US2023041016A1PendingUtilityA1
Anti-slc6a1 oligonucleotides and related methods
Individually held — no corporate assignee on recordPriority: Dec 4, 2019Filed: Dec 4, 2020Published: Feb 9, 2023
Est. expiryDec 4, 2039(~13.4 yrs left)· nominal 20-yr term from priority
C12N 2320/33C12N 2310/3341C12Q 1/6834C12N 2310/322C12N 2310/11A61P 43/00C12N 2310/315C12N 15/1138C12N 2310/346C12Q 1/6806
59
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Claims
Abstract
The present disclosure provides antisense oligonucleotides that bind to a splice modulatory element target region in an SLC6A1 RNA transcript. The present disclosure provides antisense oligonucleotides that increase the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell (i.e., GABA Transporter 1, GAT-1). The present disclosure also provides methods of treating a disease or disorder associated with non-productive SLC6A1 RNA transcripts.
Claims
exact text as granted — not AI-modifiedWhat is claimed is:
1 . An antisense oligonucleotide that binds to a target region in an SLC6A1 RNA transcript, wherein the target region comprises a splice modulatory element.
2 . The antisense oligonucleotide of claim 1 , wherein binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.
3 . The antisense oligonucleotide of claim 2 , wherein the protein comprises GABA Transporter 1 (GAT-1).
4 . The antisense oligonucleotide of claim 2 , wherein the cell comprises an SLC6A1 expressing cell.
5 . The antisense oligonucleotide of claim 4 , wherein the cell comprises a neuronal cell and/or an astrocyte.
6 . The antisense oligonucleotide of claim 5 , wherein the neuronal cell comprises a GABAergic neuronal cell.
7 . The antisense oligonucleotide of any of claims 1 or 2 , wherein the splice modulatory element comprises one or more of a non-productive splice site, a exonic splicing enhancer, an exonic splicing silencer, an intronic splicing enhancer, or an intronic splicing silencer.
8 . The antisense oligonucleotide of any of claims 1 - 7 , comprising a region of complementarity to a target region of an RNA transcript corresponding to a nucleotide sequence of any one of SEQ ID NOs: 1-108.
9 . The antisense oligonucleotide of any of claims 1 - 8 , wherein the antisense oligonucleotide comprises 8 to 80 nucleotides in length.
10 . The antisense oligonucleotide of any of claims 1 - 9 , wherein the antisense oligonucleotide comprises 15 to 25 nucleotides in length.
11 . The antisense oligonucleotide of any of claims 1 - 10 , wherein the antisense oligonucleotide comprises 18 to 20 nucleotides in length.
12 . The antisense oligonucleotide of any of claims 1 - 11 , wherein the antisense oligonucleotide comprises one or more modified nucleotides.
13 . The antisense oligonucleotide of claim 12 , wherein the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
14 . The antisense oligonucleotide of claim 13 , wherein the modification of the ribose group comprises 2′-O-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH 2 , a constrained nucleotide, or a combination thereof.
15 . The antisense oligonucleotide of claim 14 , wherein the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA NC ), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof.
16 . The antisense oligonucleotide of claim 15 , wherein the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).
17 . The antisense oligonucleotide of claim 13 , wherein the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof.
18 . The antisense oligonucleotide of claim 17 , wherein the modification of the phosphate group comprises phosphorothioate.
19 . The antisense oligonucleotide of claim 13 , wherein the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N 6 -methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof.
20 . The antisense oligonucleotide of claim 19 , wherein the modification of the nucleobase group comprises 5-methylcytosine.
21 . The antisense oligonucleotide of any of claims 1 - 20 , wherein the antisense oligonucleotide further comprises a ligand.
22 . The antisense oligonucleotide of any one of claims 1 - 21 , comprising a sequence modification pattern of
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
or
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
wherein:
s represents a phosphorothioate internucleoside linkage; and
X represents an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
23 . The antisense oligonucleotide of any one of claims 1 - 22 , comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
24 . The antisense oligonucleotide of any one of claims 1 - 22 , wherein the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contains the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.
25 . An antisense oligonucleotide comprising a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.
26 . The antisense oligonucleotide of claim 25 , wherein the target region comprises a non-productive splice site.
27 . The antisense oligonucleotide of claim 25 , wherein binding of the antisense oligonucleotide to the target region increases the expression of a functional protein encoded by the SLC6A1 RNA transcript in a cell.
28 . The antisense oligonucleotide of claim 27 , wherein the protein comprises GABA Transporter 1 (GAT-1).
29 . The antisense oligonucleotide of claim 27 , wherein the cell comprises an SLC6A1 expressing cell.
30 . The antisense oligonucleotide of claim 29 , wherein the cell comprises a neuronal cell and/or an astrocyte.
31 . The antisense oligonucleotide of claim 30 , wherein the neuronal cell comprises a GABAergic neuronal cell.
32 . The antisense oligonucleotide of any of claims 25 - 31 , wherein the antisense oligonucleotide comprises 8 to 80 nucleotides in length.
33 . The antisense oligonucleotide of any of claims 25 - 32 , wherein the antisense oligonucleotide comprises 15 to 25 nucleotides in length.
34 . The antisense oligonucleotide of any of claims 25 - 33 , wherein the antisense oligonucleotide comprises 18 to 20 nucleotides in length.
35 . The antisense oligonucleotide of any of claims 25 - 34 , wherein the antisense oligonucleotide comprises one or more modified nucleotides.
36 . The antisense oligonucleotide of claim 35 , wherein the one or more modified nucleotides comprise a modification of a ribose group, a phosphate group, a nucleobase, or a combination thereof.
37 . The antisense oligonucleotide of claim 36 , wherein the modification of the ribose group comprises 2′-methyl, 2′-fluoro, 2′-deoxy, 2′-O-(2-methoxyethyl) (MOE), 2′-O-alkyl, 2′-O-alkoxy, 2′-O-alkylamino, 2′-NH 2 , a constrained nucleotide, or a combination thereof.
38 . The antisense oligonucleotide of claim 37 , wherein the constrained nucleotide comprises a locked nucleic acid (LNA), an ethyl-constrained nucleotide, a 2′-(S)-constrained ethyl (S-cEt) nucleotide, a constrained MOE, a 2′-O,4′-C-aminomethylene bridged nucleic acid (2′,4′-BNA NC ), an alpha-L-locked nucleic acid, a tricyclo-DNA, or a combination thereof.
39 . The antisense oligonucleotide of claim 38 , wherein the modification of the ribose group comprises 2′-O-(2-methoxyethyl) (MOE).
40 . The antisense oligonucleotide of claim 36 , wherein the modification of the phosphate group comprises a phosphorothioate, a phosphonoacetate (PACE), a thiophosphonoacetate (thioPACE), an amide, a triazole, a phosphonate, a phosphotriester modification, or a combination thereof.
41 . The antisense oligonucleotide of claim 40 , wherein the modification of the phosphate group comprises phosphorothioate.
42 . The antisense oligonucleotide of claim 36 , wherein the modification of the nucleobase group comprises 2-thiouridine, 4-thiouridine, N 6 -methyladenosine, pseudouridine, 2,6-diaminopurine, inosine, thymidine, 5-methylcytosine, 5-substituted pyrimidine, isoguanine, isocytosine, halogenated aromatic groups, or a combination thereof.
43 . The antisense oligonucleotide of claim 42 , wherein the modification of the nucleobase group comprises 5-methylcytosine.
44 . The antisense oligonucleotide of any of claims 25 - 43 , wherein the antisense oligonucleotide further comprises a ligand.
45 . The antisense oligonucleotide of any of claims 25 - 44 , comprising a sequence modification pattern of
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
or
XsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXsXs ,
wherein:
s represents a phosphorothioate internucleoside linkage; and
X represents an adenosine, a guanosine, a cytidine, or a thymine comprising a 2′-O-(2-methoxyethyl) modification.
46 . The antisense oligonucleotide of any one of claims 25 - 45 , comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
47 . The antisense oligonucleotide of any one of claims 25 - 46 , wherein the antisense oligonucleotide increases the level of a functional SLC6A1 RNA transcript in a cell that contain the antisense oligonucleotide by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that does not contain the antisense oligonucleotide.
48 . A multimeric antisense oligonucleotide compound comprising two or more antisense oligonucleotides of any of claims 1 - 47 , wherein the two or more antisense oligonucleotides are linked together through a linker.
49 . The multimeric antisense oligonucleotide compound of claim 48 , wherein the linker comprises a cleavable linker.
50 . The multimeric antisense oligonucleotide compound of claim 49 , wherein the cleavable linker degrades when cleaved.
51 . The multimeric antisense oligonucleotide compound of claims 49 or 50 , wherein the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage.
52 . The multimeric antisense oligonucleotide compound of claim 51 , wherein the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides.
53 . The multimeric antisense oligonucleotide compound of claims 51 or 52 , wherein the nuclease-cleavable linker comprises about 6 nucleotides.
54 . The multimeric antisense oligonucleotide compound of any one of claims 48 - 53 , wherein the cleavable linker is cleaved under reducing conditions or changing pH conditions.
55 . The multimeric antisense oligonucleotide compound of any one of claims 48 - 54 , wherein the cleavable linker is cleaved by an intracellular or endosomal nuclease.
56 . The multimeric antisense oligonucleotide compound of any one of claims of claims 48 - 55 , wherein the cleavable linker is cleaved by an intracellular or endosomal protease.
57 . The multimeric antisense oligonucleotide compound of any one of claims of claims 48 - 55 , wherein at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
58 . A combination comprising two or more antisense oligonucleotides that bind to two or more target regions in an SLC6A1 RNA transcript, wherein the two or more target regions comprise a splice modulatory element.
59 . The combination of claim 58 , wherein two or more antisense oligonucleotides are linked together through a linker.
60 . The combination of claim 59 , wherein the linker comprises a cleavable linker.
61 . The combination of claim 60 , wherein the cleavable linker degrades when cleaved.
62 . The combination of claims 60 or 61 , wherein the cleavable linker comprises a nuclease-cleavable linker comprising a phosphodiester linkage.
63 . The combination of claim 62 , wherein the nuclease-cleavable linker comprises from about 2 to about 8 nucleotides.
64 . The combination of claims 62 or 63 , wherein the nuclease-cleavable linker comprises about 6 nucleotides.
65 . The combination of any one of claims 58 - 64 , wherein the cleavable linker is cleaved under reducing conditions or changing pH conditions.
66 . The combination of any one of claims 58 - 65 , wherein the cleavable linker is cleaved by an intracellular or endosomal nuclease.
67 . The combination of any one of claims 58 - 66 , wherein the cleavable linker is cleaved by an intracellular or endosomal protease.
68 . The combination of any one of claims 58 - 67 , wherein at least one antisense oligonucleotide comprises a region of complementarity to a SLC6A1 RNA transcript target region corresponding to a nucleotide sequence of any of SEQ ID NOs: 1-108.
69 . The combination of any one of claims 58 - 68 , wherein at least one of the antisense oligonucleotides comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 110-127.
70 . A method of treating a disease or disorder characterized by haploinsufficiency of a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1 - 69 , and treating the disease or disorder.
71 . A method of treating a disease or disorder characterized by reduced expression of a functional GAT-1 protein encoded by a SLC6A1 gene, comprising administering to a subject in need thereof the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1 - 69 , and treating the disease or disorder.
72 . The method of claims 70 or 71 , wherein the disease or disorder comprises myoclonic-atonic epilepsy (MAE), epilepsy, epileptic encephalopathy, seizures, autism spectrum disorders, intellectual disability, or a combination thereof.
73 . The method of claims 70 or 71 , comprising administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides to a brain of the subject.
74 . The method of claims 70 or 71 , comprising administering the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides by intrathecal, intraventricular, intrastriatal injection or infusion, or a combination thereof.
75 . The method of claim 74 , wherein the injection or infusion comprises administration using an Ommaya reservoir, an intrathecal catheter, or a combination thereof.
76 . A method of increasing expression of a functional SLC6A1 RNA transcript in a cell, the method comprising contacting the cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1 - 69 , thereby increasing the expression of a functional transcript of the SLC6A1 RNA transcript in a cell.
77 . The method of claim 76 , wherein expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.
78 . A method of increasing expression of a protein encoded by a SLC6A1 RNA transcript in a cell, the method comprising contacting a cell with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides of any of claims 1 - 69 , thereby increasing expression of the protein.
79 . The method of claim 78 , wherein expression is increased by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, or more, relative to a cell that is not contacted with the antisense oligonucleotide, the multimeric antisense oligonucleotide compound, or combination of antisense oligonucleotides.
80 . The method of claim 78 , wherein the protein comprises GABA Transporter 1 (GAT-1).
81 . The method of any of claims 77 - 80 , wherein the cell comprises an SLC6A1 expressing cell.
82 . The method of any of claims 77 - 80 , wherein the cell comprises a neuronal cell and/or an astrocyte.
83 . The method of claim 82 , wherein the neuronal cell comprises a GABAergic neuronal cell.
84 . An antisense oligonucleotide that increases expression of a functional protein from the SLC6A1 gene by targeting the 5′- and/or 3′-untranslated regions of the SLC6A1 transcript.
85 . The antisense oligonucleotide of claim 85 , wherein the antisense oligonucleotide inhibits translation initiation from an upstream open reading frame to increase translation efficiency from the primary open reading frame.
86 . The antisense oligonucleotide of claim 85 , wherein the antisense oligonucleotide increases mRNA stability.Join the waitlist — get patent alerts
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