPCR primer set was selected. Assay specificity was validated by evaluating melting curves and a “subtract RT” control. Human (Hs) and mouse (Mm) assays were performed using dilutions of cDNA templates from HeLa and Hepa1-6 cells (10-fold serial dilutions from 20 ng to 0.02 ng per reaction). qPCR assays were set up in 384-well plates, covered with MicroAmp membranes, and run on an Applied Biosystems 7900HT. Reagent concentrations and cycling conditions included the following: 2x SYBR mix, 10 μM forward primer, 10 μM reverse primer, DD H2O, and cDNA template to a total volume of 10 μL.

[0191] clone

[0192] According to the manufacturer's instructions, the PCR amplicon exhibiting a single melting curve was ligated to a Promega... In the vector kit, JM109 high-efficiency cells were transformed with the newly ligated vector, following the manufacturer's instructions. The cells were then plated on LB plates containing ampicillin and incubated overnight at 37°C for colony growth.

[0193] PCR screening and small-scale plasmid preparation

[0194] PCR was used to identify *E. coli* colonies transformed with vectors containing ligated target amplicones. Vector-specific primers with side-insertions were used in the PCR reactions. All PCR products were then run on 1% agarose gels and imaged via transilluminator after staining. The gels were qualitatively evaluated to determine which plasmids appeared to contain ligated amplicones of the expected size (approximately 300 bp, including both the amplicon and side-insertion vector sequence specific to the primers used).

[0195] Colonies confirmed as transformants by PCR were then incubated overnight at 37°C with shaking in a culture consisting of 2 mL of LB liquid medium containing ampicillin. E. coli cells were then lysed, and the target plasmid was isolated using a Promega mini-preparation kit. Plasmid concentration was determined by UV absorbance at 260 nm.

[0196] Plasmid sequencing and quantification

[0197] use The terminator sequencing kit was used to sequence the purified plasmid. Vector-specific primer T7 was used to obtain the read length across the insert. The following reagents were used in the sequencing reaction: water, 5X sequencing buffer, BigDye terminator mixture, T7 primers, and plasmid (100 ng / μL), to a volume of 10 μL. The mixture was incubated at 96°C for 1 minute, followed by 15 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 1 minute and 15 seconds; 5 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 1 minute and 30 seconds; and 5 cycles of 96°C for 10 seconds, 50°C for 5 seconds, and 60°C for 2 minutes. Sequencing was then performed using an Applied Biosystems capillary electrophoresis sequencer to terminate the dye reaction.

[0198] The plasmids were then quantitatively sequenced and validated. They were linearized using a single-cutting restriction endonuclease. Linearity was confirmed by agarose gel electrophoresis. All plasmids were diluted in TE buffer (pH 7.5) with 100 μg tRNA / mL buffer to reduce nonspecific binding of plasmids to polypropylene vials.

[0199] The linearized plasmid was then serially diluted from 1,000,000 copies / μL to 0.1 copies / μL and qPCR was performed. Assay efficiency was calculated, and an assay was considered acceptable if the efficiency was in the range of 90-110%.

[0200] Multiplexing determination

[0201] For each target, mRNA levels were quantified using two 5' nuclease assays. Typically, several assays are screened for each target. The two selected assays demonstrated a good combination of efficiency, low detection limits, and wide 5'→3' coverage of the target gene (GOI). Two assays targeting a single GOI can be combined in a single reaction when different fluorophores are used on the corresponding probes. Therefore, the final step in assay validation is to determine the efficiency of the selected assays when they are combined or "multiplexed" in the same qPCR.

[0202] Linearized plasmids from both assays were combined in a 10-fold dilution and qPCR was performed. The efficiency of each assay was determined as described above. Acceptable efficiencies were 90-110%. cDNA was also used as a template to evaluate the C-value of the target when validating the multiplexing reaction using linearized plasmid standards. q Values. For human or mouse targets, HeLa and Hepa1-6 cDNA were used, respectively. In this case, the cDNA was derived from RNA isolated from untransfected cells on Corbett (~5 ng / μl in water). In this manner, C values ​​were observed from the cDNA of this sample. q The value represents the expected C from 96-well plate transfection. q Value. In which C q For values ​​greater than 30, other cell lines exhibiting higher expression levels of the target gene were sought. Libraries of total RNA isolated from each human and mouse line on Corbett using high-throughput methods were generated and used for screening against acceptable levels of target expression.

[0203] Description of oligonucleotide nomenclature

[0204] All oligonucleotides described herein are named as any SN1-ASN2-MN3. The following names apply:

[0205] • N1: Sequence identifier number of the sense chain sequence

[0206] • N2: Sequence identifier number of the antisense chain sequence

[0207] • N3: Reference number for the modification pattern, where each number represents the pattern of the modified nucleotide in the oligonucleotide.

[0208] For example, S27-AS219-M1 represents an oligonucleotide having a sense sequence as shown in SEQ ID NO:27, an antisense sequence as shown in SEQ ID NO:219, and suitable for the modification pattern identified as M1.

[0209] Table 4. GYS2 RNAi oligonucleotides

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] The disclosures described herein may be suitably implemented where no one or more elements or limitations not specifically disclosed herein are present. Thus, for example, in each case herein, any of the terms “comprising,” “substantially consisting of,” and “consisting of” may be replaced by any of the other two terms. The terms and expressions used are descriptive rather than limiting, and the use of such terms and expressions is not intended to exclude any equivalents of the features or portions thereof shown and described, but it should be recognized that various modifications may be possible within the scope of the claimed invention. Therefore, it should be understood that although the invention has been specifically disclosed with regard to preferred embodiments, those skilled in the art may apply optional features, modifications, and variations of the concepts disclosed herein, and such modifications and variations are considered to be within the scope of the invention as defined in the specification and appended claims.

[0230] Furthermore, when features or aspects of the invention are described in terms of the Markush group or other alternative groups, those skilled in the art will recognize that the invention is also described in terms of any individual member or subgroup of the Markush group or other groups.

[0231] It should be understood that in some embodiments, the sequence presented in the sequence listing may be referenced in describing the structure of the oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and / or one or more modified nucleotides and / or one or more modified internucleotide bonds and / or one or more other modifications compared to the specified sequence, while retaining complementary properties that are substantially the same or similar to those of the specified sequence.

[0232] In the context of describing the invention (especially in the context of the following claims), the terms “a” and “an” and “the” and similar references are to be construed as encompassing both the singular and the plural, unless otherwise indicated herein or obviously contradicted by the context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to”), unless otherwise stated. Unless otherwise stated herein, the description of ranges of values ​​herein is intended merely as a shorthand for individually referring to each individual value falling within that range, and each individual value is incorporated into this specification as if it were individually described herein. All methods described herein may be performed in any suitable order unless otherwise indicated herein or otherwise obviously contradicted by the context. The use of any and all embodiments or exemplary language (e.g., “such as”) provided herein is merely intended to better illustrate the invention and does not constitute a limitation on the scope of the invention, unless otherwise required. No language in this specification should be construed as indicating any unclaimed element essential to the implementation of the invention.

[0233] This document describes embodiments of the present invention. Variations of these embodiments will become apparent to those skilled in the art upon reading the above description.

[0234] The inventors expect those skilled in the art to employ such variations where appropriate, and intend for the invention to be practiced in other ways different from those specifically described herein. Therefore, as permitted by applicable law, the invention includes all modifications and equivalents of the subject matter set forth in its appended claims. Furthermore, the invention covers any combination of the foregoing elements in all its possible variations, unless otherwise specified herein or otherwise obviously contradicted by the context. Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the invention described herein using only conventional experimentation. Such equivalents are intended to be covered by the following claims.

Claims

1. An oligonucleotide for reducing GYS2 expression, said oligonucleotide comprising an antisense strand and a sense strand, wherein The antisense strand consists of a sequence as shown in SEQ ID NO:595 and has a region complementary to the target sequence of GYS2, wherein the complementary region is at least 15 consecutive nucleotides in length; and The meaningful chain consists of a sequence as shown in SEQ ID NO:

583.

2. The oligonucleotide of claim 1, wherein the complementary region is completely complementary to the target sequence of GYS2.

3. The oligonucleotide of claim 1 or 2, wherein the antisense strand is 22 nucleotides in length.

4. The oligonucleotide of claim 1 or 2, wherein the sense strand is 36 nucleotides in length.

5. The oligonucleotide of claim 1 or 2, wherein the sense strand forms a double-stranded region with the antisense strand.

6. The oligonucleotide of claim 1 or 2, wherein the antisense strand and the sense strand form a double-stranded region of 20 nucleotides in length.

7. The oligonucleotide of claim 1 or 2, wherein the sense strand comprises a stem-loop at its 3' end as shown below: S1-L-S2, wherein S1 is complementary to S2, and wherein L forms a loop of 4 nucleotides in length between S1 and S2.

8. The oligonucleotide of claim 7, wherein L is a sequence as shown in GAAA.

9. The oligonucleotide of claim 6, wherein the 3'-protrusion sequence on the antisense strand is two nucleotides in length.

10. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one modified nucleotide.

11. The oligonucleotide of claim 10, wherein the modified nucleotide comprises a 2'-modification.

12. The oligonucleotide of claim 11, wherein the 2'-modification is selected from the group consisting of 2'-fluorine, 2'-O-methyl, 2'-O-methoxyethyl, and 2'-deoxy-2'-fluoro-β-d-arabinose.

13. The oligonucleotide of claim 10, wherein all nucleotides of the oligonucleotide are modified.

14. The oligonucleotide of claim 1 or 2, wherein the oligonucleotide comprises at least one modified internucleotide bond.

15. The oligonucleotide of claim 14, wherein the internucleotide bond of the at least one modified nucleotide is a phosphate thioester bond.

16. The oligonucleotide of claim 1 or 2, wherein at least one nucleotide of the oligonucleotide is conjugated to one or more targeting ligands.

17. The oligonucleotide of claim 16, wherein each targeting ligand comprises a carbohydrate.

18. The oligonucleotide of claim 16, wherein each targeting ligand comprises an amino sugar.

19. The oligonucleotide of claim 16, wherein each targeting ligand comprises cholesterol.

20. The oligonucleotide of claim 16, wherein each targeting ligand comprises a polypeptide.

21. The oligonucleotide of claim 16, wherein each targeting ligand comprises a lipid.

22. The oligonucleotide of claim 18, wherein each targeting ligand comprises an N-acetylgalactosamine (GalNAc) moiety.

23. The oligonucleotide of claim 22, wherein the GalNac moiety is a monovalent GalNAc moiety, a divalent GalNAc moiety, a trivalent GalNAc moiety, or a tetravalent GalNAc moiety.

24. An oligonucleotide for reducing GYS2 expression, said oligonucleotide comprising an antisense strand and a sense strand. The antisense strand is 22 nucleotides in length, consists of a sequence as shown in SEQ ID NO:595, and has a region complementary to GYS2; The sense strand is 36 nucleotides in length and consists of a sequence as shown in SEQ ID NO:583; The oligonucleotide comprises at least one modified nucleotide and at least one modified internucleotide bond; The 3'-protrusion sequence on the antisense strand is two nucleotides in length; and The sense strand comprises a stem-loop as shown below: S1-L-S2, wherein S1 and S2 are complementary, wherein L is a sequence as shown in GAAA, and wherein up to four nucleotides of L in the stem-loop are each conjugated to a monovalent GalNAc portion.

25. A composition for reducing the expression of GYS2, comprising an oligonucleotide and an excipient of any of the preceding claims.

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