Antisense oligonucleotide conjugates targeting glycogen synthase 1 (GYS1) and methods of use thereof

Abstract

The present invention provides antisense oligonucleotide conjugates targeting the GYS1 gene, and methods of using such antisense oligonucleotide conjugates to inhibit expression of GYS1 and to treat subjects having a disease, such as glycogen storage diseases (GSD), e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe's disease.

Description

[0001] Attorney Docket: 140101-00220

[0002] ANTISENSE OLIGONUCLEOTIDE CONJUGATES TARGETING GLYCOGEN SYNTHASE 1 (GYS1) AND METHODS OF USE THEREOF

[0003] RELATED APPLICATIONS

[0004] This application claims the benefit of priority to U. S. Provisional Application No.

[0005] 63 / 788,941, filed on April 15, 2025, the entire contents of which are incorporated herein by reference.

[0006] SEQUENCE LISTING

[0007] This application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on April 13, 2026, is named 140101-00220-SL.xml and is is 428,845 bytes in size.

[0008] FIELD OF THE INVENTION

[0009] The present invention relates to antisense oligonucleotide conjugates, compositions comprising such conjugates, and methods of use thereof.

[0010] BACKGROUND OF THE INVENTION

[0011] Glycogen Synthase 1 (GYS1) is a protein that is encoded by the GYS1 gene in humans and is the central enzyme in muscle glycogen biosynthesis. It catalyzes the addition of glucose monomers to the growing glycogen molecule through the formation of alpha- 1,4-glycoside linkages. GYS1 exits as a dimer in the asymmetric unit, with each monomer comprised of two Rossmann-fold domains located near the N-terminus and C-terminus, separated by an inter-domain cleft that contains the active site. The C-terminus typically folds back onto the N-terminal Rossmann-fold domain, such that the two domains are linked by a two-stranded hinge (Buschiazzo A et al., EMBO J. 2004 Aug 18;23(16):3196-205; Palm DC etal, FEBSJ. 2013 Jan;280(l):2-27).

[0012] Mammalian GYS is the rate-limiting enzyme in glycogen biosynthesis, and its activity is regulated posttranslationally through activation by the effector Glc6P and inhibition by reversible phosphorylation. Dephosphorylation, performed by glycogen-associated phosphatases of type 1 (PPI), substantially alters kinetic properties of GYS, including increased affinity for uridine diphosphate glucose (UDP-Glc) and sensitivity to the Glucose-6-phosphate (Glc6P) activator. Glc6P binds to an allosteric site equipped with an arginine cluster (e.g., R579, R580, R588 and R591), overcomes phosphorylation-dependent inhibition, and increases the enzyme’s susceptibility to PPI -mediated dephosphorylation (McCorvie, T. J. et al., Nat Struct Mol Biol 29, 628-638 (2022)).Attorney Docket: 140101-00220

[0013] Studies have been done to explore the relationship between GYS1 and several glycogen storage diseases (GSDs), including Lewis' disease (Kollberg G et al., N Engl J Med.

[0014] 2007 Oct 11;357(15): 1507-14), GSD type II e.g., Pompe disease) (Clayton NP etal., Mol Ther Nucleic Acids. 2014 Oct 28;3(10):e206), GSD type IV (e.g., Andersen disease and adult polyglucosan body disease) (Chown EE et al., Ann Clin Transl Neurol. 2020

[0015] Nov;7(l 1):2186-2198) and Lafora disease (Varea O et al., Neurobiol Dis. 2021 Jan;147:105173). Mutations in GYS1 cause defective glycogen synthesis or breakdown, which can result in the accumulation of aberrant or normal glycogen in the affected tissues. Currently, the goal of treatment for GSDs is to maintain glucose homeostasis by dietary management and the use of uncooked cornstarch. In addition to nutritional interventions, pharmacological treatment, physical and supportive therapies, enzyme replacement therapy (ERT) and organ transplantation are other approaches that are used for treatment of disease manifestations and long-term complications (Gurmi§ E etal., World J Gastroenterol. 2023 Jul 7;29(25):3932-3963).

[0016] The lack of a specific therapy for GSDs has prompted efforts to develop new treatment strategies. While inhibition of GYS1 activity has therapeutic potential, the development of GYS1 inhibitors has not progressed rapidly. Accordingly, there remains a need in the art to develop novel and effective therapies for inhibition of GYS1 and treatment of glycogen storage diseases.

[0017] SUMMARY OF THE INVENTION

[0018] The present invention provides antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, comprising an antisense oligonucleotide targeting Glycogen Synthase 1 (GYS1) conjugated to a cell penetrating peptide, and compositions comprising such conjugates, which target nucleic acids encoding GYS1, and interfere with the function of the targeted nucleic acid. The GYS1 nucleic acid may be within a cell, e.g., a cell within a subject, such as a human subject.

[0019] The present invention also provides effective methods and combination therapies for treating a subject having glycogen storage disease (GSD) using the antisense oligonucleotide conjugates and compositions of the invention. The specific design of the antisense oligonucleotide conjugates of the present invention allows effective delivery of the conjugates to a target cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a fibroblast, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell). The methods of the present invention are both simple and efficient, and provide a great advantage when used inAttorney Docket: 140101-00220

[0020] treating GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease).

[0021] Accordingly, in one aspect, the present invention provides an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for inhibiting expression of Glycogen Synthase 1 (GYS1), comprising an antisense oligonucleotide targeting GYS1 conjugated to a cell penetrating peptide, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO), and wherein the cell penetrating peptide comprises at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, wherein the cell penetrating peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a beta-alanine residue in combination with arginine and / or histidine residues.

[0022] In another aspect, the present invention provides an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for inhibiting expression of Glycogen Synthase 1 (GYS1), comprising an antisense oligonucleotide targeting GYS1 conjugated to a cell penetrating peptide, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising at least 10 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 106-283, and wherein the cell penetrating peptide comprises at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, wherein the cell penetrating peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a beta-alanine residue in combination with arginine and / or histidine residues.

[0023] In another aspect, the present invention provides an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for inhibiting expression of Glycogen Synthase 1 (GYS1), comprising an antisense oligonucleotide targeting GYS1 conjugated to a cell penetrating peptide, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO) comprising at least 10 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 106, 120-128, 150-157, and 166-171, and wherein the cell penetrating peptide comprises at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, wherein the cell penetrating peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a beta-alanine residue in combination with arginine and / or histidine residues.

[0024] In some embodiments, the antisense oligonucleotide is 10 to 40 nucleotides in length, 10 to 30 nucleotides in length, 10 to 20 nucleotides in length, 18 to 30 nucleotides in length, 10 to 25 nucleotides in length, or 18 to 25 nucleotides in length.Attorney Docket: 140101-00220

[0025] In some embodiments, the antisense oligonucleotide is 15 nucleotides in length, 20 nucleotides in length, or 25 nucleotides in length.

[0026] In some embodiments, the antisense oligonucleotide is 25 nucleotides in length.

[0027] In some embodiments, the antisense oligonucleotide comprises at least 10, at least 15, at least 20, or at least 25 contiguous nucleotides complementary to a target sequence in a human GYS1 gene as set forth in SEQ ID NO: 284, or a GYS1 mRNA selected from any one of the nucleotide sequences of SEQ ID NOs: 285-286.

[0028] In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 106-283.

[0029] In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NOs: 106-283.

[0030] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106-283.

[0031] In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 113-295.

[0032] In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NOs: 106, 120-128, 150-157, and 166-171.

[0033] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106, 120-128, 150-157, and 166-171.

[0034] In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106, 120-128, 150-157, and 166-171.

[0035] In some embodiments, the target sequence is located in an exon, an intron, an exonintron junction, the 5’UTR, or the 3’UTR of the GYSI gene.

[0036] In some embodiments, all the intemucleoside linkages of the PMO are -P(O)(NMe2)O-.

[0037] In some embodiments, each cationic domain is 4 to 12 amino acid residues in length, or 4 to 7 amino acid residues in length.

[0038] In some embodiments, each cationic domain comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids.

[0039] In some embodiments, each cationic domain comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues.

[0040] In some embodiments, each cationic domain comprises a sequence selected from the group consisting of RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ IDAttorney Docket: 140101-00220

[0041] NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19), and any combination thereof.

[0042] In some embodiments, each cationic domain consists of a sequence selected from the group consisting of: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19), and any combination thereof.

[0043] In some embodiments, the cell penetrating peptide comprises two cationic domains. In some embodiments, each hydrophobic domain is 3 to 6 amino acid residues in length.

[0044] In some embodiments, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids.

[0045] In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, Isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues.

[0046] In some embodiments, the cell penetrating peptide comprises one hydrophobic domain.

[0047] In some embodiments, the hydrophobic domain comprises a sequence selected from the group consisting of YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26), and any combination thereof.

[0048] In some embodiments, the hydrophobic domain consists of a sequence selected from the group consisting of YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26), and any combination thereof.

[0049] In some embodiments, the cell penetrating peptide comprises two cationic domains and one hydrophobic domain.

[0050] In some embodiments, the cell penetrating peptide comprises one hydrophobic domain flanked by two cationic domains.

[0051] In some embodiments, the cell penetrating peptide comprises one hydrophobic domain comprising a sequence selected from the group consisting of YQFLI (SEQ ID NO:Attorney Docket: 140101-00220

[0052] 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26); flanked by two cationic domains, each comprising a sequence selected from the group consisting of RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19).

[0053] In some embodiments, the cell penetrating peptide comprises a sequence selected from the group consisting of RBRRB RRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRYQFLIRBRBR (SEQ ID NO: 31), RBRRB RRILFQYRBRBR (SEQ ID NO: 32), RBRRB RFQILYBRBR (SEQ ID NO: 35), RBRRB RRFQILYRBHBH (SEQ ID NO: 37), RBRRB RRFQILYHBHBR (SEQ ID NO: 38), and RBRRB RFQILYRBHBH (SEQ ID NO: 44).

[0054] In some embodiments, the cell penetrating peptide comprises the amino acid sequence RBRRB RFQILYBRBR (SEQ ID NO: 35).

[0055] In some embodiments, the cell penetrating peptide comprises the amino acid sequence RBRRB RRFQILYRBHBH (SEQ ID NO: 37).

[0056] In some embodiments, the cell penetrating peptide comprises the amino acid sequence RBRRB RFQILYRBHBH (SEQ ID NO: 44).

[0057] In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its N-terminus.

[0058] In some embodiments, the C-terminus of the cell penetrating peptide is -NH2.

[0059] In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its C-terminus.

[0060] In some embodiments, the cell penetrating peptide is acylated at its N-terminus. In some embodiments, the cell penetrating peptide is conjugated to one or more internal nucleotides of the antisense oligonucleotide.

[0061] In some embodiments, the antisense oligonucleotide is conjugated to the cell penetrating peptide via a linker.

[0062] In some embodiments, the antisense oligonucleotide is conjugated to the linker at its 3’ terminus.

[0063] In some embodiments, the conjugate comprises a structure selected from the group consisting of:

[0064] [cell penetrating peptide] — [oligonucleotide];

[0065] [oligonucleotide] — [cell penetrating peptide];Attorney Docket: 140101-00220

[0066] [cell penetrating peptide] — [linker] — [oligonucleotide]; and [oligonucleotide] — [linker] — [cell penetrating peptide].

[0067] In some embodiments, the conjugate is of the following structure:

[0068] [oligonucleotide]

[0069] [cell penetrating peptide] '

[0070] ^•[oligonucleotide]

[0071] or

[0072] [oligonucleotide]-- penetrating peptide]

[0073]

[0074] [oligonucleotide]

[0075] or

[0076] [oligonucleotide]

[0077] [cell penetrating peptide] - [linker]

[0078] ^•[oligonucleotide]

[0079] or

[0080] [linker] - [oligonucleotide]

[0081] [cell penetrating peptide]

[0082] ^•[linker] - [oligonucleotide]

[0083] or

[0084] [oligonucleotide]-^,,.,,, „x,

[0085] [linker] - [cell penetrating peptide] [oligonucleotide]

[0086] or

[0087] [oligonucleotide] - [linker]

[0088]

[0089] .,,,

[0090]

[0091] [cell penetrating peptide] [oligonucleotide] - [linker]

[0092] In some embodiments, the conjugate is of the following structure:

[0093] [cell penetrating peptide] — [linker] — [cell penetrating peptide] — [linker] — [oligonucleotide]

[0094] In some embodiments, the linker is of formula (I):

[0095] T1-(CR1R2)n-T2.

[0096] (I)

[0097] wherein

[0098] T1is a divalent group for attachment to the cell penetrating peptide and is selected from the group consisting of -NH- and carbonyl;Attorney Docket: 140101-00220

[0099] T2 is a divalent group for attachment to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl;

[0100] n is 1, 2 or 3;

[0101] each R1is independently -Y1-X1-Z1,

[0102] wherein

[0103] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and RA2are each independently hydrogen, OH, or (1-2C)alkyl;

[0104] X1is absent, -O-, -C(O)-, -O(O)O-, -OC(O)-, -CH(0RA3)-, -N(RA3)-, -N(RA3)- C(O)-, -N(RA3)-C(0)0-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(NRA3)N(RA3)-, -SO-, -S-, -SO2-, -S(O)2N(RA3)-, or -N(RA3)SO2-, wherein each RA3is independently selected from the group consisting of hydrogen and methyl; and

[0105] Z1is a further oligonucleotide or is hydrogen, (l-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl,

[0106] wherein each (1 -6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRA4RA5, and (1-4C)alkoxy, wherein RA4and RA5are each independently selected from the group consisting of hydrogen and (1-4C)alkyl; and each R2is independently -Y2-X2-Z2, wherein

[0107] Y2is absent or a group of the formula -[CRB1RB2]m- in which m is an integer selected from 1, 2, 3 or 4, and RB1and RB2are each independently selected from hydrogen, OH or (1 -2C)alkyl;

[0108] X2is absent, -O-, -C(O)-, -O(O)O-, -OC(O)-, -CH(0RB3)-, -N(RB3)-, -N(RB3)- C(O)-, - N(RB3)-C(0)0-, -C(0)-N(RB3)-, -N(RB3)C(O)N(RB3)-, -N(RB3)C(NRB3)N(RB3)-, -SO-, -S- -SO2-, - S(O)2N(RB3)-, or -N(RB3)SO2-, wherein each RB3is independently selected from hydrogen or methyl; and

[0109] Z2is selected from hydrogen, (1 -6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, wherein each (1 -6C)alkyl, (2- 6C)alkenyl, (2- 6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRB4RB5, and (1-4C)alkoxy, wherein RB4and RB5are each independently hydrogen or (l-2C)alkyl; with the proviso that; when n=l and Ti and T2 are different to one another, then R1and R2are not both H; when n=l, Ti and T2 are different to one another and one of R1and R2is H then theAttorney Docket: 140101-00220

[0110] other of R1and R2is not methyl; or when n=2 and each occurrence of R1and R2is H, then Ti and T2 are both -C(O)- or are both -NH-.

[0111] In some embodiments, T2 is -C(O)-.

[0112] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0113] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and RA2are each hydrogen or (1- 2C)alkyl;

[0114] X1is absent, -O-, -C(O)-, -O(O)O-, -N(RA3)-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(N RA3)N(RA3)- or -S-, wherein each RA3is independently hydrogen or methyl; and

[0115] Z1is a further oligonucleotide or is hydrogen, (l-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3- 6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRA4RA5, and (l-4C)alkoxy, wherein RA4and RA5are each independently hydrogen or (1-2C)alkyl.

[0116] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0117] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3, or 4, and RA1and RA2are each independently hydrogen or (1-2C)alkyl;

[0118] X1is absent, -O-, -C(O)-, -C(O)O-, -N(RA3)-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(NRA3)N(RA3)-, or -S-, wherein each RA3is independently hydrogen or methyl; and

[0119] Z1is a further oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.

[0120] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0121] Y1is absent or a group of the formula -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and

[0122] RA2are each independently hydrogen or (l-2C)alkyl;

[0123] X1is absent, -C(O)-, -C(O)O-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, wherein each RA3is hydrogen or methyl; and

[0124] Z1is a further oligonucleotide or is hydrogen, (1 - 6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (l-6C)alkyl, aryl, (3- 6C)cycloalkyl, and heteroaryl is optionallyAttorney Docket: 140101-00220

[0125] substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.

[0126] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0127] Y1is absent, -(CH2)-, or -(CH2CH2)-;

[0128] X1is absent, -N(RA3)-C(O)-, -C(O)-N(RA3)-, wherein each RA3is independently hydrogen or methyl; and

[0129] Z1is hydrogen or (1-2C)alkyl.

[0130] In some embodiments, each R2is independently -Y2-Z2, wherein Y2is absent or -(CRB1RB2)m-, wherein m is 1, 2, 3 or 4, and RB1and RB2are each independently hydrogen or (l-2C)alkyl; and Z2is hydrogen or (1-6C)alkyl.

[0131] In some embodiments, each R2is hydrogen.

[0132] In some embodiments, n is 2 or 3.

[0133] In some embodiments, n is 1.

[0134] In some embodiments, the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and gamma-aminobutyric acid residues.

[0135] In some embodiments, the linker is:

[0136]

[0137] In some embodiments, the linker is:

[0138]

[0139] In some embodiments, the linker is:

[0140]

[0141] In some embodiments, the linker is:

[0142]

[0143] In some embodiments, the linker is:Attorney Docket: 140101-00220

[0144]

[0145] In some embodiments, the linker is:

[0146]

[0147] In some embodiments, the conjugate is of the following structure:

[0148] [cell penetrating peptide] [oligonucleotide]

[0149]

[0150] In some embodiments, the conjugate is of the following structure:

[0151] O

[0152] [cell penetrating peptide]

[0153] N

[0154]

[0155] [oligonucleotide]

[0156] In some embodiments, the conjugate is of the following structure:

[0157] [cell penetrating peptide] [oligonucleotide]

[0158]

[0159] In some embodiments, the conjugate is of the following structure:

[0160] [cell penetrating peptide] [oligonucleotide]

[0161]

[0162] In some embodiments, the conjugate is of the following structure:

[0163]

[0164] Attorney Docket: 140101-00220

[0165] In another aspect, the present invention provides a pharmaceutical composition comprising one or more of the antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, described herein.

[0166] In yet another aspect, the present invention provides a method of inhibiting GYS1 expression in a cell, the method comprising contacting the cell with one or more of the antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, and / or one or more of the pharmaceutical compositions described herein, thereby inhibiting GYS1 expression in the cell.

[0167] In some embodiments, the method comprises contacting and / or exposing the cell with one or more of the antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, and / or one or more of the pharmaceutical compositions described herein, thereby inhibiting GYSI expression in the cell.

[0168] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYSI protein expression in the cell.

[0169] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYSI mRNA and / or pre-mRNA expression in the cell.

[0170] In some embodiments, the cell is within a subject.

[0171] In some embodiments, the subject is a human.

[0172] In some embodiments, the cell is selected from the group consisting of a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cell, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, an induced pluripotent stem cell, and any combination thereof.

[0173] In some embodiments, the cell is any cell type of the central nervous system or the peripheral nervous system.

[0174] In some embodiments, the cell is a muscle cell.

[0175] In some embodiments, the GYS1 expression is inhibited by at least about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0176] In some embodiments, contacting the cell with the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits expression of the GYS1 gene by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%; reduces GYS1 protein levelsAttorney Docket: 140101-00220

[0177] by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%; and / or reduces glycogen levels in the cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

[0178] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression with an IC50 between 0.1-10 pM.

[0179] In another aspect, the present invention provides a method of treating glycogen storage disease (GSD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of one or more of the antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, and / or one or more of the pharmaceutical compositions described herein, thereby treating the GSD in the subject.

[0180] In some embodiments, the GSD is selected from the group consisting of Lewis' disease, von Gierke’s disease, Pompe’s disease (GSD Ila and lib), Danon disease, Forbes disease / Cori disease, Andersen's disease, Adult Polyglucosan Body Disease (APBD), Lafora Disease, McArdle's disease, Hers’ disease, Tarui’s disease, phosphorylase kinase deficiency, type IX of GSD, type X of GSD, Fanconi -Bickel syndrome, type XIV of GSD, type XII of GSD, type XIII of GSD, type XV of GSD, and subtypes thereof.

[0181] In some embodiments, the GSD is Lafora disease.

[0182] In some embodiments, the GSD is APBD.

[0183] In some embodiments, the GSD is Pompe’s disease.

[0184] In some embodiments, the GSD affects one or more tissues selected from the group consisting of liver, kidney, intestine, blood, heart, muscle, respiratory, and nervous system.

[0185] In some embodiments, the GSD affects the nervous system in the subject.

[0186] In some embodiments, the GSD affects a muscle in the subject.

[0187] In some embodiments, the subject is human.

[0188] In some embodiments, the conjugate or the pharmaceutical composition is administered to the subject via an intramedullary route, an intrathecal route, an intracerebroventricular route, an intraventricular route, an intravitreal route, an enteral route, a parenteral route, an intravenous route, an intra-arterial route, an intramuscular route, an intratumoral route, a subcutaneous route, an oral route, or a nasal route.

[0189] In some embodiments, the conjugate is administered at a dose of about 5 mg / kg to about 60 mg / kg to the subject.

[0190] In some embodiments, the conjugate is administered to the subject at a frequency that is weekly, biweekly, monthly, quarterly, semi-annually, annually, or a combination thereof.

[0191] In some embodiments, the methods of the present invention further comprise administering an additional therapeutic to the subject.Attorney Docket: 140101-00220

[0192] In some embodiments, the additional therapeutic is selected from the group consisting of medications for decreasing uric acid levels (e.g., Allopurinol, Febuxostat, or Diuretics), medications for decreasing cholesterol levels (e.g., Statins, Ezetimibe, Phytosterols, or citrate lyase inhibitors), cornstarch therapy, high-protein diet, calcium, vitamin D, iron supplements, glucose, nutrition supplements, concomitant surgery, occupational therapy, physical therapy, exercise program, enzyme replacement therapies, gene replacement therapies, and any combination thereof.

[0193] In another aspect, the present invention provides a kit comprising one or more of the antisense oligonucleotide conjugates and / or one or more of the pharmaceutical compositions described herein, and instructions for use thereof.

[0194] In another aspect, the present invention provides a syringe comprising one or more of the antisense oligonucleotide conjugates and / or one or more of the pharmaceutical compositions described herein.

[0195] BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1 is a histogram depicting inhibition of human GYS1 expression in U-251 MG cells transfected with 5pM of antisense phosphorodiamidate morpholino oligomers (PMO) targeting GYS1.

[0196] FIGURE 2 is a histogram depicting inhibition of human GYS1 expression in U-251 MG cells transfected with 25pM of antisense phosphorodiamidate morpholino oligomers (PMO) targeting GYS1.

[0197] FIGURE 3 is a histogram depicting inhibition of mouse GYS1 expression in C2C12 myoblasts transfected with 5pM of antisense phosphorodiamidate morpholino oligomers (PMO) targeting GYS1.

[0198] FIGURE 4 is a histogram depicting inhibition of mouse GYS1 expression in C2C12 myoblasts transfected with 25pM of antisense phosphorodiamidate morpholino oligomers (PMO) targeting GYS1.

[0199] FIGURE 5A is a plot showing inhibition of human GYS1 expression in human myotubes transfected with 0.003 - 50 pM of PMO conjugates PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162.

[0200] FIGURE 5B is a plot showing cell viability of human myotubes transfected with 0.003 - 30 pM of PMO conjugates PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162.Attorney Docket: 140101-00220

[0201] FIGURE 6A is a representative Western blot image showing GYS1 protein levels in human myotubes transfected with 6.25 pM of PMO conjugates PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162, PGN-200146.

[0202] FIGURE 6B is a plot showing % glycogen decrease as a function of %GYS1 protein knockdown levels (bottom panel) in human myotubes transfected with 6.25 pM of PMO conjugates PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162.

[0203] FIGURE 7 shows plots of differential gene expression in human myotubes transfected with 10 uM of PMO conjugates PGN-200085 (left panel), PGN-200154 (middle panel) and PGN-200159 (right panel).

[0204] FIGURE 8 is a plot showing human GYS2 expression in human hepatocytes transfected with 0.195 - 25 uM of PMO conjugates PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162.

[0205] FIGURE 9A is a plot showing inhibition of mouse GYS1 expression in mouse myotubes transfected with 0 - 30 pM of PMO conjugates PGN-200171, PGN-200166, PGN-200167, PGN-200168, PGN-200169, PGN-200170, PGN-200172, PGN-200173.

[0206] FIGURE 9B is a plot showing cell viability of mouse myotubes transfected with 0 -30 pM of PMO conjugates PGN-200171, PGN-200166, PGN-200167, PGN-200168, PGN-200169, PGN-200170, PGN-200172, PGN-200173.

[0207] FIGURE 10A is a plot showing glycogen levels in mouse myotubes transfected with 0 - 30 pM of PMO conjugates PGN-200171 and PGN-200173.

[0208] FIGURE 10B is a plot showing GYS1 protein levels in mouse myotubes transfected with 0 - 30 pM of PMO conjugates PGN-200171 and PGN-200173.

[0209] FIGURE 11 is a plot showing mouse GYS2 expression in mouse hepatocytes transfected with 0.1 - 30 pM of PMO conjugates PGN-200171, PGN-200166, PGN-200167, PGN-200168, PGN-200169, PGN-200170, PGN-200172, PGN-200173.

[0210] FIGURE 12A is a representative Western blot image and a plot showing GAA protein levels in human fibroblast cell lines GSD-0443 and GSD-11661.

[0211] FIGURE 12B is a plot showing GAA protein activity in human fibroblast cell lines GSD-0443 and GSD-11661 relative to cell lines CTL-010 and CTL-012.

[0212] FIGURE 12C is a plot showing glycogen levels in human fibroblast cell lines GSD-0443 and GSD-11661 relative to cell lines CTL-010 and CTL-012.Attorney Docket: 140101-00220

[0213] FIGURE 13A is a representative Western blot image showing GYS1 protein levels in human GSD fibroblast cell line transfected with 10 pM or 25 pM of PGN-200085, PGN-200154 and PGN-200159. ** = p<0.01; *** = p<0.005; **** = p<0.001

[0214] FIGURE 13B is a plot showing GYS1 protein levels in human GSD fibroblast cell line transfected with 10 pM or 25 pM of PGN-200085, PGN-200154, and PGN-200162.

[0215] FIGURE 13C is a plot showing glycogen levels in human GSD fibroblast cell line transfected with 10 pM or 25 pM of PGN-200085, PGN-200154 and PGN-200159. * = p<0.05; ** = p<0.01; *** = p<0.005; **** = p<0.001

[0216] DETAILED DESCRIPTION OF THE INVENTION

[0217] The present invention provides antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, and compositions comprising such conjugates which target nucleic acids encoding Glycogen Synthase 1 (GYSI) and interfere with the normal function of the targeted nucleic acid. The GYS1 nucleic acid may be within a cell, e.g., a cell within a subject, such as a human subject.

[0218] The present invention also provides effective methods and combination therapies for treating a subject having glycogen storage disease (GSD) using the antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, and compositions of the invention. In particular, the specific design of the antisense oligonucleotide conjugates of the present invention allows effective delivery of the conjugates to a target cell (including, but not limited to, a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a fibroblast, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell). The methods of the present invention are simple, efficient and effective, and provide a great advantage when used in treating a glycogen storage disease (GSD), (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease).

[0219] The antisense oligonucleotide conjugates of the invention include an antisense oligonucleotide targeting GYSI (e.g., a phosphorodiamidate morpholino oligomer (PMO)) which is about 10 to about 40 nucleotides or less in length, and which comprises at least 10, at least 15, at least 20, or at least 25 contiguous nucleotides complementary to a target sequence in a human GYS1 gene (SEQ ID NO: 284) or GYS1 mRNA (any of SEQ ID NOs: 285-286). The use of these antisense oligonucleotide conjugates enables the targeted inhibition of protein and / or RNA expression and / or activity of a GYS1 gene. The GYS1 gene may be within a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, aAttorney Docket: 140101-00220

[0220] muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a fibroblast, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), e.g., a cell within a subject, such as a human.

[0221] The present inventors have demonstrated that antisense oligonucleotide conjugates targeting GYS1 can mediate antisense inhibition resulting in significant inhibition of expression of a GYS1 gene in a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a fibroblast, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell). The methods of the invention inhibit GYS1 expression levels in the cell e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) by targeting GYS1 with an antisense oligonucleotide conjugate of the invention and, accordingly, allow treatment of a subject in need thereof, e.g., a subject having glycogen storage disease (GSD), (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease). In some embodiments, the methods of the invention reduce GYS1 protein levels in the cell (e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) by targeting the GYS1 gene with an antisense oligonucleotide conjugate of the invention, or pharmaceutically acceptable salts thereof. In some embodiments, the methods of the invention reduce glycogen levels in the cell (e.g., by about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100%) by targeting the GYS1 gene with an antisense oligonucleotide conjugate of the invention.

[0222] In some embodiments, antisense oligonucleotide conjugates of the invention, or pharmaceutically acceptable salts thereof, are specific for GYS1 and do not inhibit GYS2 expression. In some embodiments, administration of antisense oligonucleotide conjugates of the invention to a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a fibroblast, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell) inhibits GYS2 expression in the cell by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.Attorney Docket: 140101-00220

[0223] In some embodiments, antisense oligonucleotide conjugates of the invention, or pharmaceutically acceptable salts thereof, have minimal off-target effects. In some embodiments, less than 1%, 5%, 10%, 15%, 20%, 25% or 30% of the genes in a cell are differentially expressed following administration of the antisense oligonucleotide conjugates of the invention to the cell compared to their expression in the absence of the antisense oligonucleotide conjugates.

[0224] The present invention also provides methods and combination therapies for treating a subject having GSD using the antisense oligonucleotide conjugates and compositions of the invention. The combination therapies of the present invention include administering to the subject, e.g., a subject having GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease), an antisense oligonucleotide conjugate comprising an antisense oligonucleotide targeting GYS1 conjugated to a cell penetrating peptide, and an additional therapeutic, such as medications for decreasing uric acid levels (e.g., Allopurinol, Febuxostat, or Diuretics), medications for decreasing cholesterol levels (e.g., Statins, Ezetimibe, Phytosterols, or citrate lyase inhibitors), cornstarch therapy, high-protein diet, calcium, vitamin D, iron supplements, glucose, nutrition supplements, concomitant surgery, occupational therapy, physical therapy, exercise program, enzyme replacement therapies, gene replacement therapies, and any combination thereof, or any therapeutic that may prevent and / or reduce a symptom associated with GSD, for example, by modulating GYSI expression directly and / or indirectly.

[0225] The following detailed description discloses how to make and use antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, to inhibit the mRNA and / or protein expression of a GYS1 gene, as well as compositions, uses, and methods for treating subjects having diseases and disorders that would benefit from inhibition and / or reduction of the expression of this gene.

[0226] I. Definitions

[0227] In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

[0228] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

[0229] The term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to".

[0230] The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly indicates otherwise.Attorney Docket: 140101-00220

[0231] As used herein, the term “GYSI,” used interchangeably with the term “Glycogen Synthase 1” refers to the well-known gene and polypeptide. GYSI is responsible for the biosynthesis of glycogen from phosphorylated glucose units. It catalyzes the addition of glucose monomers to the growing glycogen molecule through the formation of alpha- 1,4-glycoside linkages. The molecular mass of GYSI is approximately 85 kDa. It is predominantly expressed in skeletal muscle tissue and brain providing energy storage primarily in the form of glycogen.

[0232] In humans, the GYSI gene is located on chromosome 19ql3.33, spanning approximately 25kb, and contains 16 exons. The sequence of a human GYSI mRNA transcript can be found at, for example, NCBI Reference Sequences (RefSeq) (NM_002103.5 or NM_001161587.2). Additional examples of GYSI mRNA sequences are readily available using publicly available databases, e.g., GenBank. The term “GYSI,” as used herein, also refers to naturally occurring DNA sequence variations of the GYSI gene, such as a single nucleotide polymorphism in the GYSI gene. Numerous SNPs within the GYSI gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., ncbi.nlm.nih.gov / snp).

[0233] As used herein, a “cell penetrating peptide,” refers to a relatively short peptide capable of promoting uptake of antisense oligonucleotides, such as PMOs, by cells, thereby delivering the antisense oligonucleotides to the interior (cytoplasm) of the cells. The cell penetrating peptide typically is about 12 to about 40 amino acids long. The length of the cell penetrating peptide is not particularly limited and varies in different embodiments. In some embodiments, the cell penetrating peptide comprises from 4 to 40 amino acid subunits. In some embodiments, the cell penetrating peptide comprises from 6 to 30, from 6 to 20, from 8 to 25 or from 10 to 20 amino acid subunits. In some embodiments, the cell penetrating peptide comprises a sequence selected from the group consisting of RBRRBRRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRYQFLIRBRBR (SEQ ID NO: 31), RBRRBRRILFQYRBRBR (SEQ ID NO: 32), RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), RBRRBRRFQILYHBHBR (SEQ ID NO: 38), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0234] In some embodiments, the cell penetrating peptide, when conjugated to an antisense oligonucleotide having a substantially uncharged backbone, is effective to enhance the activity of the antisense oligonucleotide, relative to the antisense oligonucleotide in unconjugated form, as evidenced by a decrease in expression of an encoded protein, relative to that provided by the unconjugated oligonucleotide, when binding of the antisense oligonucleotide to its target sequence is effective to block a translation start codon for the encoded protein. In some embodiments, conjugation of the cell penetrating peptide provides this activity in a cell-free translation assay. In some embodiments, activity is enhanced by a factor of at least two, a factor of at least five or a factor of at least ten. Alternatively or inAttorney Docket: 140101-00220

[0235] addition, the cell penetrating peptide is effective to enhance the transport of a nucleic acid analog into a cell, relative to the analog in unconjugated form. In some embodiments, transport is enhanced by a factor of at least two, a factor of at least two, a factor of at least five or a factor of at least ten.

[0236] As used herein, the terms “antisense oligonucleotide conjugate,” or “conjugate,” as used interchangeably herein, refer to a conjugate comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide. In some embodiments, the antisense oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). The PMO conjugated to the cell penetrating peptide, either directly or via a linker, is referred to herein as a peptide conjugated PMO (also referenced herein as a PPMO or a PMO conjugate). The cell penetrating peptide promotes uptake of the antisense oligonucleotide, such as a PMO, by cells (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), thereby delivering the antisense oligonucleotide to the interior (cytoplasm) of the cells.

[0237] In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its N-terminus. In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its C-terminus. In some embodiments, the cell penetrating peptide is conjugated to one or more internal nucleotides of the antisense oligonucleotide.

[0238] A cell penetrating peptide can be generally effective or it can be specifically or selectively effective for PMO delivery to a particular type or particular types of cells. The antisense oligonucleotide and the cell penetrating peptide are typically linked at their ends, e.g., the C-terminal end of the cell penetrating peptide can be linked to the 5' end of the antisense oligonucleotide, or the 3' end of the antisense oligonucleotide can be linked to the N-terminal end of the cell penetrating peptide. In some embodiments, the cell penetrating peptide is conjugated to an internal nucleotide of the antisense oligonucleotide. Antisense oligonucleotide conjugates can include uncharged antisense oligonucleotides, charged (e.g., cationic) antisense oligonucleotides, and mixtures thereof. In some embodiments, the cell penetrating peptide may be linked to the antisense oligonucleotide either directly or via an optional linker, e.g., one or more linkers described herein.

[0239] The antisense oligonucleotide conjugates comprise antisense oligonucleotides that specifically bind to the target nucleic acid molecules via hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) and interfere with the normal function of the targeted nucleic acid (e.g., by an antisense mechanism of action). ThisAttorney Docket: 140101-00220

[0240] interference with or modulation of the function of a target nucleic acid by the antisense oligonucleotide conjugates of the present invention is referred to as “antisense inhibition.” The functions of the target nucleic acid molecule to be interfered with may include functions such as, for example, translocation of the RNA to the site of protein translation, translation of protein from the RNA, splicing of the RNA to yield one or more mRNA species, and catalytic activity which may be engaged in or facilitated by the RNA.

[0241] In some embodiments, antisense inhibition refers to “inhibiting the expression” of target nucleic acid levels and / or target protein levels in a cell, e.g., a cell within a subject, such as a human, in the presence of the antisense oligonucleotide conjugate complementary to a target nucleic acid as compared to target nucleic acid levels and / or target protein levels in the absence of the antisense oligonucleotide conjugate.

[0242] As used herein, “target sequence,” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a GYSI gene, including mRNA that is a product of RNA processing of a primary transcription product. A target sequence may be from about 4-50 nucleotides in length, e.g., 8-45, 10-45, 10-40, 10-35, 10-30, 10-20, 11-45, 11-40, 11-35, 11-30, 11-20, 12-45, 12-40, 12-35, 12-30, 12-25, 12-20, 13- 45, 13-40, 13-35, 13-30, 13-25, 13-20, 14-45, 14-40, 14-35, 14-30, 14-25, 14-20, 15-45, 15- 40, 15-35, 15-30, 15-25, 15-20, 16-45, 16-40, 16-35, 16-30, 16-25, 16-20, 17-45, 17-40, 17- 35, 17-30, 17-25, 17-20, 18-45, 18-40, 18-35, 18-30, 18-25, 18-20, 19-45, 19-40, 19-35, 19- 30, 19-25, 19-20, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 contiguous nucleotides of the nucleotide sequence of an mRNA molecule formed during the transcription of a GYSI gene. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

[0243] The terms “complementary,” “fully complementary” and “substantially complementary,” are used herein with respect to the base matching between an antisense oligonucleotide and a target sequence. As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with the second nucleotide sequence, as will be understood by the skilled person. Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. As used herein, and unless otherwise indicated, the term “fully complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide comprising the first nucleotide sequence to completely hybridize (i.e., be 100% complentary without any mismatches) and form a duplex structure under certain conditions with the second nucleotide sequence, as will be understood by the skilled person. The skilled person will be able to determine the set of conditions mostAttorney Docket: 140101-00220

[0244] appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the nucleotides.

[0245] “Complementary” sequences, as used herein, include Watson-Crick base pairs formed from natural and / or modified nucleobases, and can also include, or be formed entirely from, non-Watson-Crick base pairs and / or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, wobble base pairs (guanosine-uracil, hypoxanthine-uracil, hypoxanthine-adenine, and hypoxanthinecytosine) and Hoogstein base pairing.

[0246] As used herein, “target nucleic acid” refers to a nucleic acid molecule to which an antisense oligonucleotide specifically hybridizes.

[0247] References to “X” throughout the disclosure denote any form of the amino acid aminohexanoic acid, such as 6- aminohexanoic acid.

[0248] References to “B” throughout the disclosure denote the amino acid beta-alanine. Refences to “[Hyp]” throughout the disclosure denote the amino acid hydroxyproline. References to “Ac” throughout the disclosure denote an acetyl group (CH3-C(O)-). References to other capital letters throughout the disclosure denote the relevant genetically encoded amino acid residue in accordance with the accepted alphabetic amino acid code.

[0249] The term “alkyl,” as used herein, refers to a straight or branched chain hydrocarbon group containing a total of one to twenty carbon atoms, unless otherwise specified (e.g., (1-6C) alkyl, (1-4C) alkyl, (1-3C) alkyl, or (1-2C) alkyl). Non-limiting examples of alkyls include methyl, ethyl, 1 -methylethyl, propyl, 1 -methylbutyl, 1 -ethylbutyl, etc. References to individual alkyl groups such as “propyl” are specific for the straight chain version only, and references to individual branched chain alkyl groups such as “isopropyl” are specific for the branched chain version only.

[0250] The term "alkenyl," as used herein, refers to an aliphatic group containing one, two, or three carbon-carbon double bonds and containing a total of two to twenty carbon atoms, unless otherwise specified (e.g., (2-6C) alkenyl, (2-4C) alkenyl, or (2-3C) alkenyl). Nonlimiting examples of alkenyl include vinyl, allyl, homoallyl, isoprenyl, etc. Unless otherwise specified, alkenyl may be optionally substituted by one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0251] The term "alkynyl," as used herein, refers to an aliphatic group containing one, two, or three carbon-carbon triple bonds and containing a total of two to twenty carbon atoms, unless otherwise specified (e.g., (2-6C) alkynyl, (2-4C) alkynyl, or (2-3C) alkynyl). Nonlimiting examples of alkynyl include ethynyl, propargyl, homopropargyl, but-2-yn-l-yl, 2-methyl-prop-2-yn-l-yl, etc. Unless otherwise specified, alkynyl may be optionally substitutedAttorney Docket: 140101-00220

[0252] by one, two, three, four, or five groups selected from the group consisting of carbocyclyl, aryl, heterocyclyl, heteroaryl, oxo, halogen, and hydroxyl.

[0253] By “arginine rich” with respect to a cationic domain is meant that at least 40% of the cationic domain is formed of arginine residues.

[0254] The term “artificial amino acid,” as used herein, refers to an abiogenic amino acid (e.g., non- proteinogenic). For example, artificial amino acids may include synthetic amino acids, modified amino acids (e.g., those modified with sugars), non-natural amino acids, manmade amino acids, spacers, and non-peptide bonded spacers. Synthetic amino acids may be those that are chemically synthesized by man. For the avoidance of doubt, aminohexanoic acid (X) is an artificial amino acid in the context of the present invention. For the avoidance of doubt, beta-alanine (B) and hydroxyproline (Hyp) occur in nature and therefore are not artificial amino acids in the context of the present invention but are natural amino acids. Artificial amino acids may include, for example, 6-aminohexanoic acid (X), tetrahydroisoquinoline- 3-carboxylic acid (TIC), l-(amino)cyclohexanecarboxylic acid (Cy), 3-azetidine-carboxylic acid (Az), and 11-aminoundecanoic acid.

[0255] The term “aryl,” as used herein, refers to a carbocyclic ring system containing one, two, or three rings, at least one of which is aromatic. An unsubstituted aryl contains a total of 6 to 14 carbon atoms. The term aryl includes both monovalent species and divalent species. Examples of aryl groups include, but are not limited to, phenyl, naphthyl, indanyl, and the like. In particular embodiments, an optionally substituted aryl is optionally substituted phenyl.

[0256] The term “bridged ring systems,” as used herein, refers to meant ring systems in which two rings share more than two atoms, see for example Advanced Organic Chemistry, by Jerry March, 4th Edition, Wiley Interscience, pages 131 -133, 1992. Examples of bridged heterocyclyl ring systems include, aza- bicydo[2.2.1]heptane, 2-oxa-5-azabicyclo[2.2.1]heptane, aza-bicyclo[2.2.2]octane, aza- bicyclo[3.2.1]octane, quinuclidine, etc.

[0257] The term “carbonyl,” as used herein, refers to a group of the following structure -C(O)-. Nonlimiting examples of carbonyl groups include those found, e.g, in acetone, ethyl acetate, proteinogenic amino acids, acetamide, etc.

[0258] References made herein to “cationic” denote an amino acid or domain of amino acids having an overall positive charge at physiological pH.

[0259] The term "(m-nC)" or "(m-nC) group" used alone or as a prefix, refers to a group having a total of m to n carbon atoms, when unsubstituted.

[0260] The term “cycloalkyl,” as used herein, refers to a saturated carbocyclic ring system containing one or two rings, and containing a total of 3 to 10 carbon atoms, unless otherwise specified. The two-ring cycloalkyls may be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to one another), bridged ring systems (two bridgeheadAttorney Docket: 140101-00220

[0261] carbon atoms are linked to one another via a covalent linker containing at least one carbon atom), and spiro-ring (two rings are fused at the same cabron atom) systems. Non-limiting examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, etc.

[0262] The term “cycloalkenyl,” as used herein, refers to a non-aromatic, unsaturated, carbocyclic ring system containing one or two rings; containing one, two, or three endocyclic double bonds; and containing a total of 3 to 10 carbon atoms, unless otherwise specified. The two-ring cycloalkenyls may be arranged as fused ring systems (two bridgehead carbon atoms are directly bonded to one another), bridged ring systems (two bridgehead carbon atoms are linked to one another via a covalent linker containing at least one carbon atom), and spiro-ring (two rings are fused at the same cabron atom) systems. Non-limiting examples of cycloalkenyl include cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, 3-cyclohexen-l-yl, cyclooctenyl, etc.

[0263] An “exon” refers to a defined section of nucleic acid that encodes for a protein, or a nucleic acid sequence that is represented in the mature form of an RNA molecule after either portions of a pre- processed (or precursor) RNA have been removed by splicing. The mature RNA molecule can be a messenger RNA (mRNA) or a functional form of a non-coding RNA, such as rRNA or tRNA.

[0264] “Exon skipping” refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein.

[0265] The term “halo” or “halogeno,” as used herein, refer to fluoro, chloro, bromo, and iodo.

[0266] By “histidine rich” with respect to a cationic domain it is meant that at least 40% of the cationic domain is formed of histidine residues.

[0267] The terms “heteroaryl” or “heteroaromatic,” as used interchangeably herein, refer to a ring system containing one, two, or three rings, at least one of which is aromatic and containing one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur. An unsubstituted heteroaryl group contains a total of one to nine carbon atoms. The term heteroaryl includes both monovalent species and divalent species. Examples of heteroaryl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heteroaryl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10- membered bicyclic ring, for example, a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to aboutAttorney Docket: 140101-00220

[0268] four heteroatoms typically selected from nitrogen, sulfur and oxygen. Typically, the heteroaryl ring will contain up to 3 heteroatoms, more usually up to 2, for example, a single heteroatom. In some embodiments, the heteroaryl ring contains at least one ring nitrogen atom. The nitrogen atoms in the heteroaryl rings can be basic, as in the case of an imidazole or pyridine, or essentially non-basic as in the case of an indole or pyrrole nitrogen. In general, the number of basic nitrogen atoms present in the heteroaryl group, including any amino group substituents of the ring, will be less than five.

[0269] Examples of heteroaryl include furyl, pyrrolyl, thienyl, oxazolyl, isoxazolyl, imidazolyl, pyrazolyl, thiazolyl, isothiazolyl, oxadiazolyl, thiadiazolyl, triazolyl, tetrazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, 1,3, 5 -tri azenyl, benzofuranyl, indolyl, isoindolyl, benzothienyl, benzoxazolyl, benzimidazolyl, benzothiazolyl, benzothiazolyl, indazolyl, purinyl, benzofurazanyl, quinolyl, isoquinolyl, quinazolinyl, quinoxalinyl, cinnolinyl, pteridinyl, naphthyridinyl, carbazolyl, phenazinyl, benzisoquinolinyl, pyridopyrazinyl, thieno[2,3-b]furanyl, 2H-furo[3,2-b]-pyranyl, 5H-pyrido[2,3-d]-o-oxazinyl, 1 H- pyrazolo[4,3-d]-oxazolyl, 4H-imidazo[4,5-d]thiazolyl, pyrazino[2,3-d]pyridazinyl, imidazo[2,l-b]thiazolyl, imidazofl,2-b][l,2,4]triazinyl. “Heteroaryl” also covers partially aromatic bi- or polycyclic ring systems wherein at least one ring is an aromatic ring and one or more of the other ring(s) is a non-aromatic, saturated or partially saturated ring, provided at least one ring contains one or more heteroatoms selected from nitrogen, oxygen or sulfur. Examples of partially aromatic heteroaryl groups include for example, tetrahydroisoquinolinyl, tetrahydroquinolinyl, 2-oxo-l,2.3.4-tetrahydroquinolinyl, dihydrobenzthienyl, dihydrobenzfuranyl, 2,3-dihydro- benzofl,4]dioxinyl, benzofl,3]dioxolyl, 2,2-dioxo-l,3-dihydro-2- benzothienyl, 4, 5,6,7- tetrahydrobenzofuranyl, indolinyl, 1,2,3, 4-tetrahydro-l,8-naphthyridinyl,l.2.3.4- tetrahydropyrido[2,3-b]pyrazinyl and 3,4-dihydro-2W-pyrido[3,2-b][l,4]oxazinyl. Examples of five membered heteroaryl groups include but are not limited to pyrrolyl, furanyl, thienyl, imidazolyl, furazanyl, oxazolyl, oxadiazolyl, oxatriazolyl, isoxazolyl, thiazolyl, isothiazolyl, pyrazolyl, triazolyl and tetrazolyl groups. Examples of six membered heteroaryl groups include but are not limited to pyridyl, pyrazinyl, pyridazinyl, pyrimidinyl and triazinyl. A bicyclic heteroaryl group may be, for example, a group selected from: a benzene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; a pyridine ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; a pyrimidine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrrole ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; a pyrazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a pyrazine ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an imidazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an oxazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isoxazole ring fused to a 5- or 6- membered ringAttorney Docket: 140101-00220

[0270] containing 1 or 2 ring heteroatoms; a thiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; an isothiazole ring fused to a 5- or 6-membered ring containing 1 or 2 ring heteroatoms; a thiophene ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; a furan ring fused to a 5- or 6-membered ring containing 1, 2 or 3 ring heteroatoms; a cyclohexyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2 or 3 ring heteroatoms; and a cyclopentyl ring fused to a 5- or 6-membered heteroaromatic ring containing 1, 2 or 3 ring heteroatoms. Particular examples of bicyclic heteroaryl groups containing a six membered ring fused to a five membered ring include but are not limited to benzofuranyl, benzothiophenyl, benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzothiazolyl, benzisothiazolyl, isobenzofuranyl, indolyl, isoindolyl, indolizinyl, indolinyl, isoindolinyl, purinyl (e.g., adeninyl, guaninyl), indazolyl, benzodi oxolyl and pyrazolopyridinyl groups. Particular examples of bicyclic heteroaryl groups containing two fused six membered rings include but are not limited to quinolinyl, isoquinolinyl, chromanyl, thiochromanyl, chromenyl, isochromenyl, chromanyl, isochromanyl, benzodi oxanyl, quinolizinyl, benzoxazinyl, benzodiazinyl, pyridopyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, naphthyridinyl and pteridinyl groups.

[0271] The terms “heterocyclyl,” as used herein, refer to a ring system containing one, two, or three rings, at least one of which containing one to four (e.g., one, two, or three) heteroatoms selected from the group consisting of nitrogen, oxygen, and sulfur, provided that the ring system does not contain aromatic rings that also include an endocyclic heteroatom. An unsubstituted heterocyclyl group contains a total of two to nine carbon atoms. The term heterocyclyl includes both monovalent species and divalent species. Examples of heterocyclyl groups are monocyclic and bicyclic groups containing from five to twelve ring members, and more usually from five to ten ring members. The heterocyclyl group can be, for example, a 5- or 6-membered monocyclic ring or a 9- or 10-membered bicyclic ring, for example, a bicyclic structure formed from fused five and six membered rings or two fused six membered rings. Each ring may contain up to about four heteroatoms typically selected from nitrogen, sulfur and oxygen. Non-limiting examples of heterocyclyl groups include, e.g., pyrrolidine, piperazine, piperidine, azepane, 1,4-diazepane, tetrahydrofuran, tetrahydropyran, oxepane, 1,4-dioxepane, tetrahydrothiophene, tetrahydrothiopyran, indoline, benzopyrrolidine, 2,3 -dihydrobenzofuran, phthalan, isochroman, and 2,3-dihy drob enzothi ophene.

[0272] The term “intemucleoside linkage,” as used herein, represents a group or bond that forms a covalent linkage between adjacent nucleosides in an oligonucleotide. An internucleoside linkage is an unmodified internucleoside linkage or a modified internucleoside linkage. An “unmodified intemucleoside linkage” is a phosphate (-O-P(O)(OH)-O-) intemucleoside linkage (“phosphate phosphodiester”). A “modified intemucleoside linkage” is an intemucleoside linkage other than a phosphate phosphodiester.Attorney Docket: 140101-00220

[0273] The two main classes of modified internucleoside linkages are defined by the presence or absence of a phosphorus atom. Non-limiting examples of phosphorus-containing internucleoside linkages include phosphodiester linkages, phosphotriester linkages, phosphorothioate diester linkages, phosphorothioate triester linkages, morpholino internucleoside linkages such as phosphorodiamidate linkages of the formula (-O-(P(O)-N(CH3)2-), methylphosphonates, and phosphoramidate. Non- limiting examples of nonphosphorus internucleoside linkages include methylenemethylimino ( — CH2 — N(CH3) — O — CH2 — ), thiodiester ( — O — C(O) — S — ), thionocarbamate ( — O — C(O)(NH) — S — ), siloxane ( — O — Si(H)2 — O — ), and N, N'-dimethylhydrazine ( — CH2 — N(CH3) — N(CH3) — ). Phosphorothioate linkages are phosphodiester linkages and phosphotriester linkages in which one of the non-bridging oxygen atoms is replaced with a sulfur atom. In some embodiments, an internucleoside linkage is a group of the following structure:

[0274]

[0275] where

[0276] Z is O, S, or Se;

[0277] Y is -X-L-Rl; each X is independently -O-, -S-, -N(-L-Rl)-, or L; each L is independently a covalent bond or a linker (e.g., optionally substituted Cl -60 aliphatic linker or optionally substituted C2-60 heteroaliphatic linker); each R1 is independently hydrogen, -S-S-R2, -O-CO-R2, -S-CO-R2, optionally substituted Cl -9 heterocyclyl, or a hydrophobic moiety; and each R2 is independently optionally substituted Cl-10 alkyl, optionally substituted C2-10 heteroalkyl, optionally substituted C6-10 aryl, optionally substituted C6-10 aryl Cl -6 alkyl, optionally substituted Cl -9 heterocyclyl, or optionally substituted Cl -9 heterocyclyl Cl -6 alkyl.

[0278] When L is a covalent bond, R1 is hydrogen, Z is oxygen, and all X groups are -O-, the internucleoside group is known as a phosphate phosphodiester. When L is a covalent bond, R1 is hydrogen, Z is sulfur, and all X groups are -O-, the internucleoside group is known as a phosphorothioate diester. When Z is oxygen, all X groups are -O-, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphotriester. When Z is sulfur, all X groups are -O-, and either (1) L is a linker or (2) R1 is not a hydrogen, the internucleoside group is known as a phosphorothioate triester. Non- limiting examples of phosphorothioate triester linkages and phosphotriester linkages are described in US 2017 / 0037399, the disclosure of which is incorporated herein by reference.

[0279] An “intron” refers to a nucleic acid region (within a gene) that is not translated into a protein. An intron is a non-coding section that is transcribed into a precursor mRNA (pre-Attorney Docket: 140101-00220

[0280] mRNA), and subsequently removed by splicing during formation of the mature RNA. An intron is also a region that might be fully or partially retained in a coding mRNA (cryptic exaon or exonized intron) due to splicing changes.

[0281] The term “phosphorodiamidate morpholino oligomer (PMO),” used interchangeably with the term “morpholino,” is used herein in reference to a class of oligonucleotides, represents an oligomer of at least 5 morpholino monomer units interconnected by morpholino internucleoside linkages. A morpholino includes a 5’ group and a 3’ group.

[0282] A 5’ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, di sphorodi thioate, triphosphorodithioate, phosphonate, phosphoramidate, a bond to a cell penetrating peptide, a bond to a cell penetrating peptide / linker combination, an endosomal escape moiety, or a neutral organic polymer. A 3’ group in morpholino may be, e.g., hydrogen, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate,

[0283] di sphorodi thioate, triphosphorodithioate, phosphonate, phosphoramidate, a bond to a cell penetrating peptide, a bond to a cell penetrating peptide / linker combination, an endosomal escape moiety, or a neutral organic polymer. In a conjugate of an oligonucleotide that is a morpholino and a cell penetrating peptide that is covalently bonded or linked to the oligonucleotide, the preferred 3’ group is a bond to a cell penetrating peptide or a bond to a cell penetrating peptide / linker combination.

[0284] The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the Y position of the ribofuranose / 2’ -deoxyribofuranose of a nucleoside.

[0285] Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines, as well as synthetic and natural nucleobases, e.g., 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-m ethyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5- trifluoromethyl uracil, 5 -trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and / or 06-substituted purines. Nucleic acid duplex stability can be enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-Attorney Docket: 140101-00220

[0286] thiocytosine, 5-propynyl ( — C°C — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3- deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)- 1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7- deazaguanine, 2-aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in Merigan et al., U. S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch etal., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442- 443; the entire contents of each of which are hereby incorporated herein by reference.

[0287] The term “nucleoside,” as used herein, represents sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2’-deoxyribofuranrpose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose-nucleobase is ribofuranose having an anomeric carbon bond to an unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2’-deoxyribofuranose-nucleobase compounds are 2’ -deoxy adenosine, 2’-deoxycytidine, 2’-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2 ’-substitution, locking, carbocyclization, or unlocking. A 2’ -substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’-fluoro, 2’-methoxy, or 2’-(2-methoxy)ethoxy. Alternatively, a 2’-substitution may be a 2’-(ara) substitution, which corresponds to the following structure:

[0288]

[0289] Attorney Docket: 140101-00220

[0290] where B is a nucleobase, and R is a 2’-(ara) substituent (e.g., fluoro). 2’-(ara) substituents are known in the art and can be same as other 2 ’-substituents described herein. In some embodiments, 2’-(ara) substituent is a 2’-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’-carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides. A “nucleoside” may also refer to a morpholino subunit.

[0291] The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure -X1-P(X2)(R1)2, where X1is O, S, or NH, and X2is absent, =0, or =S, and each R1is independently -OH, -N(R2)2, or -O-CH2CH2CN, where each R2is independently an optionally substituted alkyl, or both R2groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

[0292] The term “oligonucleotide,” as used herein, represents a structure containing 5 or more contiguous nucleosides covalently bound together by internucleoside linkages; a morpholino containing 5 or more morpholino subunits; or a nucleic acid containing 10 or more morpholino subunits. Preferably, an oligonucleotide is a PMO of the structure:

[0293] Structure of

[0294]

[0295] wherein the Base is a nucleobase, preferably selected from purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T) and cytosine (C), n represents the total number of bases of the antisense oligomer sequence. Unlabeled groups attached to theAttorney Docket: 140101-00220

[0296] nitrogen atoms shown in the foregoing figure are methyl groups (-CH3). The foregoing

[0297]

[0298] structure shows a preferred 5' group:

[0299] The term "optionally substituted" refers to groups, structures, or molecules that may be substituted or unsubstituted as described for each respective group. The term “wherein a / any CH, CH2, CH3 group or heteroatom (i.e., NH) within a R1group is optionally substituted” means that (any) one of the hydrogen radicals of the R1group is substituted by a relevant stipulated group.

[0300] The term "operably linked" may include the situation where a selected nucleotide sequence and regulatory nucleotide sequence are covalently linked in such a way as to place the expression of a nucleotide coding sequence under the control of the regulatory sequence, as such, the regulatory sequence is capable of effecting transcription of a nucleotide coding sequence which forms part or all of the selected nucleotide sequence. Where appropriate, the resulting transcript may then be translated into a desired peptide.

[0301] The term “pharmaceutically acceptable,” as used herein, refers to those compounds, materials, compositions, and / or dosage forms, which are suitable for contact with the tissues of an individual (e.g., a human), without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit / risk ratio.

[0302] The term “pharmaceutical composition,” as used herein, represents a composition containing an oligonucleotide described herein, formulated with a pharmaceutically acceptable excipient, and manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a subject.

[0303] The term “pharmaceutically acceptable salt,” as used herein, means any pharmaceutically acceptable salt of a conjugate, oligonucleotide, or peptide disclosed herein. Pharmaceutically acceptable salts of any of the compounds described herein may include those that are within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1 -19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008; the entire contents of each of which are hereby incorporated herein by reference. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting a free base group with a suitable acid. Representative acid additionAttorney Docket: 140101-00220

[0304] salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, di gluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3 -phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, ethylamine, and the like.

[0305] The term “reduce” or “inhibit” may relate generally to the ability of one or more compounds of the invention to “decrease” a relevant physiological or cellular response, such as a symptom of a disease or condition described herein, as measured according to routine techniques in the diagnostic art. Relevant physiological or cellular responses (in vivo or in vitro) will be apparent to persons skilled in the art, and may include reductions in the symptoms or pathology of GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease), or reductions in the expression of defective forms of GYSI, such as the altered forms of GYSI that are expressed in individuals with GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease). A “decrease” in a response may be statistically significant as compared to the response produced by no antisense compound or a control composition, and may include a 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% decrease, including all integers in between.

[0306] The term “subject,” as used herein, represents a human or non-human animal (e.g., a mammal) that is suffering from, or is at risk of, disease, disorder, or condition, as determined by a qualified professional (e.g., a doctor or a nurse practitioner) with or without known in the art laboratory test(s) of sample(s) from the subject. Non-limiting examples of diseases, disorders, and conditions include GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease).

[0307] A “sugar” or “sugar moiety,” includes naturally occurring sugars having a furanose ring or a structure that is capable of replacing the furanose ring of a nucleoside. Sugars included in the nucleosides of the invention may be non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures include simple changes relative to the natural furanose ring (e.g., a six- membered ring). Alternative sugars may also include sugarAttorney Docket: 140101-00220

[0308] surrogates wherein the furanose ring has been replaced with another ring system such as, e.g., a morpholino or hexitol ring system. Non-limiting examples of sugar moieties useful that may be included in the oligonucleotides of the invention include b- D-ribose, P-D-2'-deoxyribose, substituted sugars (e.g., 2', 5', and bis substituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic sugar moieties (e.g., the 2'- O — CH2-4' or 2'-0 — (CH2)2-4' bridged ribose derived bicyclic sugars) and sugar surrogates (when the ribose ring has been replaced with a morpholino or a hexitol ring system).

[0309] “Treatment” and "treating," as used herein, refer to the medical management of a subject with the intent to improve, ameliorate, or stabilize a disease, disorder, or condition (e.g., GSD). This term includes active treatment (treatment directed to improve GSD); palliative treatment (treatment designed for the relief of symptoms of GSD); and supportive treatment (treatment employed to supplement another therapy).

[0310] The term “Glycogen storage disease,” or “GSD,” as used interchangeably herein, refers to a metabolic disorder of glycogen metabolism caused by deficiency of enzymes or transporters involved in the synthesis or degradation of glycogen leading to aberrant storage and / or utilization. GSDs are multisystemic diseases that can present at any age from the neonatal period to adulthood. The overall GSD incidence is approximately 1 case per 20, GOO-43, 000 live births. GSDs primarily affect liver, or muscle, or both, as glycogen is particularly abundant in these tissues. GSDs may also affect other organs including heart, kidney and / or brain. Hypoglycemia is the hallmark of hepatic GSDs. Muscle GSDs can present with exercise intolerance, muscle cramps / pain, rhabdomyolysis, and muscle weakness and in the case of cardiac involvement, cardiomyopathy.

[0311] There are over 20 types of GSD, including, for example, Lewis' disease (type 0), von Gierke’s disease (type I), Pompe’s disease (type II), Danon disease (type-IIb), Forbes disease / Cori disease (type III), Andersen's disease (type IV), Adult Polyglucosan Body Disease (APBD) (type IV), Lafora Disease, McArdle's disease (type V), Hers’ disease (type VI), Tarui’s disease (type VII), phosphorylase kinase deficiency (type IX), type IX of GSD, type X of GSD, Fanconi -Bickel syndrome (type XI), type XI of GSD, type XII of GSD, type XIII of GSD, type XV of GSD, and subtypes thereof.

[0312] In some embodiments, the GSD is Lafora disease. In some embodiments, the GSD is APBD. In some embodiments, the GSD is Pompe’s disease.

[0313] Lafora’s disease is a neurodegenerative disorder caused by recessive loss-of-function mutations in the EPM2A (laforin glycogen phosphatase) or EPM2B (malin E3 ubiquitin ligase) genes. Neuropathology is characterized by malformed precipitated glycogen aggregates termed Lafora bodies. Symptoms usually begin during adolescence and include jerking seizures, a loss of balance and difficulty speaking.Attorney Docket: 140101-00220

[0314] Adult polyglucosan body disease (APBD) is a type IV GSD caused by inherits loss-of-function mutations in the GBE1 gene. Symptoms include walking problems due to reduced sensation in their legs (peripheral neuropathy) and progressive muscle weakness and stiffness (spasticity) and neurogenic bladder. Some people with the disease experience dementia.

[0315] Pompe’s disease is a lysosomal storage disease. The clinical manifestations of Pompe disease are variable, predominantly due to the varying amounts of residual acid alphaglucosidase (GAA) activity linked with distinct mutations in the causative gene (GAA).

[0316] Enzyme deficiency results in intra-lysosomal storage of glycogen especially in skeletal and cardiac muscles. Symptoms of infantile form of Pompe’s disease may begin in the first months of life, with feeding problems, poor weight gain, trouble breathing, muscle weakness, enlarged heart, floppiness, and head lag. Many infants with Pompe disease also have enlarged tongues. Symptoms of juvenile / adult form of Pompe’s disease may begin as early as the first decade of childhood or well into adulthood. Muscle weakness progresses to death from respiratory failure after several years.

[0317] Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to,” and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0318] All references to “conjugates” also refer to solvates thereof, including pharmaceutically acceptable solvates thereof.

[0319] All references to “oligonucleotides” also refer to salts and / or solvates thereof, including pharmaceutically acceptable salts and / or solvates thereof.

[0320] Unless otherwise specified, all peptides are shown herein in N-terminus to C-terminus direction (left to right). Unless otherwise specified, all oligonucleotides are shown herein in 5’ to 3’ direction (left to right).

[0321] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0322] All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive.

[0323] II. Antisense Oligonucleotide Conjugates of the InventionAttorney Docket: 140101-00220

[0324] The present invention provides antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide, and compositions comprising such conjugates, which target a GYSI gene and inhibit the expression of the GYSI gene (e.g., inhibit GYSI transcript expression, and / or GYSI protein expression). In one embodiment, the antisense oligonucleotide conjugates inhibit the expression of a GYSI gene in a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), which is for example, within a subject, e.g., a human having a disease, e.g, GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease).

[0325] The antisense oligonucleotide conjugates of the invention comprise an antisense oligonucleotide (e.g., a PMO) targeting GYSI that includes a region of complementarity, which is complementary to a target sequence in a human GYSI gene (SEQ ID NO: 284) or GYSI mRNA (SEQ ID NOs: 285-286), for example, at least a part of an mRNA formed in the expression of a GYSI gene. The region of complementarity may be about 50 nucleotides or less in length (e.g., about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, or 4 nucleotides or less in length). Upon contact with a cell expressing the GYSI gene, the antisense oligonucleotide inhibits the expression of the GYSI gene (e.g., a human GYSI gene) by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 98%, or about 100%.

[0326] The target sequence can be derived from the sequence of an mRNA formed during the expression of a GYSI gene. Accordingly, in one aspect, an antisense oligonucleotide of the invention specifically hybridizes to a target nucleic acid molecule, such as the mRNA encoding GYSI, and comprises a contiguous nucleotide sequence which corresponds to the reverse complement of a nucleotide sequence of GYSI mRNA, or a fragment thereof.

[0327] In some embodiments, the antisense oligonucleotide of the invention may be substantially complementary to the target sequence. For example, an antisense oligonucleotide that is substantially complementary to the target sequence may include a contiguous nucleotide sequence comprising no more than 5 mismatches (e.g., no more than 1, no more than 2, no more than 3, no more than 4, or no more than 5 mismatches) when hybridizing to a target sequence, such as to the corresponding region of a nucleic acid which encodes a GYSI mRNA. In some embodiments, the contiguous nucleotide sequenceAttorney Docket: 140101-00220

[0328] comprises no more than a single mismatch when hybridizing to the target sequence, such as the corresponding region of a nucleic acid which encodes a GYSI mRNA.

[0329] In some embodiments, the antisense oligonucleotides of the invention that are substantially complementary to the target sequence comprise a contiguous nucleotide sequence which is at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 80% complementary over its entire length to the equivalent region of the nucleotide sequence of any one of SEQ ID NOs: 284-286, or a fragment thereof, such as about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% complementary.

[0330] In some embodiments, the antisense oligonucleotide comprises at least 10, at least 15, at least 20, or at least 25 contiguous nucleotides complementary to a target sequence in a human GYSI gene (SEQ ID NO: 284) or GYSI mRNA (SEQ ID NOs: 285-286).

[0331] In some embodiments, the antisense oligonucleotide targets the 5’ UTR, the 3’ UTR, an exon, an intron, or an exon-intron junction of the GYSI pre-mRNA. In some embodiments, the antisense oligonucleotide targets the 5’ UTR of the GYSI pre-mRNA. In some embodiments, the antisense oligonucleotide targets the 3’ UTR of the GYSI pre-mRNA. In some embodiments, the antisense oligonucleotide targets an exon of the GYSI pre-mRNA. In some embodiments, the antisense oligonucleotide targets an intron of the GYSI pre-mRNA. In some embodiments, the antisense oligonucleotide targets an exonintronjunction of the GYSI pre-mRNA.

[0332] In some embodiments, the antisense oligonucleotide of the invention inhibits GYSI expression (e.g., transcript expression and / or protein expression) by inducing one or more of exon skipping, steric clocking regulatory regions, nonsense-mediated decay, destabilizing full length and / or pre-mRNA transcript, inhibiting protein production etc., all leading to a decrease in the expression of GYSI full-length mRNA and or functional protein, or expression of healthy, non-mutant or mutant functional protein.

[0333] In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, induces exon skipping in the GYSI mRNA. “Exon skipping” refers generally to the process by which an entire exon, or a portion thereof, is removed from a given pre-processed RNA, and is thereby excluded from being present in the mature RNA, such as the mature mRNA that is translated into a protein. Hence, the portion of the protein that is otherwise encoded by the skipped exon is not present in the expressed form of the protein, typically creating an altered, though still functional, form of the protein. As described herein, the exon being skipped is one or more of exon la, exon lb, exon 2, exon 3, exon 4, or exon 5 of the human GYSI gene. In some embodiments, the exon is exon la. In some embodiments, the exon is exon lb. In someAttorney Docket: 140101-00220

[0334] embodiments, the exon is exon 2. In some embodiments, the exon is exon 3. In some embodiments, the exon is exon 4. In some embodiments, the exon is exon 5.

[0335] In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein, or a pharmaceutically acceptable salt thereof, targets one or more of the exon-intron junction of exon 5 and the exon-intron junction of exon 6 of the mouse GYSI gene. In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein targets the exon-intron junction of exon 5 of the mouse GYSI gene. In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein targets the exon-intron junction of exon 6 of the mouse GYSI gene. In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein targets the exon-intron junction of exon 5 and intron 5 of the mouse GYSI gene. In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein targets the exon-intron junction of intron 5 and exon 6 of the mouse GYSI gene. In some embodiments, the antisense oligonucleotide or the antisense oligonucleotide conjugate disclosed herein targets the exon-intron junction of exon 6 and intron 6 of the mouse GYSI gene. In some embodiments, the exon being skipped is one or more of exon 5 or exon 6 of the mouse GYSI gene.

[0336] An antisense oligonucleotide may comprise a contiguous nucleotide sequence of about 4 to about 50 nucleotides in length, e.g., 8-49, 8-48, 8-47, 8-46, 8-45, 8-44, 8-43, 8-42, 8-41, 8-40, 8-39, 8-38, 8-37, 8-36, 8-35, 8-34, 8-33, 8-32, 8-31, 8-30, 8-29, 8-28, 8-27, 8-26, 8-25, 8-24, 8-23, 8-22, 8-21, 8-20, 8-19, 8-18, 8-17, 8-16, 8-15, 8-14, 8-13, 8-12, 8-11, 8-10, 8-9, 10-49, 10-48, 10-47, 10-46, 10-45, 10-44, 10-43, 10-42, 10-41, 10-40, 10-39, 10-38, 10-37, 10-36, 10-35, 10-34, 10-33, 10-32, 10-31, 10-30, 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 10-11,11-49, 11-48, 11-47, 11-46, 11-45, 11-44, 11-43, 11-42, 11-41, 11-40, 11-39, 11-38, 11-37, 11-36, 11-35, 11-34, 11-33, 11-32, 11-31, 11-30, 11-29, 11-28, 11-27, 11-26, 11-25, 11- 24, 11-23, 11-22, 11-21, 11-20, 11-19, 11-18, 11-17, 11-16, 11-15, 11-14, 11-13, 11-12, 12- 49, 12-48, 12-47, 12-46, 12-45, 12-44, 12-43, 12-42, 12-41, 12-40, 12-39, 12-38, 12-37, 12- 36, 12-35, 12-34, 12-33, 12-32, 12-31, 12-30, 12-29, 12-28, 12-27, 12-26, 12-25, 12-24, 12- 23, 12-22, 12-21, 12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, 12-13, 13-49, 13-48, 13- 47, 13-46, 13-45, 13-44, 13-43, 13-42, 13-41, 13-40, 13-39, 13-38, 13-37, 13-36, 13-35, 13- 34, 13-33, 13-32, 13-31, 13-30, 13-29, 13-28, 13-27, 13-26, 13-25, 13-24, 13-23, 13-22, 13- 21, 13-20, 13-19, 13-18, 13-17, 13-16, 13-15, 13-14, 14-49, 14-48, 14-47, 14-46, 14-45, 14-44, 14-43, 14-42, 14-41, 14-40, 14-39, 14-38, 14-37, 14-36, 14-35, 14-34, 14-33, 14-32, 14- 31, 14-30, 14-29, 14-28, 14-27, 14-26, 14-25, 14-24, 14-23, 14-22, 14-21, 14-20, 14-19, 14- 18, 14-17, 14-16, 14-15, 15-49, 15-48, 15-47, 15-46, 15-45, 15-44, 15-43, 15-42, 15-41, 15- 40, 15-39, 15-38, 15-37, 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15- 27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 15-16,16-49, 16-Attorney Docket: 140101-00220

[0337] 48, 16-47, 16-46, 16-45, 16-44, 16-43, 16-42, 16-41, 16-40, 16-39, 16-38, 16-37, 16-36, 16- 35, 16-34, 16-33, 16-32, 16-31, 16-30, 16-29, 16-28, 16-27, 16-26, 16-25, 16-24, 16-23, 16- 22, 16-21, 16-20, 16-19, 16-18, 16-17, 17-49, 17-48, 17-47, 17-46, 17-45, 17-44, 17-43, 17- 42, 17-41, 17-40, 17-39, 17-38, 17-37, 17-36, 17-35, 17-34, 17-33, 17-32, 17-31, 17-30, 17- 29, 17-28, 17-27, 17-26, 17-25, 17-24, 17-23, 17-22, 17-21, 17-20, 17-19, 17-18, 18-49, 18- 48, 18-47, 18-46, 18-45, 18-44, 18-43, 18-42, 18-41, 18-40, 18-39, 18-38, 18-37, 18-36, 18- 35, 18-34, 18-33, 18-32, 18-31, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18- 22, 18-21, 18-20, 19-49, 19-48, 19-47, 19-46, 19-45, 19-44, 19-43, 19-42, 19-41, 19-40, 19- 39, 19-38, 19-37, 19-36, 19-35, 19-34, 19-33, 19-32, 19-31, 19-30, 19-29, 19-28, 19-27, 19- 26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-49, 20-48, 20-47, 20-46, 20-45, 20-44, 20- 43, 20-42, 20-41, 20-40, 20-39, 20-38, 20-37, 20-36, 20-35, 20-34, 20-33, 20-32, 20-31, 20- 30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-49, 21-48, 21-47, 21- 46, 21-45, 21-44, 21-43, 21-42, 21-41, 21-40, 21-39, 21-38, 21-37, 21-36, 21-35, 21-34, 21- 33, 21-32, 21-31, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, 21-22, 22-49, 22- 48, 22-47, 22-46, 22-45, 22-44, 22-43, 22-42, 22-41, 22-40, 22-39, 22-38, 22-37, 22-36, 22- 35, 22-34, 22-33, 22-32, 22-31, 22-30, 22-29, 22-28, 22-27, 22-26, 22-25, 22-24, 22-23, 23- 49, 23-48, 23-47, 23-46, 23-45, 23-44, 23-43, 23-42, 23-41, 23-40, 23-39, 23-38, 23-37, 23- 36, 23-35, 23-34, 23-33, 23-32, 23-31, 23-30, 23-29, 23-28, 23-27, 23-26, 23-25, 23-24, 24- 49, 24-48, 24-47, 24-46, 24-45, 24-44, 24-43, 24-42, 24-41, 24-40, 24-39, 24-38, 24-37, 24- 36, 24-35, 24-34, 24-33, 24-32, 24-31, 24-30, 24-29, 24-28, 24-27, 24-26, 24-25, 25-49, 25- 48, 25-47, 25-46, 25-45, 25-44, 25-43, 25-42, 25-41, 25-40, 25-39, 25-38, 25-37, 25-36, 25- 35, 25-34, 25-33, 25-32, 25-31, 25-30, 25-29, 25-28, 25-27, 25-26,26-49, 26-48, 26-47, 26- 46, 26-45, 26-44, 26-43, 26-42, 26-41, 26-40, 26-39, 26-38, 26-37, 26-36, 26-35, 26-34, 26- 33, 26-32, 26-31, 26-30, 26-29, 26-28, 26-27, 27-49, 27-48, 27-47, 27-46, 27-45, 27-44, 27- 43, 27-42, 27-41, 27-40, 27-39, 27-38, 27-37, 27-36, 27-35, 27-34, 27-33, 27-32, 27-31, 27- 30, 27-29, 27-28, 28-49, 28-48, 28-47, 28-46, 28-45, 28-44, 28-43, 28-42, 28-41, 28-40, 28- 39, 28-38, 28-37, 28-36, 28-35, 28-34, 28-33, 28-32, 28-31, 28-30, 28-29, 29-49, 29-48, 29- 47, 29-46, 29-45, 29-44, 29-43, 29-42, 29-41, 29-40, 29-39, 29-38, 29-37, 29-36, 29-35, 29- 34, 29-33, 29-32, 29-31, 29-30, 30-49, 30-48, 30-47, 30-46, 30-45, 30-44, 30-43, 30-42, 30- 41, 30-40, 30-39, 30-38, 30-37, 30-36, 30-35, 30-34, 30-33, 30-32, or 30-31 nucleotides in length, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length.

[0338] In some embodiments, the antisense oligonucleotide may comprise a contiguous nucleotide sequence of no more than 22 nucleotides, such as no more than 21 nucleotides, 20 nucleotides, 19 nucleotides, or no more than 18 nucleotides. In some embodiments the antisense oligonucleotides of the invention comprises less than 20 nucleotides. In otherAttorney Docket: 140101-00220

[0339] embodiments, the antisense oligonucleotides of the invention comprise 20 nucleotides. In other embodiments, the antisense oligonucleotides of the invention comprise 25 nucleotides.

[0340] In some embodiments, the antisense oligonucleotide comprises at least 10 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 106-283. In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the nucleotide sequences of SEQ ID NOs: 106-283. In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleotides of any one of the nucleotide sequences of SEQ ID NOs: 106-283. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106-283. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106-283.

[0341] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 106. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 107. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 108. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 109. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 110. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 111. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 112. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 113. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 114. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 115. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 116. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 117. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 118. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 119. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 120. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 121. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 122. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 123. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 124. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 125. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 126. In some embodiments, the antisense oligonucleotide comprises aAttorney Docket: 140101-00220

[0342] nucleotide sequence of SEQ ID NO: 127. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 128. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 129. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 130. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 131. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 132. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 133. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 134. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 135. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 136. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 137. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 138. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 139. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 140. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 141. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 142. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 143. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 144. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 145. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 146. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 147. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 148. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 149. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 150. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 151. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 152. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 153. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 154. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 155. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 156. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 157. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 158. In some embodiments,Attorney Docket: 140101-00220

[0343] the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 159. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 160. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 161. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 162. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 163. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 164. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 165. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 166. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 167. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 168. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 169. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 170. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 171. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 172. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 173. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 174. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 175. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 176. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 177. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 178. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 179. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 180. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 181. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 182. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 183. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 184. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 185. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 186. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 187. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 188. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 189. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO:Attorney Docket: 140101-00220

[0344] 190. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 191. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 192. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 193. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 194. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 195. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 196. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 197. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 198. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 199. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 200. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 201. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 202. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 203. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 204. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 205. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 206. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 207. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 208. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 209. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 210. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 211. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 212. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 213. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 214. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 215. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 216. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 217. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 218. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 219. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 220. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 221. In some embodiments, the antisense oligonucleotide comprises aAttorney Docket: 140101-00220

[0345] nucleotide sequence of SEQ ID NO: 222. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 223. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 224. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 225. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 226. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 227. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 228. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 229. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 230. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 231. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 232. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 233. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 234. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 235. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 236. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 237. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 238. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 239. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 240. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 241. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 242. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 243. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 244. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 245. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 246. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 247. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 248. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 249. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 250. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 251. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 252. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 253. In some embodiments,Attorney Docket: 140101-00220

[0346] the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 254. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 255. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 256. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 257. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 258. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 259. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 260. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 261. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 262. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 263. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 264. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 265. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 266. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 267. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 268. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 269. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 270. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 271. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 272. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 273. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 274. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 275. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 276. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 277. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 278. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 279. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 280. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 281. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 282. In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of SEQ ID NO: 283.

[0347] In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 106. In some embodiments, the antisense oligonucleotide consists of aAttorney Docket: 140101-00220

[0348] nucleotide sequence of SEQ ID NO: 107. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 108. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 109. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 110. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 111. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 112. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 113. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 114. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 115. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 116. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 117. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 118. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 119. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 120. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 121. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 122. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 123. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 124. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 125. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 126. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 127. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 128. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 129. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 130. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 131. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 132. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 133. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 134. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 135. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 136. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 137. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 138. In some embodiments,Attorney Docket: 140101-00220

[0349] the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 139. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 140. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 141. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 142. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 143. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 144. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 145. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 146. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 147. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 148. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 149. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 150. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 151. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 152. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 153. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 154. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 155. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 156. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 157. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 158. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 159. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 160. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 161. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 162. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 163. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 164. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 165. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 166. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 167. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 168. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 169. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO:Attorney Docket: 140101-00220

[0350] 170. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 171. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 172. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 173. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 174. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 175. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 176. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 177. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 178. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 179. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 180. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 181. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 182. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 183. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 184. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 185. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 186. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 187. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 188. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 189. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 190. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 191. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 192. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 193. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 194. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 195. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 196. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 197. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 198. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 199. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 200. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 201. In some embodiments, the antisense oligonucleotide consists of aAttorney Docket: 140101-00220

[0351] nucleotide sequence of SEQ ID NO: 202. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 203. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 204. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 205. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 206. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 207. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 208. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 209. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 210. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 211. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 212. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 213. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 214. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 215. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 216. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 217. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 218. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 219. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 220. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 221. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 222. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 223. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 224. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 225. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 226. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 227. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 228. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 229. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 230. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 231. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 232. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 233. In some embodiments,Attorney Docket: 140101-00220

[0352] the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 234. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 235. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 236. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 237. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 238. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 239. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 240. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 241. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 242. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 243. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 244. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 245. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 246. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 247. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 248. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 249. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 250. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 251. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 252. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 253. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 254. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 255. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 256. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 257. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 258. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 259. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 260. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 261. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 262. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 263. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 264. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO:Attorney Docket: 140101-00220

[0353] 265. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 266. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 267. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 268. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 269. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 270. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 271. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 272. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 273. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 274. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 275. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 276. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 277. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 278. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 279. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 280. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 281. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 282. In some embodiments, the antisense oligonucleotide consists of a nucleotide sequence of SEQ ID NO: 283.

[0354] Further, antisense oligonucleotides having a sequence of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Table 1A and differing in their ability to inhibit the expression of a GYSI gene by not more than about 5%, 10%, 15%, 20%, 25%, or 30% inhibition from an antisense oligonucleotide comprising the full sequence, are contemplated to be within the scope of the present invention.

[0355] In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). PMO refers to to a class of oligonucleotides, represents an oligomer of at least 5 morpholino monomer units interconnected by morpholino intemucleoside linkages. A morpholino includes a 5’ group and a 3’ group. For example, a morpholino may be of the following structure:

[0356]

[0357] Attorney Docket: 140101-00220

[0358] n is an integer of at least 10 (e.g, 12 to 30) indicating the number of morpholino subunits and associated groups L; each B is independently a nucleobase;

[0359] R1is a 5’ group (R1may be referred to herein as a 5’ terminus);

[0360] R2is a 3’ group (R2may be referred to herein as a 3’ terminus); and L is (i) a morpholino intemucleoside linkage or, (ii) if L is attached to R2, a covalent bond. In certain embodiments, L is a phosphorodiamidate linkage of the formula (-O-(P(O)-N(CH3)2-).

[0361] A 5’ group in morpholino may be, e.g., hydroxyl, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, di sphorodi thioate, triphosphorodithioate, phosphonate, phosphoramidate, a bond to a cell penetrating peptide, a bond to a cell penetrating peptide / linker combination, an endosomal escape moiety, or a neutral organic polymer. In some embodiments, the 5’ group is of the following structure:

[0362]

[0363] Preferred 5’ group are hydroxyl and groups of the following structure:

[0364]

[0365] A more preferred 5’ group is of the following structure:

[0366]

[0367] A 3’ group in morpholino may be, e.g., hydrogen, a hydrophobic moiety, phosphate, diphosphate, triphosphate, phosphorothioate, diphosphorothioate, triphosphorothioate, phosphorodithioate, di sphorodi thioate, triphosphorodithioate, phosphonate, phosphoramidate, a bond to a cell penetrating peptide, a bond to a cell penetrating peptide / linker combination, an endosomal escape moiety, or a neutral organic polymer. In a conjugate of an oligonucleotide that is a morpholino and a cell penetrating peptide that is covalently bondedAttorney Docket: 140101-00220

[0368] or linked to the oligonucleotide, the preferred 3’ group is a bond to a cell penetrating peptide or a bond to a cell penetrating peptide / linker combination.

[0369] In some embodiments, all the intemucleoside linkages of the PMO are -P(O)(NMe2)O-.

[0370] The term “intemucleoside linkage of the PMO,” as used herein, represents a divalent group of the following structure:

[0371]

[0372] where

[0373] Z is O or S;

[0374] X1is a bond, -CH2-, or -O-;

[0375] X2is a bond, -CH2-O-, or -O-; and

[0376] Y is -NR2, where each R is independently H or C1-6alkyl (e.g., methyl), or both R combine together with the nitrogen atom to which they are attached to form a C2-9heterocyclyl (e.g, N-piperazinyl); provided that both X1and X2are not simultaneously a bond.

[0377] The term “morpholino subunit,” as used herein, refers to the following structure:

[0378]

[0379] where B is a nucleobase.

[0380] The term “nucleobase,” as used herein, represents a nitrogen-containing heterocyclic ring found at the Y position of the ribofuranose / 2’ -deoxyribofuranose of a nucleoside.

[0381] Nucleobases are unmodified or modified. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and O-6 substituted purines, as well as synthetic and natural nucleobases, e.g, 5-methylcytosine, 5 -hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g, 6-m ethyl) adenine and guanine, 2-alkyl (e.g., 2-propyl) adenine and guanine, 2-thiouracil, 2- thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 5- trifluoromethyl uracil, 5 -trifluoromethyl cytosine, 7-methyl guanine, 7-methyl adenine, 8-azaguanine, 8- azaadenine, 7-deazaguanine, 7-deazaadenine, 3 -deazaguanine, 3 -deazaadenine. Certain nucleobases are particularly useful for increasing the binding affinity of nucleic acids, e g., 5-substituted pyrimidines; 6-azapyrimidines; N2-, N6-, and / or 06-substituted purines. Nucleic acid duplex stability can beAttorney Docket: 140101-00220

[0382] enhanced using, e.g., 5-methylcytosine. Non-limiting examples of nucleobases include: 2-aminopropyladenine, 5- hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-N-methylguanine, 6-N- methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl ( — C°C — CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5-trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F-adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3- deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3-diazaphenothiazine-2-one and 9-(2- aminoethoxy)- 1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example, 7-deazaadenine, 7- deazaguanine, 2-aminopyridine, or 2-pyridone. Further nucleobases include those disclosed in Merigan et aL, U. S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch etal., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. and Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442- 443; the entire contents of each of which are hereby incorporated herein by reference.

[0383] The term “nucleoside,” as used herein, represents sugar-nucleobase compounds and groups known in the art, as well as modified or unmodified 2’-deoxyribofuranrpose-nucleobase compounds and groups known in the art. The sugar may be ribofuranose. The sugar may be modified or unmodified. An unmodified ribofuranose-nucleobase is ribofuranose having an anomeric carbon bond to an unmodified nucleobase. Unmodified ribofuranose-nucleobases are adenosine, cytidine, guanosine, and uridine. Unmodified 2’-deoxyribofuranose-nucleobase compounds are 2’ -deoxy adenosine, 2’-deoxycytidine, 2’-deoxyguanosine, and thymidine. The modified compounds and groups include one or more modifications selected from the group consisting of nucleobase modifications and sugar modifications described herein. A nucleobase modification is a replacement of an unmodified nucleobase with a modified nucleobase. A sugar modification may be, e.g., a 2 ’-substitution, locking, carbocyclization, or unlocking. A 2’ -substitution is a replacement of 2’-hydroxyl in ribofuranose with 2’-fluoro, 2’-methoxy, or 2’-(2-methoxy)ethoxy. Alternatively, a 2’-substitution may be a 2’-(ara) substitution, which corresponds to the following structure:Attorney Docket: 140101-00220

[0384]

[0385] where B is a nucleobase, and R is a 2’-(ara) substituent (e.g., fluoro). 2’-(ara) substituents are known in the art and can be same as other 2 ’-substituents described herein. In some embodiments, 2’-(ara) substituent is a 2’-(ara)-F substituent (R is fluoro). A locking modification is an incorporation of a bridge between 4’-carbon atom and 2’-carbon atom of ribofuranose. Nucleosides having a locking modification are known in the art as bridged nucleic acids, e.g., locked nucleic acids (LNA), ethylene-bridged nucleic acids (ENA), and cEt nucleic acids. The bridged nucleic acids are typically used as affinity enhancing nucleosides. A “nucleoside” may also refer to a morpholino subunit.

[0386] The term “nucleotide,” as used herein, represents a nucleoside bonded to an internucleoside linkage or a monovalent group of the following structure -X1-P(X2)(R1)2, where X1is O, S, or NH, and X2is absent, =0, or =S, and each R1is independently -OH, -N(R2)2, or -O-CH2CH2CN, where each R2is independently an optionally substituted alkyl, or both R2groups, together with the nitrogen atom to which they are attached, combine to form an optionally substituted heterocyclyl.

[0387] Representative patent documents that teach the structure and / or preparation of the antisense oligonucleotides, such as PMOs, of the present invention include, but are not limited to, International PCT Publication No. WO 2022192749; International PCT Publication No. WO 2022192754; and US Patent Publication No. 2021 / 0299264, the entire contents of each of which are hereby incorporated herein by reference.

[0388] III. Cell Penetrating Peptides of the Invention

[0389] In one aspect, the present invention provides antisense oligonucleotide conjugates, or pharmaceutically acceptable salts thereof, for inhibiting expression of GYSI, comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide, wherein the cell penetrating peptide comprises at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, wherein the cell penetrating peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a beta-alanine residue in combination with arginine and / or histidine residues.

[0390] Preferably, peptides included in the conjugates described herein include no artificial amino acid residues.

[0391] In some embodiments, the cell penetrating peptide does not contain aminohexanoic acid residues. In some embodiments, the cell penetrating peptide does not contain any formAttorney Docket: 140101-00220

[0392] of aminohexanoic acid residues. In some embodiments, the cell penetrating peptide does not contain 6-aminohexanoic acid residues.

[0393] In some embodiments, the cell penetrating peptide contains only natural amino acid residues, and therefore consists of natural amino acid residues.

[0394] In some embodiments, artificial amino acids such as 6-aminohexanoic acid that are typically used in cell-penetrating peptides are replaced by natural amino acids. In some embodiments, the artificial amino acids such as 6-aminohexanoic acid that are typically used in cell penetrating peptides are replaced by amino acids selected from beta-alanine, serine, proline, arginine and histidine or hydroxyproline.

[0395] In some embodiments, aminohexanoic acid is replaced by beta-alanine. In some embodiments, 6-aminohexanoic acid is replaced by beta-alanine

[0396] In some embodiments, aminohexanoic acid is replaced by histidine. In some embodiments, 6- aminohexanoic acid is replaced by histidine.

[0397] In some embodiments, aminohexanoic acid is replaced by hydroxyproline. In some embodiments, 6-aminohexanoic acid is replaced by hydroxyproline.

[0398] In some embodiments, the artificial amino acids such as 6-aminohexanoic acid that are typically used in cell penetrating peptides may be replaced by a combination of any of beta-alanine, serine, proline, arginine and histidine or hydroxyproline, e.g., a combination of any of beta-alanine, histidine, and hydroxyproline.

[0399] In some embodiments, the present invention provides a cell penetrating peptide having a total length of 40 amino acid residues or less, the cell penetrating peptide comprising: two or more cationic domains each comprising at least 4 amino acid residues; and one or more hydrophobic domains each comprising at least 3 amino acid residues; wherein at least one cationic domain comprises histidine residues. In some embodiments, wherein at least one cationic domain is histidine rich.

[0400] In some embodiments, what is meant by histidine rich is defined herein in relation to the cationic domains.

[0401] Cationic Domain

[0402] The present invention relates to short cell penetrating peptides having a particular structure in which there are at least two cationic domains having a certain length.

[0403] In some embodiments, the cell penetrating peptide comprises up to 4 cationic domains, up to 3 cationic domains.

[0404] In some embodiments, the cell penetrating peptide comprises 2 cationic domains. As defined above, the cell penetrating peptide comprises two or more cationic domains each having a length of at least 4 amino acid residues.

[0405] In some embodiments, each cationic domain has a length of between 4 to 12 amino acid residues, e.g., a length of between 4 to 7 amino acid residues.Attorney Docket: 140101-00220

[0406] In some embodiments, each cationic domain has a length of 4, 5, 6, or 7 amino acid residues.

[0407] In some embodiments, each cationic domain is of similar length, e.g., each cationic domain is the same length.

[0408] In some embodiments, each cationic domain comprises cationic amino acids and may also contain polar and or nonpolar amino acids.

[0409] Non-polar amino acids may be selected from: alanine, beta-alanine, proline, glycine, cysteine, valine, leucine, isoleucine, methionine, tryptophan, phenylalanine. In some embodiments, non-polar amino acids do not have a charge.

[0410] Polar amino acids may be selected from: serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine. In some embodiments, the selected polar amino acids do not have a negative charge.

[0411] Cationic amino acids may be selected from: arginine, histidine, and lysine. In some embodiments, cationic amino acids have a positive charge at physiological pH.

[0412] In some embodiments, each cationic domain does not comprise anionic or negatively charged amino acid residues. In some embodiments, each cationic domain comprises arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues.

[0413] In some embodiments, each cationic domain comprises or consists of arginine, histidine, beta-alanine, hydroxyproline, and / or serine residues.

[0414] In some embodiments, each cationic domain comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% cationic amino acids.

[0415] In some embodiments, each cationic domain comprises a majority of cationic amino acids. In some embodiments, each cationic domain comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% cationic amino acids. In some embodiments, each cationic domain comprises an isoelectric point (pi) of at least 7.5, at least 8.0, at least 8.5, at least 9.0, at least 9.5, at least 10.0, at least 10.5, at least 11.0, at least 11.5, or at least 12.0.

[0416] In some embodiments, each cationic domain comprises an isoelectric point (pi) of at least 10.0.

[0417] In some embodiments, each cationic domain comprises an isoelectric point (pi) of between 10.0 and 13.0

[0418] In some embodiments, each cationic domain comprises an isoelectric point (pi) of between 10.4 and 12.5.

[0419] In some embodiments, the isoelectric point of a cationic domain is calculated at physiological pH by any suitable means available in the art, such as by using the I PC (isoelectric.org) a web-based algorithm developed by Lukasz Kozlowski, Biol Direct. 2016; 11:55; the entire contents of which are incorporated herein by reference.Attorney Docket: 140101-00220

[0420] In some embodiments, each cationic domain comprises at least 1 cationic amino acid, e.g., 1-5 cationic amino acids. In some embodiments, each cationic domain comprises at least 2 cationic amino acids, e.g., 2-5 cationic amino acids.

[0421] In some embodiments, each cationic domain is arginine rich and / or histidine rich. In some embodiments, a cationic domain may contain both histidine and arginine.

[0422] In some embodiments, each cationic domain comprises a majority of arginine and / or histidine residues.

[0423] In some embodiments, each cationic domain comprises at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues. In some embodiments, a cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine residues.

[0424] In some embodiments, a cationic domain may comprise at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% histidine residues.

[0425] In some embodiments, a cationic domain may comprise a total of between 1-5 histidine and 1-5 arginine residues. In some embodiments, a cationic domain may comprise between 1-5 arginine residues. In some embodiments, a cationic domain may comprise between 1-5 histidine residues. In some embodiments, a cationic domain may comprise a total of between 2-5 histidine and 3-5 arginine residues. In some embodiments, a cationic domain may comprise between 3-5 arginine residues. In some embodiments, a cationic domain may comprise between 2-5 histidine residues.

[0426] In some embodiments, each cationic domain comprises one or more beta-alanine residues. In some embodiments, each cationic domain may comprise a total of between 2-5 beta-alanine residues, e.g., a total of 2 or 3 beta-alanine residues.

[0427] In some embodiments, a cationic domain may comprise one or more hydroxyproline residues or serine residues.

[0428] In some embodiments, a cationic domain may comprise between 1-2 hydroxyproline residues. In some embodiments, a cationic domain may comprise between 1-2 serine residues.

[0429] In some embodiments, all of the cationic amino acids in a given cationic domain may be histidine, alternatively, e.g., all of the cationic amino acids in a given cationic domain may be arginine.

[0430] In some embodiments, the cell penetrating peptide may comprise at least one histidine rich cationic domain. In some embodiments, the cell penetrating peptide may comprise at least one arginine rich cationic domain. In some embodiments, the cell penetrating peptide may comprise at least one arginine rich cationic domain and at least one histidine rich cationic domain.Attorney Docket: 140101-00220

[0431] In some embodiments, the cell penetrating peptide comprises two arginine rich cationic domains.

[0432] In some embodiments, the cell penetrating peptide comprises two histidine rich cationic domains.

[0433] In some embodiments, the cell penetrating peptide comprises two arginine and histidine rich cationic domains.

[0434] In some embodiments, the cell penetrating peptide comprises one arginine rich cationic domain and one histidine rich cationic domain. In some embodiments, each cationic domain comprises no more than 3 contiguous arginine residues, e.g., no more than 2 contiguous arginine residues.

[0435] In some embodiments, each cationic domain comprises no contiguous histidine residues.

[0436] In some embodiments, each cationic domain comprises arginine, histidine and / or beta-alanine residues. In some embodiments, each cationic domain comprises a majority of arginine, histidine and / or beta-alanine residues. In some embodiments, each cationic domain comprises at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 60%, at least 65%, or at least 70% arginine and / or histidine residues. In some embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the amino acid residues in each cationic domain are arginine, histidine and / or beta-alanine residues. In some embodiments, each cationic domain comprises or consists of arginine, histidine and / or beta-alanine residues.

[0437] In some embodiments, the cell penetrating peptide comprises a first cationic domain comprising arginine and beta-alanine residues and a second cationic domain comprising arginine and beta-alanine residues.

[0438] In some embodiments, the cell penetrating peptide comprises a first cationic domain comprising arginine and beta-alanine resides, and a second cationic domain comprising histidine, beta-alanine, and optionally arginine residues.

[0439] In some embodiments, the cell penetrating peptide comprises a first cationic domain comprising arginine and beta-alanine resides, and a second cationic domain comprising histidine and beta-alanine residues.

[0440] In some embodiments, the cell penetrating peptide comprises a first cationic domain consisting of arginine and beta-alanine residues and a second cationic domain consisting of arginine and beta-alanine residues.

[0441] In some embodiments, the cell penetrating peptide comprises a first cationic domain consisting of arginine and beta-alanine residues and a second cationic domain consisting of arginine, histidine and beta- alanine residues.

[0442] In some embodiments, the cell penetrating peptide comprises at least two cationic domains, e.g., these cationic domains form the arms of the cell penetrating peptide. In someAttorney Docket: 140101-00220

[0443] embodiments, the cationic domains are located at the N and C terminus of the cell penetrating peptide. In some embodiments, therefore, the cationic domains may be known as the cationic arm domains.

[0444] In some embodiments, the cell penetrating peptide comprises two cationic domains, wherein one is located at the N-terminus of the cell penetrating peptide and one is located at the C-terminus of the cell penetrating peptide. In some embodiments, at either end of the cell penetrating peptide. In some embodiments, no further amino acids or domains are present at the N- terminus and C-terminus of the cell penetrating peptide, with the exception of other groups such as a terminal modification, linker and / or oligonucleotide. For the avoidance of doubt, such other groups may be present in addition to ‘the cell penetrating peptide’ described and claimed herein. In some embodiments, therefore each cationic domain forms the terminus of the cell penetrating peptide. In some embodiments, this does not preclude the presence of a further linker group as described herein.

[0445] In some embodiments, the cell penetrating peptide may comprise up to 4 cationic domains. In some embodiments, the cell penetrating peptide comprises two cationic domains.

[0446] In some embodiments, the cell penetrating peptide comprises two cationic domains that are both arginine rich. In some embodiments, the cell penetrating peptide comprises one cationic domain that is arginine rich.

[0447] In some embodiments, the cell penetrating peptide comprises two cationic domains that are both arginine and histidine rich.

[0448] In some embodiments, the cell penetrating peptide comprises one cationic domain that is arginine rich and one cationic domain that is histidine rich.

[0449] In some embodiments, the cationic domains comprise amino acid units selected from the following: R, H, B, RR, HH, BB, RH, HR, RB, BR, HB, BH, RBR, RBB, BRR, BBR, BRB, RBH, RHB, HRB, BRH, HRR, RRH, HRH, HBB, BBH, RHR, BHB, HBH, or any combination thereof.

[0450] In some embodiments, a cationic domain may also include serine, proline and / or hydroxyproline residues. In some embodiments, the cationic domains may further comprise amino acid units selected from the following: RP, PR, RPR, RRP, PRR, PRP, Hyp; R[Hyp]R, RR[Hyp], [Hyp]RR, [Hyp]R[Hyp], [Hyp][Hyp]R, R[Hyp][Hyp], SB, BS, or any combination thereof, or any combination with the above listed amino acid units.

[0451] In some embodiments, each cationic domain comprises any one of the following sequences: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16),Attorney Docket: 140101-00220

[0452] BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19), or any combination thereof.

[0453] In some embodiments, each cationic domain comprises or consists of any one of the following sequences: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B, R[Hyp]H[Hyp]HB, R[Hyp]RR[Hyp]R (SEQ ID NO: 19), or any combination thereof.

[0454] In some embodiments, each cationic domain comprises or consists of one of the following sequences: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRRBR (SEQ ID NO: 4), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), or HBHBR (SEQ ID NO: 9).

[0455] In some embodiments, each cationic domain in the cell penetrating peptide may be identical or different. In some embodiments, each cationic domain in the cell penetrating peptide is different.

[0456] Hydrophobic Domain

[0457] The present invention relates to short cell penetrating peptides having a particular structure in which there is at least one hydrophobic domain having a certain length.

[0458] References to ‘hydrophobic’ herein denote an amino acid or domain of amino acids having the ability to repel water or which do not mix with water.

[0459] In some embodiments, the cell penetrating peptide comprises up to 3 hydrophobic domains or up to 2 hydrophobic domains. In some embodiments, the cell penetrating peptide comprises 1 hydrophobic domain.

[0460] As defined above, the cell penetrating peptide comprises one or more hydrophobic domains each having a length of at least 3 amino acid residues. In some embodiments, each hydrophobic domain has a length of between 3-6 amino acids. In some embodiments, each hydrophobic domain has a length of 5 amino acids.

[0461] In some embodiments, each hydrophobic domain may comprise nonpolar, polar, and hydrophobic amino acid residues.

[0462] Hydrophobic amino acid residues may be selected from: alanine, valine, leucine, isoleucine, phenylalanine, tyrosine, methionine, and tryptophan.

[0463] Non-polar amino acid residues may be selected from: proline, glycine, cysteine, alanine, valine, leucine, isoleucine, tryptophan, phenylalanine, and methionine.

[0464] Polar amino acid residues may be selected from: serine, asparagine, hydroxyproline, histidine, arginine, threonine, tyrosine, and glutamine.Attorney Docket: 140101-00220

[0465] In some embodiments, the hydrophobic domains do not comprise hydrophilic amino acid residues.

[0466] In some embodiments, each hydrophobic domain comprises a majority of hydrophobic amino acid residues. In some embodiments, each hydrophobic domain comprises at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% hydrophobic amino acids. In some embodiments, each hydrophobic domain consists of hydrophobic amino acid residues.

[0467] In some embodiments, each hydrophobic domain comprises a hydrophobicity of at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.8, at least 1.0, at least 1.1, at least 1.2, or at least 1.3.

[0468] In some embodiments, each hydrophobic domain comprises a hydrophobicity of at least 0.3, at least 0.35, at least 0.4, or at least 0.45.

[0469] In some embodiments, each hydrophobic domain comprises a hydrophobicity of at least 1.2, at least 1.25, at least 1.3, or at least 1.35.

[0470] In some embodiments, each hydrophobic domain comprises a hydrophobicity of between 0.4 and 1.4

[0471] In some embodiments, each hydrophobic domain comprises of a hydrophobicity of between 0.45 and 0.48.

[0472] In some embodiments, each hydrophobic domain comprises a hydrophobicity of between 1.27 and 1.39

[0473] In some embodiments, hydrophobicity is as measured by White and Wimley: W. C. Wimley and S. H. White, " Experimentally determined hydrophobicity scale for proteins at membrane interfaces" Nature Struct Biol 3:842 (1996); the entire contents of which are incorporated herein by reference.

[0474] In some embodiments, each hydrophobic domain comprises at least 3 or at least 4 hydrophobic amino acid residues.

[0475] In some embodiments, each hydrophobic domain comprises phenylalanine, leucine, Isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues. In some embodiments, each hydrophobic domain comprises or consists of phenylalanine, leucine, isoleucine, tyrosine, tryptophan, proline, and / or glutamine residues.

[0476] In some embodiments, each hydrophobic domain comprises or consists of phenylalanine, leucine, isoleucine, tyrosine and / or glutamine residues.

[0477] In some embodiments, each hydrophobic domain comprises or consists of tryptophan and / or proline residues. In some embodiments, the cell penetrating peptide comprises one hydrophobic domain. In some embodiments, the or each hydrophobic domain is located in the center of the cell penetrating peptide. In some embodiments, therefore, the hydrophobic domain may be known as a core hydrophobic domain. In some embodiments, the or each hydrophobic core domain is flanked on either side by an arm domain. In some embodiments,Attorney Docket: 140101-00220

[0478] the arm domains may comprise one or more cationic domains and one or more further hydrophobic domains. In some embodiments, each arm domain comprises a cationic domain.

[0479] In some embodiments, the cell penetrating peptide comprises two arm domains flanking a hydrophobic core domain, wherein each arm domain comprises a cationic domain.

[0480] In some embodiments, the cell penetrating peptide comprises or consists of two cationic arm domains flanking a hydrophobic core domain.

[0481] In some embodiments, the or each hydrophobic domain comprises one of the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26), or any combination thereof.

[0482] In some embodiments, the or each hydrophobic domain comprises or consists of one of the following sequences: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26), or any combination thereof.

[0483] In some embodiments, the or each hydrophobic domain comprises or consists of one of the following sequences FQILY (SEQ ID NO: 21), YQFLI (SEQ ID NO: 20), or ILFQY (SEQ ID NO: 22).

[0484] In some embodiments, the or each hydrophobic domain comprises or consists of FQILY (SEQ ID NO: 21).

[0485] In some embodiments, each hydrophobic domain in the cell penetrating peptide may have the same sequence or a different sequence.

[0486] The present invention relates to short cell penetrating peptides for use in transporting therapeutic molecules in the treatment of medical conditions.

[0487] The cell penetrating peptide has a sequence that is a contiguous single molecule, therefore the domains of the cell penetrating peptide are contiguous. In some embodiments, the cell penetrating peptide comprises several domains in a linear arrangement between the N-terminus and the C-terminus. In some embodiments, the domains are selected from cationic domains and hydrophobic domains described above. In some embodiments, the cell penetrating peptide comprises or consists of cationic domains and hydrophobic domains wherein the domains are as defined above.

[0488] Each domain has common sequence characteristics as described in the relevant sections above, but the exact sequence of each domain is capable of variation and modification. Thus, a range of sequences is possible for each domain. The combination of each possible domain sequence yields a range of peptide structures, each of which form part of the present invention. Features of the cell penetrating peptide structures are described below.Attorney Docket: 140101-00220

[0489] In some embodiments, a hydrophobic domain separates any two cationic domains. In some embodiments, each hydrophobic domain is flanked by cationic domains on either side thereof.

[0490] In some embodiments, no cationic domain is contiguous with another cationic domain.

[0491] In some embodiments, the cell penetrating peptide comprises one hydrophobic domain flanked by two cationic domains in the following arrangement:

[0492] [cationic domain] - [hydrophobic domain] - [cationic domain]

[0493] In some embodiments, the hydrophobic domain may be known as the core domain and each of the cationic domains may be known as an arm domain. In some embodiments, the hydrophobic arm domains flank the cationic core domain on either side thereof.

[0494] In some embodiments, the cell penetrating peptide comprises or consists of two cationic domains and one hydrophobic domain.

[0495] In some embodiments, the cell penetrating peptide comprises or consists of one hydrophobic core domain flanked by two cationic arm domains.

[0496] In some embodiments, the cell penetrating peptide comprises or consists of one hydrophobic core domain comprising a sequence selected from: YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26), flanked by two cationic arm domains each comprising a sequence selected from: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19).

[0497] In some embodiments, the cell penetrating peptide comprises or consists of one hydrophobic core domain comprising a sequence selected from: FQILY (SEQ ID NO: 21), YQFLI (SEQ ID NO: 20), and ILFQY (SEQ ID NO: 22), flanked by two cationic arm domains comprising a sequence selected from: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRRBR (SEQ ID NO: 4), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), and HBHBR (SEQ ID NO: 9). In some embodiments, the cell penetrating peptide comprises or consists of one hydrophobic core domain comprising the sequence: FQILY (SEQ ID NO: 21), flanked by two cationic arm domains comprising a sequence selected from: RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRRBR (SEQ ID NO: 4), BRBR (SEQ ID NO: 7), and RBHBH (SEQ ID NO: 8).

[0498] In any such embodiment, further groups may be present such as a linker, terminal modification and / or oligonucleotide.Attorney Docket: 140101-00220

[0499] In some embodiments, the cell penetrating peptide is N-terminally modified.

[0500] In some embodiments, the cell penetrating peptide is N-acetylated, N-methylated, N-trifluoroacetylated, N- trifluoromethylsulfonylated, or N-methylsulfonylated. In some embodiments, the cell penetrating peptide is N-acetylated.

[0501] Optionally, the N-terminus of the cell penetrating peptide may be unmodified.

[0502] In some embodiments, the cell penetrating peptide is N-acetylated.

[0503] In some embodiments, the cell penetrating peptide is C-terminal modified.

[0504] In some embodiments, the cell penetrating peptide comprises a C-terminal modification selected from: carboxy-, thioacid-, aminooxy-, hydrazino-, thioester-, azide, strained alkyne, strained alkene, aldehyde-, thiol or haloacetyl -group.

[0505] Advantageously, the C-terminal modification provides a means for linkage of the cell penetrating peptide to the oligonucleotide.

[0506] Accordingly, the C-terminal modification may comprise the linker and vice versa. In some embodiments, the C-terminal modification may consist of the linker or vice versa. Suitable linkers are described herein elsewhere. In some embodiments, the cell penetrating peptide comprises a C-terminal carboxyl group.

[0507] In some embodiments, the C-terminal carboxyl group is provided by a glycine or beta-alanine residue.

[0508] In some embodiments, the C terminal carboxyl group is provided by a beta-alanine residue. In some embodiments, the C terminal beta-alanine residue is a linker.

[0509] In some embodiments, therefore each cationic domain may further comprise an N or C terminal modification. In some embodiments, the cationic domain at the C terminus comprises a C-terminal modification. In some embodiments, the cationic domain at the N terminus comprises aN-terminal modification. In some embodiments, the cationic domain at the C terminus comprises a linker group, In some embodiments, the cationic domain at the C terminus comprises a C-terminal beta-alanine. In some embodiments, the cationic domain at the N terminus is N-acetylated.

[0510] The cell penetrating peptide of the present invention is defined as having a total length of 40 amino acid residues or less. The cell penetrating peptide may therefore be regarded as an oligopeptide.

[0511] In some embodiments, the cell penetrating peptide has a total length of 3-30 amino acid residues, e.g., of 5-25 amino acid residues, of 10-25 amino acid residues, of 13-23 amino acid residues, or of 15-20 amino acid residues.

[0512] In some embodiments, the cell penetrating peptide has a total length of at least 12, at least 13, at least 14, at least 15, at least 16, or at least 17 amino acid residues.

[0513] In some embodiments, the cell penetrating peptide is capable of penetrating cells. The cell penetrating peptide may therefore be regarded as a cell penetrating peptide.Attorney Docket: 140101-00220

[0514] In some embodiments, the cell penetrating peptide is for attachment to an oligonucleotide. In some embodiments, the cell penetrating peptide is for transporting an oligonucleotide into a target cell. In some embodiments, the cell penetrating peptide is for delivering an oligonucleotide into a target cell. The cell penetrating peptide may therefore be regarded as a cell penetrating peptide.

[0515] In some embodiments, the cell penetrating peptide is capable of penetrating into cells and tissues, e.g., into the nucleus of cells. In some embodiments, into muscle tissues.

[0516] In some embodiments, the cell penetrating peptide may comprise or consist of a cell penetrating peptide selected from any one of the following sequences:

[0517] RBRRBRRFQILYRBRBR (SEQ ID NO: 27)

[0518] RBRRBRRFQILYRBRR (SEQ ID NO: 28)

[0519] RBRRBRFQILYRRBRBR (SEQ ID NO: 29)

[0520] RBRBRFQILYRBRRBRR (SEQ ID NO: 30)

[0521] RBRRBRRYQFLIRBRBR (SEQ ID NO: 31)

[0522] RBRRBRRI LFQYRBRBR (SEQ ID NO: 32)

[0523] RBRRBRFQILYRBRBR (SEQ ID NO: 33)

[0524] RBRRBFQILYRBRRBR (SEQ ID NO: 34)

[0525] RBRRBRFQILYBRBR (SEQ ID NO: 35)

[0526] RBRRBFQILYRBRBR (SEQ ID NO: 36)

[0527] RBRRBRRFQILYRBHBH (SEQ ID NO: 37)

[0528] RBRRBRRFQILYHBHBR (SEQ ID NO: 38)

[0529] RBRRBRRFQILYHBRBH (SEQ ID NO: 39)

[0530] RBRRBRRYQFLIRBHBH (SEQ ID NO: 40)

[0531] RBRRBRRILFQYRBHBH (SEQ ID NO: 41)

[0532] RBRHBHRFQILYRBRBR (SEQ ID NO: 42)

[0533] RBRBBHRFQILYRBHBH (SEQ ID NO: 43)

[0534] RBRRBRFQILYRBHBH (SEQ ID NO: 44)

[0535] RBRRBRFQILYHBHBH (SEQ ID NO: 45)

[0536] RBRRBHFQILYRBHBH (SEQ ID NO: 46)

[0537] HBRRBRFQILYRBHBH (SEQ ID NO: 47)

[0538] RBRRBFQILYRBHBH (SEQ ID NO: 48)

[0539] RBRRBRFQILYBHBH (SEQ ID NO: 49)

[0540] RBRRBRYQFLIHBHBH (SEQ ID NO: 50)

[0541] RBRRBRILFQYHBHBH (SEQ ID NO: 51)

[0542] RBRRBRRFQILYHBHBH (SEQ ID NO: 52)

[0543] In some embodiments, the cell penetrating peptide may comprise or consist of a cell penetrating peptide selected from any one of the following additional sequences:

[0544] RBRRBRFQILYBRBS (SEQ ID NO: 53)Attorney Docket: 140101-00220

[0545] RBRRBRFQILYBRBR[Hyp] (SEQ ID NO: 54) RBRRBRFQILYBR[Hyp]R (SEQ ID NO: 55) RRBRRBRFQILYBRBR (SEQ ID NO: 56) BRRBRRFQILYBRBR (SEQ ID NO: 57) RBRRBRWWWBRBR (SEQ ID NO: 58) RBRRBRWWPWWBRBR (SEQ ID NO: 59) RBRRBRWPWWBRBR (SEQ ID NO: 60) RBRRBRWWPWBRBR (SEQ ID NO: 61) RBRRBRRWWWRBRBR (SEQ ID NO: 62) RBRRBRRWWPWWRBRBR (SEQ ID NO: 63) RBRRBRRWPWWRBRBR (SEQ ID NO: 64) RBRRBRRWWPWRBRBR (SEQ ID NO: 65) RBRRBRRFQILYBRBR (SEQ ID NO: 66) RBRRBRRFQILYRBR (SEQ ID NO: 67) BRBRBWWPWWRBRRBR (SEQ ID NO: 68) RBRRBRRFQILYBHBH (SEQ ID NO: 69) RBRRBRRFQIYRBHBH (SEQ ID NO: 70) RBRRBRFQILYBRBH (SEQ ID NO: 71) RBRRBRFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 72) R[Hyp]RR[Hyp]RFQILYRBHBH (SEQ ID NO: 73) R[Hyp]RR[Hyp]RFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 74) RBRRBRWWWRBHBH (SEQ ID NO: 75) RBRRBRWWPRBHBH (SEQ ID NO: 76) RBRRBRPWWRBHBH (SEQ ID NO: 77) RBRRBRWWPWWRBHBH (SEQ ID NO: 78) RBRRBRWWPWRBHBH (SEQ ID NO: 79) RBRRBRWPWWRBHBH (SEQ ID NO: 80) RBRRBRRWWWRBHBH (SEQ ID NO: 81) RBRRBRRWWPWWRBHBH (SEQ ID NO: 82) RBRRBRRWPWWRBHBH (SEQ ID NO: 83) RBRRBRRWWPWRBHBH (SEQ ID NO: 84) RRBRRBRFQILYRBHBH (SEQ ID NO: 85) BRRBRRFQILYRBHBH (SEQ ID NO: 86) RRBRRBRFQILYBHBH (SEQ ID NO: 87) BRRBRRFQILYBHBH (SEQ ID NO: 88) RBRRBHRFQILYRBHBH (SEQ ID NO: 89) RBRRBRFQILY[Hyp]R[Hyp]R (SEQ ID NO: 101) R[Hyp]RR[Hyp]RFQILYBRBR (SEQ ID NO: 102)Attorney Docket: 140101-00220

[0546] R[Hyp]RR[Hyp]RFQILY[Hyp]R[Hyp]R (SEQ ID NO: 103) RBRRBRWWWBRBR (SEQ ID NO: 104)

[0547] RBRRBRWWPWWBRBR (SEQ ID NO: 105)

[0548] In some embodiments, the cell penetrating peptide may comprise or consist of a cell penetrating peptide selected from one of the following sequences:

[0549] RBRRBRRFQILYRBRBR (SEQ ID NO: 27)

[0550] RBRRBRRYQFLIRBRBR (SEQ ID NO: 31)

[0551] RBRRBRRI LFQYRBRBR (SEQ ID NO: 32)

[0552] RBRRBRFQILYBRBR (SEQ ID NO: 35)

[0553] RBRRBRRFQILYRBHBH (SEQ ID NO: 37)

[0554] RBRRBRRFQILYHBHBR (SEQ ID NO: 38)

[0555] RBRRBRFQILYRBHBH (SEQ ID NO: 44)

[0556] In some embodiments, the cell penetrating peptide comprises or consists of the following sequence: RBRRBRFQILYBRBR (SEQ ID NO: 35).

[0557] In some embodiments, the cell penetrating peptide comprises or consists of the following sequence: RBRRBRRFQILYRBHBH (SEQ ID NO: 37).

[0558] In some embodiments, the cell penetrating peptide comprises or consists of the following sequence: RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0559] Further peptide designs suitable for use in the conjugates, compositions, and methods of the invention are disclosed in, for example, U. S. Patent Nos. 7,687,617 and 8,580,756; U. S. Patent Publication Nos. 20060128646, 20090209748, 20140128586, 20140128591, 20100210712, and 20080015162A1; and International Publication No. WO 2013 / 159108, the entire contents of each of which are incorporated herein by reference. Further, cell penetrating peptides that may be used in the conjugates described herein also include those disclosed in International Application No. PCT / US2023 / 080778, and International Publication Nos. WO 2016 / 08784, WO 2018 / 150196, WO 2020 / 030927, WO 2020 / 030928, WO 2020 / 115494, and WO 2022 / 172019; the entire contents of each of which are incorporated herein by reference.

[0560] Peptides of the invention may be produced by any standard protein synthesis method, for example chemical synthesis, semi-chemical synthesis or through the use of expression systems. Accordingly, the present invention also relates to the nucleotide sequences comprising or consisting of the DNA coding for the cell penetrating peptides, expression systems, e.g., vectors comprising said sequences accompanied by the necessary sequences for expression and control of expression, and host cells and host organisms transformed by said expression systems.

[0561] Accordingly, a nucleic acid encoding a cell penetrating peptide according to the present invention is also provided. In some embodiments, the nucleic acids may be provided in isolated or purified form.Attorney Docket: 140101-00220

[0562] An expression vector comprising a nucleic acid encoding a cell penetrating peptide according to the present invention is also provided. In some embodiments, the vector is a plasmid. In some embodiments, the vector comprises a regulatory sequence, e.g. promoter, operably linked to a nucleic acid encoding a cell penetrating peptide according to the present invention. In some embodiments, the expression vector is capable of expressing the cell penetrating peptide when transfected into a suitable cell, e.g., mammalian, bacterial, or fungal cell.

[0563] A host cell comprising the expression vector of the invention is also provided.

[0564] Expression vectors may be selected depending on the host cell into which the nucleic acids of the invention may be inserted. Such transformation of the host cell involves conventional techniques such as those taught in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, USA, 2001; the entire contents of which are incorporated herein by reference. Selection of suitable vectors is within the skills of the person knowledgeable in the field. Suitable vectors include plasmids, bacteriophages, cosmids, and viruses.

[0565] The cell penetrating peptides produced may be isolated and purified from the host cell by any suitable method, e.g., precipitation or chromatographic separation, e.g., affinity chromatography. Suitable vectors, hosts, and recombinant techniques are well known in the art.

[0566] IV. Linkers for Use in the Antisense Oligonucleotide Conjugates of the Invention

[0567] In another aspect, the present invention provides an antisense oligonucleotide conjugate, or pharmaceutically acceptable salts thereof, for inhibiting expression of GYSI, comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide, wherein the antisense oligonucleotide is conjugated to the cell penetrating peptide via a linker.

[0568] The conjugates of the present invention may include a linker covalently linking a cell penetrating peptide described herein to an oligonucleotide described herein.

[0569] Suitable linkers include, for example, a C-terminal cysteine residue that permits formation of a disulfide, thioether or thiol-maleimide linkage, a C-terminal aldehyde to form an oxime, a click reaction or formation of a morpholino linkage with a basic amino acid on the cell penetrating peptide or a carboxylic acid moiety on the cell penetrating peptide covalently conjugated to an amino group to form a carboxamide linkage.

[0570] In some embodiments, the linker is of formula (I):

[0571] T1-(CR1R2)n-T2.

[0572] (I)

[0573] whereinAttorney Docket: 140101-00220

[0574] T1is a divalent group for attachment to the cell penetrating peptide and is selected from the group consisting of -NH- and carbonyl;

[0575] T2 is a divalent group for attachment to an oligonucleotide and is selected from the group consisting of -NH- and carbonyl;

[0576] n is 1, 2 or 3;

[0577] each R1is independently -Y1-X1-Z1,

[0578] wherein

[0579] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and RA2are each independently hydrogen, OH, or (1-2C)alkyl;

[0580] X1is absent, -O-, -C(O)-, -O(O)O-, -OC(O)-, -CH(0RA3)-, -N(RA3)-, -N(RA3)- C(O)-, -N(RA3)-C(0)0-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(NRA3)N(RA3)-, -SO-, -S-, -SO2-, -S(O)2N(RA3)-, or -N(RA3)SO2-, wherein each RA3is independently selected from the group consisting of hydrogen and methyl; and

[0581] Z1is a further oligonucleotide or is hydrogen, (l-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl,

[0582] wherein each (1 -6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted with one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRA4RA5, and (1-4C)alkoxy, wherein RA4and RA5are each independently selected from the group consisting of hydrogen and (1-4C)alkyl; and each R2is independently -Y2-X2-Z2, wherein

[0583] Y2is absent or a group of the formula -[CRB1RB2]m- in which m is an integer selected from 1, 2, 3 or 4, and RB1and RB2are each independently selected from hydrogen, OH or (1 -2C)alkyl;

[0584] X2is absent, -O-, -C(O)-, -O(O)O-, -OC(O)-, -CH(0RB3)-, -N(RB3)-, -N(RB3)- C(O)-, - N(RB3)-C(0)0-, -C(0)-N(RB3)-, -N(RB3)C(O)N(RB3)-, -N(RB3)C(NRB3)N(RB3)-, -SO-, -S- -SO2-, - S(O)2N(RB3)-, or -N(RB3)SO2-, wherein each RB3is independently selected from hydrogen or methyl; and

[0585] Z2is selected from hydrogen, (1 -6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl, wherein each (1 -6C)alkyl, (2- 6C)alkenyl, (2- 6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl or heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRB4RB5, and (1-4C)alkoxy, wherein RB4and RB5are each independently hydrogen or (l-2C)alkyl; with theAttorney Docket: 140101-00220

[0586] proviso that; when n=l and Ti and T2 are different to one another, then R1and R2are not both H; when n=l, Ti and T2 are different to one another and one of R1and R2is H then the other of R1and R2is not methyl; or when n=2 and each occurrence of R1and R2is H, then Ti and T2 are both -C(O)- or are both -NH-.

[0587] In some embodiments, T2 is -C(O)-.

[0588] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0589] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and RA2are each hydrogen or (1- 2C)alkyl;

[0590] X1is absent, -O-, -C(O)-, -O(O)O-, -N(RA3)-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(N RA3)N(RA3)- or -S-, wherein each RA3is independently hydrogen or methyl; and

[0591] Z1is a further oligonucleotide or is hydrogen, (l-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3- 6C)cycloalkyl, (3-6C)cycloalkenyl, or heteroaryl, wherein each (1-6C)alkyl, (2-6C)alkenyl, (2-6C)alkynyl, aryl, (3-6C)cycloalkyl, (3-6C)cycloalkenyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, oxo, halo, cyano, nitro, hydroxy, carboxy, NRA4RA5, and (l-4C)alkoxy, wherein RA4and RA5are each independently hydrogen or (1-2C)alkyl.

[0592] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0593] Y1is absent or -(CRA1RA2)m-, wherein m is 1, 2, 3, or 4, and RA1and RA2are each independently hydrogen or (1-2C)alkyl;

[0594] X1is absent, -O-, -C(O)-, -C(O)O-, -N(RA3)-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, -N(RA3)C(O)N(RA3)-, -N(RA3)C(NRA3)N(RA3)-, or -S-, wherein each RA3is independently hydrogen or methyl; and

[0595] Z1is a further oligonucleotide or is hydrogen, (1-6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (1-6C)alkyl, aryl, (3-6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.

[0596] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0597] Y1is absent or a group of the formula -(CRA1RA2)m-, wherein m is 1, 2, 3 or 4, and RA1and

[0598] RA2are each independently hydrogen or (l-2C)alkyl;

[0599] X1is absent, -C(O)-, -C(O)O-, -N(RA3)-C(0)-, -C(0)-N(RA3)-, wherein each RA3is hydrogen or methyl; andAttorney Docket: 140101-00220

[0600] Z1is a further oligonucleotide or is hydrogen, (1 - 6C)alkyl, aryl, (3-6C)cycloalkyl, or heteroaryl, wherein each (l-6C)alkyl, aryl, (3- 6C)cycloalkyl, and heteroaryl is optionally substituted by one or more (e.g., 1, 2, 3, 4, or 5) substituent groups selected from the group consisting of (1-4C) alkyl, halo, and hydroxy.

[0601] In some embodiments, each R1is independently -Y1-X1-Z1, wherein:

[0602] Y1is absent, -(CH2)-, or -(CH2CH2)-;

[0603] X1is absent, -N(RA3)-C(O)-, -C(O)-N(RA3)-, wherein each RA3is independently hydrogen or methyl; and

[0604] Z1is hydrogen or (1-2C)alkyl.

[0605] In some embodiments, each R2is independently -Y2-Z2, wherein Y2is absent or -(CRB1RB2)m-, wherein m is 1, 2, 3 or 4, and RB1and RB2are each independently hydrogen or (l-2C)alkyl; and Z2is hydrogen or (1-6C)alkyl.

[0606] In some embodiments, each R2is hydrogen.

[0607] In some embodiments, n is 2 or 3.

[0608] In some embodiments, n is 1.

[0609] In some embodiments, the linker is an amino acid residue selected from the group consisting of glutamic acid, succinic acid, and gamma-aminobutyric acid residues.

[0610] In some embodiments, the linker is:

[0611]

[0612] In some embodiments, the linker is:

[0613]

[0614] In some embodiments, the linker is:Attorney Docket: 140101-00220

[0615]

[0616] In some embodiments, the linker is:

[0617]

[0618] In some embodiments, the linker is:

[0619]

[0620] In some embodiments, the linker is:

[0621]

[0622] In some embodiments, the linker is:

[0623]

[0624] In some embodiments, the linker is between 1-5 amino acids in length. In some embodiments, the linker may comprise any linker that is known in the art. In some embodiments, the linker is selected from any of the following sequences: G, BC, XC, C, GGC, BBC, BXC, XBC, X, XX, B, BB, BX and XB. In some embodiments, wherein X is 6-aminohexanoic acid. In some embodiments the linker is a Glu linker.

[0625] In some embodiments, the linker may be a polymer, such as for example PEG.

[0626] In some embodiments, the linker is beta-alanine.

[0627] Additional linkers useful in the present invention can be found in WO 2020 / 115494, the entire contents of which are incorporated herein by reference.

[0628] V. Antisense Oligonucleotide Conjugates of the Invention

[0629] The conjugates of the present invention may comprise any therapeutic molecule that may be used for treatment of a disease, e.g., GSD. In some embodiments, the therapeuticAttorney Docket: 140101-00220

[0630] molecule is conjugated to one or more cell penetrating pepetides described herein. Such a therapeutic molecule may be selected from: a nucleic acid, a peptide nucleic acid, an antisense oligonucleotide (such as a PNA, or a PMO), a short interfering RNA, a micro RNA, an mRNA, a gRNA (for example in the use of CRISPR / Cas9 technology), an antagomiRNA, a peptide, a cyclic peptide, aprotein, a pharmaceutical, a drug, or a nanoparticle.

[0631] In preferred embodiments, the therapeutic molecule is an antisense oligonucleotide. In one aspect the present invention provides an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for inhibiting expression of GYSI, comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide.

[0632] In some embodiments, the conjugate comprises a cell penetrating peptide selected from one of the following sequences: RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), and RBRRBRFQILYRBHBH (SEQ ID NO: 44).

[0633] In some embodiments, the cell penetrating peptide may further comprise one or more N-terminal modifications as described herein.

[0634] In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its N-terminus. In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at its C-terminus. In some embodiments, the cell penetrating peptide is conjugated to one or more internal nucleotides of the antisense oligonucleotide. In some embodiments, the cell penetrating peptide is conjugated to the antisense oligonucleotide at a side-chain of the peptide or a side-chain of a linker attached to the peptide.

[0635] In some embodiments, the antisense oligonucleotide is conjugated to the cell penetrating peptide via a linker. In some embodiments, the antisense oligonucleotide is conjugated to the linker at the 3’ terminus of the antisense oligonucleotide. In alternative embodiments, the antisense oligonucleotide is conjugated to the linker at the 5' end of the antisense oligonucleotide.

[0636] In some embodiments, the conjugate comprises a structure selected from the group consisting of:

[0637] [cell penetrating peptide] — [oligonucleotide];

[0638] [oligonucleotide] — [cell penetrating peptide];

[0639] [cell penetrating peptide] — [linker] — [oligonucleotide]; and [oligonucleotide] — [linker] — [cell penetrating peptide].

[0640] In some embodiments, the conjugate is of the following structure:Attorney Docket: 140101-00220

[0641] [oligonucleotide]

[0642] [cell penetrating peptide!

[0643] ^-[oligonucleotide]

[0644]

[0645] [oligonucleotide]-^ _tl.......

[0646] [cell penetrating peptide]

[0647] [oligonucleotide]

[0648] or

[0649] [oligonucleotide]

[0650] [cell penetrating peptide] - [linker]

[0651] ^■’[oligonucleotide]

[0652] or

[0653] [linker] - [oligonucleotide]

[0654] [cell penetrating peptide]

[0655] ^[linker] - [oligonucleotide]

[0656] or

[0657] [oligonucleotide]*^

[0658] [linker] - [cell penetrating peptide] [oligonucleotide]

[0659] or

[0660] [oligonucleotide] ■ — [linker],

[0661] : [cell penetrating peptide] [oligonucleotide] - [linker] S

[0662] In some embodiments, the conjugate is of the following structure:

[0663] [cell penetrating peptide] — [linker] — [cell penetrating peptide] — [linker] — [oligonucleotide]

[0664] With respect to the series of embodiments shown immediately below, the cell penetrating peptide is attached to the linker where shown at either its N-terminus or C-terminus and the oligonucleotide is attached to the linker where shown at either its 5' end or 3' end. In some embodiments, the cell penetrating peptide is attached to the linker where shown at its N-terminus and the oligonucleotide is attached to the linker where shown at its 5' end. In some embodiments, the cell penetrating peptide is attached to the linker where shown at its N-terminus and the oligonucleotide is attached to the linker where shown at its 3' end. In some embodiments, the cell penetrating peptide is attached to the linker where shown at its C-terminus and the oligonucleotide is attached to the linker where shown at its 5' end. In preferred embodiments, the cell penetrating peptide is attached to the linker where shown at its C-terminus and the oligonucleotide is attached to the linker where shown at its 3' end.Attorney Docket: 140101-00220

[0665] In some embodiments, the conjugate is of the following structure:

[0666] [ceil penetrating peptide]... [oligonucleotide]

[0667] N

[0668] H

[0669]

[0670] In some embodiments, the conjugate is of the following structure:

[0671] [cell penetrating peptide]

[0672] [oligonucleotide]

[0673]

[0674] In some embodiments, the conjugate is of the following structure:

[0675] [cell penetrating peptide] [oligonucleotide]

[0676]

[0677] In some embodiments, the conjugate is of the following structure:

[0678] [cell penetrating peptide] [oligonucleotide]

[0679]

[0680] In some embodiments, the conjugate is of the following structure:

[0681]

[0682] In some embodiments, the cell penetrating peptide is conjugated to the oligonucleotide through a carboxamide linkage.

[0683] The linker of the conjugate may form part of the oligonucleotide to which the cell penetrating peptide is attached. Alternatively, the attachment of the oligonucleotide may be directly linked to the C-terminus of the cell penetrating peptide. In some embodiments, no linker is required.

[0684] Alternatively, the cell penetrating peptide may be chemically conjugated to the oligonucleotide. Chemical linkage may be via a disulfide, alkenyl, alkynyl, aryl, ether, thioether, triazole, amide, carboxamide, urea, thiourea, semicarbazide, carbazide, hydrazine,Attorney Docket: 140101-00220

[0685] oxime, phosphate, phosphoramidate, thiophosphate, boranophosphate, iminophosphates, or thiol-maleimide linkage.

[0686] In some embodiments, a cysteine residue may be added at the N-terminus of a cell penetrating peptide to allow for disulfide bond formation to the cell penetrating peptide, or the N-terminus may undergo bromoacetylation for thioether conjugation to the cell penetrating peptide.

[0687] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is capable of penetrating into cells and tissues, e.g., into the nucleus of cells, e.g., into muscle tissues.

[0688] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, comprises one or more of the cell penetrating peptides described herein.

[0689] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, comprises one or more of the antisense oligonucleotides described herein. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0690] VI. Delivery of Antisense Oligonucleotide Conjugates of the Invention

[0691] The delivery of an antisense oligonucleotide conjugate of the invention, or a pharmaceutically acceptable salt thereof, to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disease, e.g, GSD disease) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an antisense oligonucleotide conjugate of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an antisense oligonucleotide conjugate of the invention to a subject.

[0692] The cell penetrating peptide component of the antisense oligonucleotide conjugates promotes uptake of antisense oligonucleotides, such as PMOs, by cells (e.g, a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), thereby delivering the antisense oligonucleotides to the interior (cytoplasm) of the cells. In some embodiments, the cell penetrating peptide, when conjugated to an antisense oligonucleotide having a substantially uncharged backbone, is effective to enhance the activity of the antisense oligonucleotide, relative to the antisense oligonucleotide in unconjugated form, as evidenced by a decrease in expression of an encoded protein, relative to that provided by the unconjugated oligonucleotide, when bindingAttorney Docket: 140101-00220

[0693] of the antisense oligonucleotide to its target sequence is effective to block a translation start codon for the encoded protein. In some embodiments, conjugation of the cell penetrating peptide provides this activity in a cell-free translation assay. In some embodiments, activity is enhanced by a factor of at least two, a factor of at least five or a factor of at least ten.

[0694] Alternatively or in addition, the cell penetrating peptide is effective to enhance the transport of a nucleic acid analog into a cell, relative to the analog in unconjugated form. In some embodiments, transport is enhanced by a factor of at least two, a factor of at least two, a factor of at least five or a factor of at least ten.

[0695] In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an antisense oligonucleotide conjugate of the invention, or a pharmaceutically acceptable salt thereof. For in vivo delivery, factors to consider in order to deliver an antisense oligonucleotide conjugate include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an antisense oligonucleotide conjugate can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the conjugate, limits the exposure of the conjugate to systemic tissues that can otherwise be harmed by the conjugate or that can degrade the conjugate, and permits a lower total dose of the antisense oligonucleotide conjugate to be administered.

[0696] In one aspect, the present invention provides methods comprising contacting a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell) with one or more antisense oligonucleotide conjugates described herein, and / or the pharmaceutical compositions described herein, thereby inhibiting GYSI expression in the cell.

[0697] The phrase “contacting a cell with an antisense oligonucleotide conjugate,” as used herein, includes contacting a cell by any possible means. Contacting a cell with an antisense oligonucleotide conjugate includes contacting a cell in vitro with the antisense oligonucleotide conjugate or contacting a cell in vivo with the antisense oligonucleotide conjugate. The contacting may be done directly or indirectly. Thus, for example, the antisense oligonucleotide conjugate may be put into physical contact with the cell by the individual performing the method, or alternatively, the antisense oligonucleotide conjugate may be put into a situation that will permit or cause it to subsequently come into contact with the cell.Attorney Docket: 140101-00220

[0698] Contacting a cell in vitro may be done, for example, by incubating the cell with the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof.

[0699] Contacting a cell in vivo may be done, for example, by injecting the antisense oligonucleotide conjugate into or near the tissue where the cell is located, or by injecting the antisense oligonucleotide conjugate into another area, e.g., the bloodstream or the subcutaneous space, such that the conjugate will subsequently reach the tissue where the cell to be contacted is located. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an antisense oligonucleotide conjugate and subsequently transplanted into a subject.

[0700] In one embodiment, contacting a cell with an antisense oligonucleotide conjugate includes “introducing” or “delivering the antisense oligonucleotide conjugate into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an antisense oligonucleotide conjugate can occur through unaided diffusive or active cellular processes, or by auxiliary conjugates or devices. Introducing an antisense oligonucleotide conjugate into a cell may be in vitro and / or in vivo. In vitro introduction into a cell includes methods known in the art such as electroporation. Further approaches are described herein below and / or are known in the art.

[0701] In order to ensure in vivo delivery of the conjugate to the target tissue (e.g., liver, kidney, intestine, blood, heart, muscle, respiratory, and nervous system) and / or cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cells, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), the conjugate can be administered to a subject via an intramedullary route, an intrathecal route, an intracerebroventricular route, an intraventricular route, an intravitreal route, an enteral route, a parenteral route, an intravenous route, an intra-arterial route, an intramuscular route, an intratumoral route, a subcutaneous route, an oral route, or a nasal route.

[0702] Further, for administering an antisense oligonucleotide conjugate systemically for the treatment of a disease, the conjugate can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the antisense oligonucleotide conjugate in vivo. Modification of the conjugate or a pharmaceutical carrier can also permit targeting of the antisense oligonucleotide conjugate composition to the target tissue and avoid undesirable off-target effects.

[0703] VII. Pharmaceutical Compositions of the InventionAttorney Docket: 140101-00220

[0704] The present invention also provides pharmaceutical compositions and formulations which include one or more antisense oligonucleotide conjugates, or a pharmaceutically acceptable salt thereof, described herein. In one embodiment, provided herein are pharmaceutical compositions containing an antisense oligonucleotide conjugate, as described herein, and a pharmaceutically acceptable carrier.

[0705] The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0706] The phrase "pharmaceutically-acceptable carrier" as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating conjugates, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering conjugates, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and / or polyanhydrides; (22) bulking conjugates, such as polypeptides and amino acids (23) serum components, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

[0707] The pharmaceutical compositions containing the antisense oligonucleotide conjugates, or a pharmaceutically acceptable salt thereof, are useful for treating a disease or disorder associated with the expression or activity of a GYSI gene, e.g. GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease). Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via an intramedullary route, an intrathecal route, an intracerebroventricular route, an intraventricular route, an intravitreal route, anAttorney Docket: 140101-00220

[0708] enteral route, a parenteral route, an intravenous route, an intra-arterial route, an intramuscular route, an intratumoral route, a subcutaneous route, an oral route, or a nasal route.

[0709] The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a GYSI gene. In general, a suitable dose of an antisense oligonucleotide conjugate of the invention, or a pharmaceutically acceptable salt thereof, will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day.

[0710] For example, subjects can be administered, e.g., via an intramedullary route, an intrathecal route, an intracerebroventricular route, an intraventricular route, an intravitreal route, an enteral route, a parenteral route, an intravenous route, an intra-arterial route, an intramuscular route, an intratumoral route, a subcutaneous route, an oral route, or a nasal route.

[0711] A single and / or a repeat-dose regimine may include administration of a therapeutically effective amount of the antisense oligonucleotide conjugate at a frequency that is weekly, biweekly, monthly, quarterly, semi-annually, annually, or a combination thereof.

[0712] The pharmaceutical composition can be administered over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21, 22, 23, 24, or about a 25 minute period. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration weekly or biweekly for three months, administration can be repeated once per month, for six months or a year or longer.

[0713] The pharmaceutical composition can be administered once daily, or the antisense oligonucleotide conjugate can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation.

[0714] The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual antisense oligonucleotide conjugates encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

[0715] The antisense oligonucleotide conjugate can be delivered in a manner to target a particular tissue, such as the nervous system (e.g., the peripheral nervous system).Attorney Docket: 140101-00220

[0716] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized.

[0717] In any event, the administering physician can adjust the amount and timing of antisense oligonucleotide conjugate administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

[0718] VIII. Methods for Inhibiting GYS1 Expression

[0719] The present invention also provides methods of inhibiting expression of GYS1 in a cell (e.g., inhibit GYS1 transcript expression, and / or GYS1 protein expression). The methods include contacting a cell with an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, of the invention in an amount effective to inhibit expression of the GYS1 in the cell, thereby inhibiting expression of GYS1 (e.g., inhibit GYS1 transcript expression, and / or GYS1 protein expression) in the cell.

[0720] A cell suitable for treatment using the methods of the invention may be any cell that expresses a GYS1 gene. In some embodiments, the cell is selected from the group consisting of a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cell, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, an induced pluripotent stem cell, and any combination thereof.

[0721] In some embodiments, the cell is any cell type of the central nervous system or the peripheral nervous system. In some embodiments, the cell is a muscle cell.

[0722] Contacting of a cell with an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, may be performed in vitro or in vivo. Contacting a cell in vivo with the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, includes contacting a cell or group of cells within a subject, e.g, a human subject, with the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof. Combinations of in vitro and in vivo methods of contacting are also possible.

[0723] Contacting may be direct or indirect, as discussed above.

[0724] The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating” and other similar terms, and includes any level of inhibition.

[0725] The phrase “inhibiting expression of GYSI” is intended to refer to inhibition of expression of any GYSI gene (such as, e.g, a mouse GYSI gene, a rat GYSI gene, a monkey GYSI gene, or a human GYSI gene) as well as variants or mutants of a GYSI gene. Thus, the GYSI gene may be a wild-type GYSI gene, a mutant GYSI gene, or a transgenic GYSI gene in the context of a genetically manipulated cell, group of cells, or organism.Attorney Docket: 140101-00220

[0726] In some embodiments, the “inhibiting expression of GYSI” refers to inhibiting GYSI transcript (e.g., pre-mRNA, processed or mature mRNA, or a combination thereof) expression, and / or GYSI protein expression. In some embodiments, the “inhibiting expression of GYSI” refers to inhibiting GYSI transcript expression. In some embodiments, the “inhibiting expression of GYSI” refers to inhibiting GYSI protein expression.

[0727] “Inhibiting expression of a GYSI gene” includes any level of inhibition of a GYSI gene, e.g., at least partial suppression of the expression of a GYSI gene. The expression of the GYSI gene may be assessed based on the level, or the change in the level, of any variable associated with GYSI gene expression, e.g., GYSI mRNA level, or GYSI protein level. This level may be assessed in an individual cell or in a group of cells, including, for example, a sample derived from a subject.

[0728] Inhibition may be assessed by a decrease in an absolute or relative level of one or more variables that are associated with GYS1 expression compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive conjugate control).

[0729] In some embodiments, the expression of a GYS1 gene is inhibited by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0730] A control cell or group of cells that may be used to assess the inhibition of the expression of a GYS1 gene includes a cell or group of cells that has not yet been contacted with an antisense oligonucleotide conjugate of the invention. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an antisense oligonucleotide conjugate described herein.

[0731] The level of GYS1 mRNA that is expressed by a cell or group of cells may be determined using any method known in the art. In one embodiment, the level of expression of GYS1 in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the GYS1 gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy RNA preparation kits (Qiagen) or PAXgene (PreAnalytix, Switzerland) or cells to cT kit (Thermofisher). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis.Attorney Docket: 140101-00220

[0732] In one embodiment, the level of expression of GYS1 is determined using a nucleic acid probe. The term "probe", as used herein, refers to any molecule that is capable of selectively binding to a specific GYS1. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0733] Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or Northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to GYS1 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the mRNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known mRNA detection methods for use in determining the level of GYS1 mRNA.

[0734] An alternative method for determining the level of expression of GYS1 in a sample involves the process of nucleic acid amplification and / or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U. S. Pat. No. 4,683,202; the entire contents of each of which are hereby incorporated herein by reference), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193; the entire contents of each of which are hereby incorporated herein by reference), self sustained sequence replication (Guatelli etal. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878; the entire contents of each of which are hereby incorporated herein by reference), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173-1177; the entire contents of each of which are hereby incorporated herein by reference), Q-Beta Replicase (Lizardi et al. (1988) Bio / Technology 6: 1197; the entire contents of each of which are hereby incorporated herein by reference), rolling circle replication (Lizardi et al., U. S. Pat. No. 5,854,033; the entire contents of each of which are hereby incorporated herein by reference) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the invention, the level of expression of GYS1 is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System).

[0735] The level of GYS1 protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC),Attorney Docket: 140101-00220

[0736] thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, electrochemiluminescence assays, and the like.

[0737] In some embodiments, the expression of a GYS1 protein is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0738] The level of glycogen may be determined using any method known in the art for measuring polysaccharides. Such methods, include, for example, enzymatic assays, colorimetry, high performance liquid chromatography, mass spectrometry, nuclear magnetic resonance, ultrasound, bioimpedance, histochemical or microscopic methods. In some embodiments, the level of glycogen is reduced by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%. at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100%.

[0739] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression in a cell with an IC₅₀ between 0.1-20 pM. In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression in a cell with an IC₅₀ between 0.1-2.5 pM, 2.5-5 pM, 5-7.5 pM, 7.5-10 pM, 10-12.5 pM, 12.5-15 pM, or 15-20 pM. In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression in a cell with an IC₅₀ between 0.1-10 pM. In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression in a cell with an IC₅₀ of about 0.5 pM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM or 10 pM. In some embodiments, the cell is a human cell and the GYS1 is human GYS1.

[0740] In some embodiments, the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is administered to a subject such that the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, is delivered to a specific site within the subject. The inhibition of expression of GYS1 may be assessed using measurements ofAttorney Docket: 140101-00220

[0741] the level or change in the level of GYS1 mRNA or GYS1 protein in a sample derived from fluid or tissue from the specific site within the subject. In some embodiments, the site is the peripheral nervous system. The site may also be a subsection or subgroup of cells from any one of the aforementioned sites. The site may also include cells, such as Schwann cells.

[0742] IX. Methods of Treatment

[0743] The present invention also provides effective methods and combination therapies for treating a subject having glycogen storage disease (GSD) using the antisense oligonucleotide conjugates, or a pharmaceutically acceptable salt thereof, and compositions of the invention. The specific design of the antisense oligonucleotide conjugates of the present invention allows effective delivery of the conjugates to a target cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a diaphragm cell, an alveolar cell, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell). The methods of the present invention are both simple and efficient, and provide a great advantage when used in treating GSD (e.g, Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease).

[0744] In some embodiments, the GSD is selected from the group consisting of Lewis' disease, von Gierke’s disease, Pompe’s disease (GSD Ila and lib), Danon disease, Forbes disease / Cori disease, Andersen's disease, Adult Polyglucosan Body Disease (APBD), Lafora Disease, McArdle's disease, Hers’ disease, Tarui’s disease, phosphorylase kinase deficiency, type IX of GSD, type X of GSD, Fanconi -Bickel syndrome, type XIV of GSD, type XII of GSD, type XIII of GSD, type XV of GSD, and subtypes thereof.

[0745] In some aspects of the invention, the methods further include administering to the subject an additional therapeutic, such as medications for decreasing uric acid levels (e.g, Allopurinol, Febuxostat, or Diuretics), medications for decreasing cholesterol levels (e.g., Statins, Ezetimibe, Phytosterols, or citrate lyase inhibitors), cornstarch therapy, high-protein diet, calcium, vitamin D, iron supplements, glucose, nutrition supplements, concomitant surgery, occupational therapy, physical therapy, exercise program, enzyme replacement therapies, gene replacement therapies, and any combination thereof.

[0746] In one aspect, the present invention provides methods of treating glycogen storage disease (GSD) in a subject in need thereof, by administering to the subject a therapeutically effective amount of the antisense oligonucleotide conjugate or the pharmaceutical composition of the invention, thereby treating the GSD in the subject.Attorney Docket: 140101-00220

[0747] In some embodiments, the GSD is selected from the group consisting of Lewis' disease, von Gierke’s disease, Pompe’s disease (GSD Ila and lib), Danon disease, Forbes disease / Cori disease, Andersen's disease, Adult Polyglucosan Body Disease (APBD), Lafora Disease, McArdle's disease, Hers’ disease, Tarui’s disease, phosphorylase kinase deficiency, type IX of GSD, type X of GSD, Fanconi -Bickel syndrome, type XIV of GSD, type XII of GSD, type XIII of GSD, type XV of GSD, and subtypes thereof.

[0748] The term "therapeutically effective amount," as used herein, is intended to include the amount of an antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, that, when administered to a subject having a disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The "therapeutically effective amount" may vary depending on the antisense oligonucleotide conjugate, how the conjugate is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

[0749] A "therapeutically effective amount" also includes an amount of an antisense oligonucleotide conjugate that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The antisense oligonucleotide conjugates employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit / risk ratio applicable to such treatment.

[0750] In another aspect, the present invention provides use of an antisense oligonucleotide conjugate of the invention targeting a GYS1 gene or a pharmaceutical composition comprising an antisense oligonucleotide conjugate targeting a GYS1 gene in the manufacture of a medicament for treating a subject, e.g., a subject a disease, e.g., GSD.

[0751] In another aspect, the present invention provides uses of an antisense oligonucleotide conjugate of the invention targeting a GYS1 gene or a pharmaceutical composition comprising an antisense oligonucleotide conjugate targeting a GYS1 gene in the manufacture of a medicament, optionally for use in combination with an additional therapeutic, for treating a subject, e.g, a subject having a disease, e.g, GSD.

[0752] In one embodiment, an antisense oligonucleotide conjugate targeting GYS1 is administered to a subject having a disease, such as GSD, such that GYS1 levels, e.g., GYS1 mRNA levels or GYS1 protein levels, e.g., in a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cell, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), tissue or fluid of the subject are reduced by at least about 10%, 11%, 12%, 13%,Attorney Docket: 140101-00220

[0753] 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0754] In one embodiment, an antisense oligonucleotide conjugate targeting GYS1, or a pharmaceutically acceptable salt thereof, is administered to a subject having a disease, such as GSD, such that glycogen levels, e.g., in a cell (e.g., a Schwann cell, a peripheral nerve axon, an endothelial cell, a muscle cell, a myocyte, a myoblast, a myocardial cell, a smooth muscle cell, a skeletal muscle cell, a fibroblast, a diaphragm cell, an alveolar cell, any type of respiratory cell, a podocyte or any type of a kidney cell, a hepatocyte or any type of a liver cell, a neuronal cell, a microglial cell, any cell type of the central nervous system and peripheral nervous system, a stem cell, an embryonic stem cell, or an induced pluripotent stem cell), tissue or fluid of the subject are reduced by at least about 10%, 11%, 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%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 62%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least about 99% or more.

[0755] In some embodiments, the levels of GYS1 mRNA, GYS1 protein or glycogen are measured in a sample obtained from a subject. In some embodiments, the sample comprises blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, samples from tissues (e.g., muscles), organs or localized regions. In some embodiments, the subject has a GSD (e.g., Lafora disease, Adult Polyglucosan Body Disease (APBD), or Pompe’s disease). In some embodiments, the subject is administered an antisense oligonucleotide conjugate targeting GYS1, or a pharmaceutically acceptable salt thereof.

[0756] In some embodiments, the methods further comprise administering an additional therapeutic, e.g., medications for decreasing uric acid levels (e.g., Allopurinol, Febuxostat, or Diuretics), medications for decreasing cholesterol levels (e.g., Statins, Ezetimibe, Phytosterols, or citrate lyase inhibitors), cornstarch therapy, high-protein diet, calcium, vitamin D, iron supplements, glucose, nutrition supplements, concomitant surgery, occupational therapy, physical therapy, exercise program, enzyme replacement therapies, gene replacement therapies, and any combination thereof, to the subject.

[0757] The additional therapeutic may be administered to the subject at the same time as the antisense oligonucleotide conjugate of the invention, or at a different time. Moreover, the additional therapeutic, may be administered to the subject in the same formulation as theAttorney Docket: 140101-00220

[0758] antisense oligonucleotide conjugate or in a different formulation as the antisense oligonucleotide conjugate.

[0759] The methods and uses of the invention include administering a composition described herein such that expression of the target GYS1 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, or about 80 hours. In one embodiment, expression of the target GYS1 gene is decreased for an extended duration, e.g., at least about two, three, four, five, six, seven days or more, e.g., about one week, two weeks, three weeks, or about four weeks or longer.

[0760] Administration of the antisense oligonucleotide conjugate according to the methods and uses of the invention may result in a reduction of the severity, signs, symptoms, and / or markers of such diseases or disorders in a patient with a disease, e.g., GSD. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

[0761] Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters.

[0762] Subjects can be administered a therapeutic amount of antisense oligonucleotide conjugate. The antisense oligonucleotide conjugate may be administered to the subject at a frequency that is weekly, biweekly, monthly, quarterly, semi-annually, annually, or a combination thereof. The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

[0763] X. Kits of the Invention

[0764] In another aspect, the present invention provides a kit comprising one or more of the antisense oligonucleotide conjugates described herein, and / or the pharmaceutical compositions described herein, and instructions for use thereof. The instructions may include instructions for administering a fixed dose of the antisense oligonucleotide conjugate or the pharmaceutical composition of the invention.

[0765] The antisense oligonucleotide conjugate may be in a vial or a pre-filled syringe. The kits may optionally further comprise means for administering the antisense oligonucleotide conjugate (e.g., an injection device, such as a pre-filled syringe), or means for measuring the inhibition of GYS1 (e.g., means for measuring the inhibition of GYS1 mRNA, GYS1 protein,Attorney Docket: 140101-00220

[0766] and / or GYS1 activity). Such means for measuring the inhibition of GYS1 may comprise a means for obtaining a sample from a subject. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The kits of the invention may optionally further comprise means for determining the therapeutically effective or prophylactically effective amount.

[0767] The present invention also provides syringes comprising one or more of the antisense oligonucleotide conjugates described herein, and / or the pharmaceutical compositions described herein. In some embodiments, the syringe is made of glass. In some embodiments the syringe is made of plastic.

[0768] The present invention further provides vials comprising the antisense oligonucleotide conjugates described herein, and / or the pharmaceutical compositions described herein.

[0769] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the antisense oligonucleotide conjugates and methods featured in the invention, suitable methods and materials are described below.

[0770] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0771] EXAMPLES

[0772] Example 1. PMO and PPMO Synthesis

[0773] PMOs targeting GYS1 were synthesized using standard synthesis methods well known in the art and were purchased. The representative process for the manufacture of the PMOs is provided below. A detailed list of PMOs targeting GYS1 is provided in Table 1A.

[0774] 1.1 Synthesis of peptide variants via Microwave Synthesizer

[0775] Peptides were synthesized on a 100 μmol scale using a CEM Liberty Blue™ microwave Peptide Synthesizer and Fmoc chemistry following manufacturer’s recommendations. Peptides synthesized with a glutamic acid linker were synthesized with a Rink amide resin to afford an amide on the carboxyl terminus of the peptide after TFA cleavage. The side chain protecting groups used were labile to TFA treatment and the peptide was synthesized using a 5-fold excess of Fmoc-protected amino acids (0.25 mmol) that were activated using PyBOP (5-fold excess) in the presence of DIPEA. Piperidine (20%Attorney Docket: 140101-00220

[0776] v / v in DMF) was used to remove N-Fmoc protecting groups. The coupling was carried out once at 75 °C for 5 min at 60-watt microwave power except for arginine residues, which were coupled twice each. Each deprotection reaction was carried out at 75°C twice, once for 30 sec and then once for 3 min at 35-watt microwave power. Once the synthesis was complete, the resin was washed with DMF (3 x 50 mL) and the N-terminus of the solid phase bound peptide was acetylated with acetic anhydride in the presence of DIPEA at room temperature for 15 min. After acetylation of the N-terminus, the peptide resin was washed with DMF (3 x 20 mL) and DCM (3 x 20 mL).

[0777] For DPEP peptides with succinic acid on the N-terminus, acetylation of the N-terminus was not performed. Instead, the free N-terminus of the peptide was treated with succinic anhydride in the presence of DIPEA at room temperature for 30 min followed by washing with DMF (3 x 20 mL).

[0778] For DPEP peptides carrying glutamic acid on the N-terminus as a linker, the N-terminus was acetylated as described, but attachment of the PMO at its 3' end was performed on the side chain carboxylic group of the peptide. For DPEP peptides carrying glutamic acid on the C-terminus as a linker, attachment of the PMO at its 3' end was performed on the side chain carboxylic group of the peptide.

[0779] 1.2 Cleavage from the solid support and purification of the peptide via Semi-Prep HPLC

[0780] The peptide was cleaved from the solid support by treatment with a cleavage cocktail consisting of TFA / H2O / triisopropylsilane (TIPS) (95:2.5:2.5, 10 mL) for 3 h at room temperature. Excess TFA was removed by sparging with nitrogen. The cleaved peptide was precipitated via the addition of ice-cold diethyl ether and centrifuged at 3000 rpm for 5 min. The crude peptide pellet was washed thrice with cold diethyl ether (3 × 40 mL) and purified by RP-HPLC using a Varian 940-LC HPLC System fitted with a 445-LC Scale-up module and 440-LC fraction collector. Peptides were purified by semi-preparative HPLC on an RP-C18 column (10 x 250 mm, Phenomenex Jupiter) using a linear gradient of CH3CN in 0.1 % TFA / H2O (0-99%, 0.1 % TFA in CH3CN) with a flow rate of 15 mL-min-1 over 15 min. Detection was performed at 220 nm and 260 nm.

[0781] 1.3 Synthesis of Peptide-conjugated PMOs

[0782] PMOs with GYSI targeting nucleotide sequences based on the antisense sequences shown in Table 1 A were purchased and were used to make the peptide-conjugated PMO conjugates (PPMOs) shown in Tables 2 and 3. The 3 ’-end of the PMO was conjugated to the carboxyl side-chain of the glutamic acid linker as described in this example, which in turn was covalently bound to the C-terminal end of the peptide (SEQ ID NO: 35). This was achieved using 2.3 and 2-fold equivalents of PyBOP and HOAt in NMP respectively in theAttorney Docket: 140101-00220

[0783] presence of 2.3 equivalents of DIPEA over peptide and a 2.5-fold excess of peptide over PMO dissolved in DMSO. In general, to a solution of peptide (10 pmol) in N-methylpyrrolidone (NMP, 100 pL) were added PyBOP (76.6 pL of 0.3 M in NMP), HOAt in (66.7 pL of 0.3 M NMP), DIPEA (4.0 pL) and PMO (4 pmol, 400 pL of 10 mM in DMSO). The mixture was left for 2 h at 40°C and the reaction was quenched by the addition of H2O (1 mL). The reaction was purified on a cation exchange chromatography column (Resource S 6 mL column, GE Healthcare) using a linear gradient of sodium chloride (0 to 1 M) in sodium phosphate buffer (25 mM, pH 7.0) containing 20% CH3CN at a flow rate of 6 mL-min-1. The removal of excess salts from the PPMO was afforded through the filtration of the fractions collected after ion exchange using an Amicon® ultra- 15 3K centrifugal filter device. The conjugate was lyophilized and analyzed by MALDI-TOF. The conjugates were dissolved in sterile water and filtered through a 0.22 pm cellulose acetate membrane before use. The concentration of PPMO was determined by the molar absorption of the conjugates at 265 nm in 0.1 M HCl solution.

[0784] A detailed list of PPMOs targeting GYSI is provided in Tables 2 and 3.

[0785] Table 1A. PMOs targeting GYSI.

[0786] All PMOs in Table 1 A were made with the following group at the 5' end:

[0787]

[0788]

[0789] Attorney Docket: 140101-00220

[0790]

[0791] Attorney Docket: 140101-00220

[0792]

[0793] Attorney Docket: 140101-00220

[0794]

[0795] Attorney Docket: 140101-00220

[0796]

[0797] Certain PMOs from Table 1 A, identified in Tables 2 and 3, were used to synthesize the PPMOs identified in Tables 2 and 3. All such PPMOs were synthesized with the following structural parameters of Table IB and Table 1C.Attorney Docket: 140101-00220

[0798] Table IB. Characteristics of Peptide and Linker Used to Make PPMOs.

[0799]

[0800] Table 1C. Sequence of Peptide Used to Make PPMOs.

[0801]

[0802] aLinkers are listed as their single amino acid abbreviations.bLinker attachment is with respect to the peptide (the PMO is attached to the side chain of the glutamic acid linker), C-term= carboxyl terminus, N-term = amino terminus. The SEQ ID NO refers to the sequence of the peptide without any additional N and C terminal modifications or the linkers.

[0803] The desired molecular weights of the PPMOs as determined by LC-MS (MS+1) were observed. Below is representative observed MS data for certain PPMOs made in accordance with this disclosure (Table ID)

[0804] Table ID. MS data

[0805]

[0806] Example 2. In vitro screening

[0807] An in vitro screening of the PMOs targeting GYSI was performed by transfecting different cell types with a single dose (at varying concentrations) of a PMO, or a PMO conjugate comprising a PMO targeting GYSI conjugated to a cell penetrating peptide using methods well known in the art.

[0808] Briefly, in the first study, a real time PCR analysis was performed to demonstrate inhibition of GYSI expression in U-251 MG cells (a human glioblastoma multiforme (GBM) cell line) transfected with 5pM (Figure 1) or 25pM (Figure 2) of PMOs targeting humanAttorney Docket: 140101-00220

[0809] GYSI. Briefly, an in vitro endogenous screen was performed, wherein U-251 MG cells were plated in a 96 well plate. Following overnight incubation, cells were treated with PMOs targeting human GYSI at 5 pM or 25 pM, with 0.9 pL per well EndoPorter reagent for 24 hours in technical triplicate. Cell were lysed using Cells-to-CT kit and mRNA expression was measured by qRT-PCR and normalized to GAPDH expression. Mean Relative Quantification (RQ) was observed as the average of technical quadruplicates (n=4).

[0810] In a further study, a real time PCR analysis was performed to demonstrate inhibition of GYSI expression in C2C12 myoblasts (a mouse myoblast cell line) transfected with 5pM (Figure 3) or 25 pM (Figure 4) of PMOs targeting mouse GYSI. Briefly, an in vitro endogenous screen was performed, wherein C2C12 myoblasts were plated in a 96 well plate. Following overnight incubation, cells were treated with PMOs targeting mouse GYSI at 5 pM or 25 pM, with 0.9 pL per well EndoPorter reagent for 24 hours in technical triplicate. Cell were lysed using Cells-to-CT kit and mRNA expression was measured by qRT-PCR and normalized to GAPDH expression. Mean Relative Quantification (RQ) was observed as the average of technical quadruplicates (n=4).

[0811] In a further study, human fibroblasts were transfected with 5 pM or 25 pM of PMO conjugates comprising a PMO targeting human GYSI conjugated to a cell penetrating peptide (PPMO) for 24 hours (Table 2). Briefly, human fibroblasts were plated in a 96 well plate. Following overnight incubation, cells were treated with PMO conjugates comprising a PPMO targeting human GYSI conjugated to a cell penetrating peptide at 5 pM or 25 pM, for 24 hours in technical triplicate. Cells were lysed using Cells-to-CT kit and mRNA expression was measured by qRT-PCR and normalized to GAPDH expression. Mean Relative Quantification (RQ) was observed as the average of technical triplicates (n=3), and calculated as the average %RQ (RNA quantity remaining) relative to mock control. The results of this study demonstrate that PMO conjugates comprising a PMO targeting human GYSI conjugated to a cell penetrating peptide inhibit GYSI expression in human fibroblasts.

[0812] In a further study, mouse C2C12 myotubes were transfected with 3pM or 30pM of PMO conjugates comprising a PMO targeting mouse GYSI conjugated to a cell penetrating peptide (PPMO) for 24 hours (Table 3). Cells were lysed using Cells-to-CT kit and mRNA expression was measured by qRT-PCR and normalized to GAPDH expression. Mean Relative Quantification (RQ) was observed as the average of technical triplicates (n=3). The results of this study demonstrate that PMO conjugates comprising a PMO targeting mouse GYSI conjugated to a cell penetrating peptide inhibit GYSI expression in mouse C2C12 myotubes.

[0813] Table 2. Human GYSI targeting PPMO screen in human fibroblasts. The activity of the PPMOs observed by the in vitro screening assay of Example 2 is provided in Tables 2 and 3 and is expressed as a RQ mean value in accordance with the following levels without consideration of the standard deviations provided:Attorney Docket: 140101-00220

[0814] - 25: +, >25 - 50: ++, >50 - 75: +++, >75 - 100: ++++, >100: ++++

[0815]

[0816] Attorney Docket: 140101-00220

[0817]

[0818] Table 3. Mouse GYSI targeting PPMO screen in C2C12 myotubes.

[0819]

[0820] Example 3. In vivo activity

[0821] In vivo activity is tested using acid a-glucosidase (GAA) knockout (Gaa_ / ) mouse model to study the effects of lowering Gysi expression on Pompe myopathy in vivo. Pompe disease is a glycogen storage disease caused by a deficiency in acid a-glucosidase (GAA), a hydrolase necessary for the degradation of lysosomal glycogen. Deficiency in GAA results in muscle and neuronal glycogen accumulation, which causes respiratory insufficiency, a symptom of Pompe disease. Gaa mouse recapitulates Pompe disease manifestations, including the accumulation of glycogen in skeletal and cardiac muscle, the autophagy defects, and the consequent myopathy. Threrefore, Gaa mouse model is selected to study the effects of decreasing GYSI expression on Pompe myopathy in vivo.

[0822] Gaa mice are bred and genotyped according to established protocols. Starting at 5-7 weeks, animals are injected intravenously or subcutaneously with repeat doses (e.g., one to three times a week) of the selected PMOs, the selected PMO conjugates comprising a PMO targeting GYSI conjugated to a cell penetrating peptide, a control PMO, or control commercially available enzyme used for enzyme replacement therapy for Pompe, or a combination of enzyme with selected PMO conjugate. Saline (vehicle control) treated Gaa mice and wildtype are included as a control. Animals are sacrificed after 2 days after the last injection and tissues are collected (serum, quadriceps muscle, diaphragm, and heart tissue, etc. and analyzed for GYSI mRNA and protein reduction using quantitative RT-PCR and western blotting.Attorney Docket: 140101-00220

[0823] To evaluate the impact of GYSI knockdown on excessive glycogen accumulation in the muscle of the Pompe mice, glycogen levels are measured by a colorimetric biochemical assay and analyzed by Periodic Acid-Schiff (PAS) staining in the muscle (e.g., quadriceps muscle, diaphragm, and heart tissue).

[0824] To assess if treatment with the selected PMOs or the selected PMO conjugates could reverse Pompe pathology, accelerating rotarod performance test is performed to evaluate the muscle function.

[0825] Example 4. In vitro activity

[0826] The activity of PMO conjugates comprising a PMO targeting either human or mouse GYSI conjugated to a cell penetrating peptide were tested in vitro in different cell types to evaluate, potency, cytotoxicity, glycogen reduction, off-target effects, and GYSI specificity.

[0827] Human skeletal muscle cells were plated in 96 well plates in growth media, and differentiation into myotubes was initiated through the addition of differentiation media for 6 days. On Day 7, the cells were treated with 10 pL of 10X PPMO targeting human GYSI (PGN-200085, PGN-200152, PGN-200154, PGN-200158, PGN-200159, PGN-200162) (Table 4)

[0828] To evaluate potency of the PMO conjugates, cells were lysed using Cells-to-CT kit and mRNA expression was analyzed by qRT-PCR. The average %RQ (RNA quantity remaining) relative to mock control of two independent replicate samples of loglO(PPMO) from 0.003 - 50 pM was calculated. The data were represented as a nonlinear regression curve fit of loglO(PPMO) vs normalized response (%RQ relative to mock) in which the data is constrained from 100 to 0 to account for variability. As shown in Figure 5A, the results of this study demonstrated that PMO conjugates comprising a PMO targeting human GYSI conjugated to a cell penetrating peptide potently inhibit GYSI expression in human myotubes.

[0829] Cell viability was measured using a Promega Cell-Titer Glo kit and the average of two experimental cell viability measurements per PPMO concentration (0.003 - 30 pM) was measured. As shown in Figure 5B, the results of this study demonstrated that treatment with PMO conjugates comprising a PMO targeting human GYSI conjugated to a cell penetrating peptide did not affect myotube cell viability.

[0830] To evaluate glycogen reduction upon treatment with PMO conjugates targeting human GYSI, differentiated muscle cells were treated with 6.25 pM of each PPMO twice over a 72h period (once at T=0h and again at T=30h) on Day 6 after initiation of differentiation in myotubes. Cells were lysed in CYTO Buffer (50 mM Tris-HCl, pH 8.0 + 150 mM NaCl with protease inhibitors), and run on Western blots using anti-GYSl and anti-Actin (control) antibodies. Glycogen levels were assayed using the Glycogen Glo Kit. For both protein analysis and glycogen assays, the average of 3 independent replicates wereAttorney Docket: 140101-00220

[0831] measured. As shown in Figures 6A and 6B, treatment of myotubes with PMO conjugates targeting human GYSI resulted in the reduction of GYSI protein expression which correlated with the reduction in glycogen in the cells.

[0832] To evaluate if PMO conjugates targeting human GYSI had off-target effects, PPMO compounds were diluted to 10X final concentration (100 pM) in PBS to 10X final concentration (0.195 - 25 uM; 2x serial dilutions) from a 1 mM stock solution, and 10 uL was added to each well of skeletal muscle cells which had differentiated into myotubes. A PPMO having the peptide and linker shown in Table 1C conjugated to a PMO with a nucleotide sequence that targets exon 23 of the mouse dystrophin gene (10 pM) was used as a control. After 24h, cells were detached, pelleted, frozen, and the frozen cell pellets were subject to RNA extraction, library generation, and RNAseq experiment / analysis. As shown in Figure 7, treatment of myotubes led to highly specific reduction of GYSI expression, with no significant off-target differentially expressed genes / pathways observed.

[0833] Human GYSI and GYS2 share 70% nucleic acid sequence identity. To evaluate specifity of PMO conjugates for GYSI over GYS2, mRNA expression of GYS2 was quantified following treatment of human primary hepatocytes with PMO conjugates targeting human GYSI. Human primary hepatocytes were plated in 96-well plates and PMO conjugates were added at concentration ranging from 0.195 - 25 pM. After 24h, cells were harvested for qPCR analysis using Cells-to-CT Kit, and mRNA expression was analyzed by qRT-PCR. Data were plotted as the average %RQ (RNA Quantity Remaining) relative to mock control + / - standard deviation of the two independent replicate samples of loglO(PPMO) from 0.195 - 25 pM. As shown in Figure 8, treatment of hepatocytes with PMO conjugates targeting GYSI did not reduce GYS2 expression. Thus, PMO conjugates targeting human GYSI show specificity for GYSI and do not target liver-specific GYS2.

[0834] In addition to PMO conjugates targeting human GYSI, the effect of PMO conjugates targeting mouse GYSI (PGN-200171, PGN-200166, PGN-200167, PGN-200168, PGN-200169, PGN-200170, PGN-200172, PGN-200173) were evaluated for potency, cytotoxicity, glycogen reduction and GYSI specificty in different mouse cell lines.

[0835] To evlaute potency of PMO conjugates targeting mouse GYSI, mouse C2C12 myoblasts were plated in 96 well plates and differentiation into myotubes was initiated. On day 6 after initation of differentiation, cells were treated with 10 uL of 10X PMO conjugates targeting mouse GYSI diluted in PBS (Table 5). Cells were lysed using Cells-to-CT kit and mRNA expression was analyzed by qRT-PCR. The average %RQ (RNA Quantity Remaining) relative to mock control + / - standard deviation of two independent replicate samples of loglO(PPMO) from 0 - 30 pM was calculated. The data were represented as a nonlinear regression curve fit of loglO(PPMO) vs normalized response (%RQ relative to mock) in which the data is constrained from 100 to 0 to account for variability. As shown in Figure 9 A, the results of this study demonstrated that PMO conjugates comprising a PMOAttorney Docket: 140101-00220

[0836] targeting mouse GYSI conjugated to a cell penetrating peptide potently inhibit mouse GYSI expression in mouse myotubes.

[0837] Cell viability was measured using a Promega Cell-Titer Glo kit and the average of two experimental cell viability measurements per PMO conjugate concentration (0 - 30 pM) was measured. As shown in Figure 9B, the results of this study demonstrated that treatment with PMO conjugates comprising a PMO targeting mouse GYSI conjugated to a cell penetrating peptide did not affect mouse myotube cell viability.

[0838] Glycogen levels were assayed in mouse myotubes treated with PMO conjugates targeting mouse GYSI using the Glycogen Glo Kit. As shown in Figures 10A and 10B, treatment of myotubes with either of two PMO conjugates targeting mouse GYSI resulted in the reduction of GYSI protein expression which correlated with the reduction in glycogen levels in the cells.

[0839] To evaluate specifity of PMO conjugates for mouse GYSI over mouse GYS2, mRNA expression of mouse GYS2 was quantified following treatment of mouse AML 12 hepatocytes with PMO conjugates targeting mouse GYSI. Mouse AML12 hepatocytes were plated in 96-well plates and PMO conjugates were added at concentrations ranging from 0.1 - 30 pM. After 24h, cells were harvested for qPCR analysis using Cells-to-CT Kit, and mRNA expression was analyzed by qRT-PCR. Data were plotted as the average %RQ (RNA Quantity Remaining) relative to mock control + / - standard deviation of the two independent replicate samples of loglO(PPMO) from 0.1 - 30 pM. As shown in Figure 11, treatment of mouse hepatocytes with PMO conjugates targeting mouse GYSI did not reduce GYS2 expression. Thus, PMO conjugates targeting mouse GYSI show specificity for GYSI and do not target liver-specific mouse GYS2.

[0840] Overall, the in vitro experiments showed that PMOs targeting either human GYSI or mouse GYSI displayed highly specific target GYSI engagement, low micromolar potency, and minimal toxicity and off-target effects.

[0841] Table 4. Potency of human GYSI tageting PPMO in human myotubes.

[0842] The IC50 values of PPMOs targeting human GYS1 measured by the in vitro assay of Example 4 is provided in Table 4 and is expressed in accordance with the following levels:

[0843] 1 - 2.5 pM: +, >2.5 - 5 pM: ++, >5 - 7.5 pM: +++, >7.5 - 10 pM: ++++, >10 pM: +++++.

[0844]

[0845] Attorney Docket: 140101-00220

[0846]

[0847] Table 5. Potency of mouse GYSI tageting PPMO in mouse myotubes. The IC50 values of PPMOs targeting mouse GYSI measured by the in vitro assay of Example 4 is provided in Table 5 and is expressed in accordance with the following levels:

[0848] 1 - 2.5 pM: +, >2.5 - 5 pM: ++, >5 - 7.5 pM: +++, >7.5 - 10 pM: ++++, >10 pM: +++++.

[0849]

[0850] Example 5. In vitro activity in cellular models of Pompe disease

[0851] The in vitro activity of PMO conjugates targeting human GYSI were tested in cellular models of Pompe disease.

[0852] Initially, human fibroblasts derived from patients suffering from late-onset Pompe disease (LOPD) were evaluated for their suitability as cellular models of Pompe disease. Primary human dermal fibroblast lines GSD-0443 and GSD-11661 (containing a mutation in the GAA gene), and control dermal fibroblast lines, PCS-201-010 neonatal and PCS-201-012 adult, were cultured in growth media (DMEM + 15%FBS + 1% PS). For protein analysis, cells were lysed in CYTO Buffer (50 mM Tris-HCl, pH 8.0 + 150 mM NaCl with protease inhibitors) and run on Western blots using anti-GAA and anti -Actin (control) primary antibodies. GAA enzyme activity was measured using the Lysosomal alpha-glucosidase fluorometric activity assay kit. Glycogen was assayed using the Glycogen Gio Kit and normalized to the total protein from each sample (measured by BCA) and reported as RLU / total protein. As shown in Figures 12A-12C, GSD-0443 and GSD-11661 cell lines have reduced GAA protein levels (Figure 12A) and protein activity (Figure 12B), as well as increased glycogen levels (Figure 12C) compared to the control fibroblast cell lines.

[0853] Having established that GSD-0443 and GSD-11661 fibroblast cell lines have relevant Pompe disease phenotypes, the activity of PMO conjugates targeting human GYSI (PGN-200085, PGN-200154 and PGN-200159) were tested in these cell lines. The fibroblast cell lines were plated in 96-well plates and treated with PPMO (diluted to 10X in PBS from 1 mM stock) for 30 hours, with an additional treatment at T=30h for a total for 72h treatmentAttorney Docket: 140101-00220

[0854] time. Protein and glycogen levels were analyzed as described above. As shown in Figures 13A-13C, treatment with all PMO conjugates targeting human GYSI resulted in a 60-80% decrease in GYSI protein levels, along with over 40% reduction in glycogen in GSD fibroblasts. Thus, PMO conjugates targeting human GYSI showed strong activity in disease-specific cellular models.

[0855] EQUIVALENTS

[0856] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims.

Claims

Attorney Docket: 140101-00220CLAIMS1. An antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, for inhibiting expression of Glycogen Synthase 1 (GYSI), comprising an antisense oligonucleotide targeting GYSI conjugated to a cell penetrating peptide,wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO), andwherein the cell penetrating peptide comprises at least one cationic domain comprising at least 4 amino acid residues and at least one hydrophobic domain comprising at least 3 amino acid residues, wherein the cell penetrating peptide comprises a total of 7 to 40 amino acid residues and the at least one cationic domain comprises a beta-alanine residue in combination with arginine and / or histidine residues.

2. The antisense oligonucleotide conjugate of claim 1, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is 10 to 40 nucleotides in length, 10 to 30 nucleotides in length, 10 to 20 nucleotides in length, 18 to 30 nucleotides in length, 10 to 25 nucleotides in length, or 18 to 25 nucleotides in length.

3. The antisense oligonucleotide conjugate of claim 1 or 2, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises at least 10, at least 15, at least 20, at least 23, or at least 25 contiguous nucleotides complementary to a target sequence in a human GYS1 gene as set forth in SEQ ID NO: 284, or a GYS1 mRNA selected from any one of the nucleotide sequences of SEQ ID NOs: 285-286.

4. The antisense oligonucleotide conjugate of any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide comprises a nucleotide sequence of any one of the nucleotide sequences of SEQ ID NOs: 106-283.

5. The antisense oligonucleotide conjugate of any one of claims 1-4, or a pharmaceutically acceptable salt thereof, wherein each cationic domain is 4 to 12 amino acid residues in length, or 4 to 7 amino acid residues in length.Attorney Docket: 140101-002206. The antisense oligonucleotide conjugate of any one of claims 1-5, or a pharmaceutically acceptable salt thereof, wherein each cationic domain comprises a sequence selected from the group consisting of RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), R[Hyp]RR[Hyp]R (SEQ ID NO: 19), and any combination thereof.

7. The antisense oligonucleotide conjugate of any one of claims 1-6, or a pharmaceutically acceptable salt thereof, wherein each hydrophobic domain comprises a sequence selected from the group consisting of YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), WWPW (SEQ ID NO: 26), and any combination thereof.

8. The antisense oligonucleotide conjugate of any one of claims 1-7, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide comprises one hydrophobic domain flanked by two cationic domains.

9. The antisense oligonucleotide conjugate of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide comprises one hydrophobic domain comprising a sequence selected from the group consisting of YQFLI (SEQ ID NO: 20), FQILY (SEQ ID NO: 21), ILFQY (SEQ ID NO: 22), FQIY (SEQ ID NO: 23), WWW, WWPWW (SEQ ID NO: 24), WPWW (SEQ ID NO: 25), and WWPW (SEQ ID NO: 26); flanked by two cationic domains, each comprising a sequence selected from the group consisting of RBRRBRR (SEQ ID NO: 1), RBRBR (SEQ ID NO: 2), RBRR (SEQ ID NO: 3), RBRRBR (SEQ ID NO: 4), RRBRBR (SEQ ID NO: 5), RBRRB (SEQ ID NO: 6), BRBR (SEQ ID NO: 7), RBHBH (SEQ ID NO: 8), HBHBR (SEQ ID NO: 9), RBRHBHR (SEQ ID NO: 10), RBRBBHR (SEQ ID NO: 11), RBRRBH (SEQ ID NO: 12), HBRRBR (SEQ ID NO: 13), HBHBH (SEQ ID NO: 14), BHBH (SEQ ID NO: 15), BRBSB (SEQ ID NO: 16), BRB[Hyp]B (SEQ ID NO: 17), R[Hyp]H[Hyp]HB (SEQ ID NO: 18), and R[Hyp]RR[Hyp]R (SEQ ID NO: 19).Attorney Docket: 140101-0022010. The antisense oligonucleotide conjugate of any one of claims 1-9, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide comprises a sequence selected from the group consisting of: RBRRBRRFQILYRBRBR (SEQ ID NO: 27), RBRRBRRFQILYRBRR (SEQ ID NO: 28), RBRRBRFQILYRRBRBR (SEQ ID NO: 29), RBRBRFQILYRBRRBRR (SEQ ID NO: 30), RBRRBRRYQFLIRBRBR (SEQ ID NO: 31), RBRRBRRI LFQYRBRBR (SEQ ID NO: 32), RBRRBRFQILYRBRBR (SEQ ID NO: 33), RBRRBFQILYRBRRBR (SEQ ID NO: 34), RBRRBRFQILYBRBR (SEQ ID NO: 35), RBRRBFQILYRBRBR (SEQ ID NO: 36), RBRRBRRFQILYRBHBH (SEQ ID NO: 37), RBRRBRRFQILYHBHBR (SEQ ID NO: 38), RBRRBRRFQILYHBRBH (SEQ ID NO: 39), RBRRBRRYQFLIRBHBH (SEQ ID NO: 40), RBRRBRRILFQYRBHBH (SEQ ID NO: 41), RBRHBHRFQILYRBRBR (SEQ ID NO: 42), RBRBBHRFQILYRBHBH (SEQ ID NO: 43), RBRRBRFQILYRBHBH (SEQ ID NO: 44), RBRRBRFQILYHBHBH (SEQ ID NO: 45), RBRRBHFQILYRBHBH (SEQ ID NO: 46), HBRRBRFQILYRBHBH (SEQ ID NO: 47), RBRRBFQILYRBHBH (SEQ ID NO: 48), RBRRBRFQILYBHBH (SEQ ID NO: 49), RBRRBRYQFLIHBHBH (SEQ ID NO: 50), RBRRBRILFQYHBHBH (SEQ ID NO: 51), RBRRBRRFQILYHBHBH (SEQ ID NO: 52), RBRRBRFQILYBRBS (SEQ ID NO: 53), RBRRBRFQILYBRBR[Hyp] (SEQ ID NO: 54), RBRRBRFQILYBR[Hyp]R (SEQ ID NO: 55), RRBRRBRFQILYBRBR (SEQ ID NO: 56), BRRBRRFQILYBRBR (SEQ ID NO: 57), RBRRBRWWWBRBR (SEQ ID NO: 58), RBRRBRWWPWWBRBR (SEQ ID NO: 59), RBRRBRWPWWBRBR (SEQ ID NO: 60), RBRRBRWWPWBRBR (SEQ ID NO: 61), RBRRBRRWWWRBRBR (SEQ ID NO: 62), RBRRBRRWWPWWRBRBR (SEQ ID NO: 63), RBRRBRRWPWWRBRBR (SEQ ID NO: 64), RBRRBRRWWPWRBRBR (SEQ ID NO: 65), RBRRBRRFQILYBRBR (SEQ ID NO: 66), RBRRBRRFQILYRBR (SEQ ID NO: 67), BRBRBWWPWWRBRRBR (SEQ ID NO: 68), RBRRBRRFQILYBHBH (SEQ ID NO: 69), RBRRBRRFQIYRBHBH (SEQ ID NO: 70), RBRRBRFQILYBRBH (SEQ ID NO: 71), RBRRBRFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 72), R[Hyp]RR[Hyp]RFQILYRBHBH (SEQ ID NO: 73), R[Hyp]RR[Hyp]RFQILYR[Hyp]H[Hyp]H (SEQ ID NO: 74), RBRRBRWWWRBHBH (SEQ ID NO: 75), RBRRBRWWPRBHBH (SEQ ID NO: 76), RBRRBRPWWRBHBH (SEQ ID NO: 77), RBRRBRWWPWWRBHBH (SEQ ID NO: 78), RBRRBRWWPWRBHBH (SEQ ID NO: 79), RBRRBRWPWWRBHBH (SEQ ID NO: 80), RBRRBRRWWWRBHBH (SEQ ID NO: 81), RBRRBRRWWPWWRBHBH (SEQ ID NO: 82), RBRRBRRWPWWRBHBH (SEQ ID NO: 83), RBRRBRRWWPWRBHBH (SEQ ID NO: 84), RRBRRBRFQILYRBHBH (SEQ ID NO: 85), BRRBRRFQILYRBHBH (SEQ ID NO: 86), RRBRRBRFQILYBHBH (SEQ ID NO: 87), BRRBRRFQILYBHBH (SEQ ID NO: 88), RBRRBHRFQILYRBHBH (SEQ ID NO: 89), RBRRBRFQILY[Hyp]R[Hyp]R (SEQ ID NO: 101), R[Hyp]RR[Hyp]RFQILYBRBR (SEQ ID NO: 102),Attorney Docket: 140101-00220R[Hyp]RR[Hyp]RFQILY[Hyp]R[Hyp]R (SEQ ID NO: 103), RBRRBRWWWBRBR (SEQ ID NO: 104), and RBRRBRWWPWWBRBR (SEQ ID NO: 105).

11. The antisense oligonucleotide conjugate of any one of claims 1-10, or a pharmaceutically acceptable salt thereof, wherein the cell penetrating peptide is acylated at its N-terminus.

12. The antisense oligonucleotide conjugate of any one of claims 1-11, or a pharmaceutically acceptable salt thereof, wherein the antisense oligonucleotide is conjugated to the cell penetrating peptide via a linker.

13. The antisense oligonucleotide conjugate of any one of claims 1-12, or a pharmaceutically acceptable salt thereof, wherein the conjugate comprises any one of the following structures:[cell penetrating peptide] — [oligonucleotide]; or[oligonucleotide] — [cell penetrating peptide]; or[cell penetrating peptide] — [linker] — [oligonucleotide]; or [oligonucleotide] — [linker] — [cell penetrating peptide]; orAttorney Docket: 140101-00220[oligonucleotide][cell penetrating peptide]^"[oligonucleotide]or[oligonucleotide]-^.; [cell penetrating peptide][oligonucleotide]or[oligonucleotide][cell penetrating peptide] - [linker]1^[oligonucleotide]or[linker] - [oligonucleotide][cell penetrating peptide]^[linker] - [oligonucleotide]or[oligonucleotide]^[linker] - [cell penetrating peptide] [oligonucleotide]or[oligonucleotide] - [linker] *^, „..,[cell penetrating peptide] [oligonucleotide] - [linker] '14. The antisense oligonucleotide conjugate of any one of claims 1-13, or a pharmaceutically acceptable salt thereof, wherein the linker comprises:

15. The antisense oligonucleotide conjugate of any one of claims 1-14, or a pharmaceutically acceptable salt thereof, wherein the conjugate is any one of the following structures:Attorney Docket: 140101-00220[cell penetrating peptide] [oligonucleotide]o[cell penetrating peptide]N[oligonucleotide][ceil penetrating peptide] [oligonucleotide], or16. A pharmaceutical composition comprising the antisense oligonucleotide conjugate of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

17. A method of inhibiting GYSI expression in a cell, the method comprising contacting the cell with the antisense oligonucleotide conjugate of any one of claims 1-15, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 16, thereby inhibiting GYSI expression in the cell.

18. The method of claim 17, wherein the cell is any cell type of the central nervous system or the peripheral nervous system.

19. The method of claim 17, wherein the cell is a muscle cell.Attorney Docket: 140101-0022020. The method of any one of claims 17-19, wherein contacting the cell with the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, results in:inhibition of expression of the GYSI gene by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%;decrease in GYSI protein levels by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%; and / ordecrease in glycogen levels in the cell by at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95%.

21. The method of any one of claims 17-20, wherein the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, inhibits GYS1 expression with an IC50 between 0.1-10 pM.

22. A method of treating glycogen storage disease (GSD) in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antisense oligonucleotide conjugate, or a pharmaceutically acceptable salt thereof, of any one of claims 1-15, or the pharmaceutical composition of claim 16, thereby treating the GSD in the subject.

23. The method of claim 22, wherein the GSD is selected from the group consisting of Lewis' disease, von Gierke’s disease, Pompe’s disease (GSD Ila and lib), Danon disease, Forbes disease / Cori disease, Andersen's disease, Adult Polyglucosan Body Disease (APBD), Lafora Disease, McArdle's disease, Hers’ disease, Tarui’s disease, phosphorylase kinase deficiency, type IX of GSD, type X of GSD, Fanconi-Bickel syndrome, type XIV of GSD, type XII of GSD, type XIII of GSD, type XV of GSD, and subtypes thereof.

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