CCCTC-Binding Factor (CTCF) RNA Interactome
This invention relates to methods and compositions for selectively reactivating or downregulating certain genes, e.g., genes regulated by zinc-finger protein CCCTC-binding factor (CTCF) on autosomes (e.g., imprinted genes, tumor suppressors, cancer) and the inactive X chromosome (Xi), e.g., genes associated with X-linked diseases, e.g., Rett Syndrome, Factor VIII or IX deficiency, Fragile X Syndrome, Duchenne muscular dystrophy, and PNH, in heterozygous females carrying a mutated allele, in addition to a functional wildtype or hypomorphic allele.
1 . A method of activating an inactive X-linked allele in a cell, preferably a cell of a female heterozygous subject, the method comprising administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA.
2 . A method of activating a repressed autosomal gene in a cell, the method comprising administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that represses the autosome or the autosomal gene.
3 . A method of downregulating an X-linked escapee gene in a cell, the method comprising administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that increases expression of the X-linked escapee gene.
4 . A method of repressing an autosomal gene in a cell, the method comprising administering to the cell an inhibitory oligonucleotide targeting a sequence within 500 nucleotides of a CTCF binding site on a CTCF-interacting RNA that increases expression of the autosomal gene.
5 . The method of claim 1 , wherein the inactive X-linked allele is associated with an X-linked disorder, and the oligonucleotide is administered in a therapeutically effective amount.
6 . The method of claim 1 , wherein the cell is in a living subject.
7 . The method of claim 1 , wherein the inhibitory oligonucleotide is identical or complementary to at least 8 consecutive nucleotides of a strong or moderate binding site nucleotide sequence as set forth in Tables 1-2, or complementary to at least 8 consecutive nucleotides of a caRNA as set forth in Tables 1-2.
8 . The method of claim 1 , wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
9 . The method of claim 1 , wherein the oligonucleotide does not comprise four or more consecutive guanosine nucleotides.
10 . The method of claim 1 , wherein the oligonucleotide is 8 to 30 nucleotides in length.
11 . The method of claim 1 , wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue or a 2′ O-methyl.
12 . The method of claim 1 , wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
13 . The method of claim 12 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
14 . The method of claim 1 , wherein one or more of the nucleotides of the oligonucleotide comprise 2′-fluoro-deoxyribonucleotides, one or more of the nucleotides of the oligonucleotide comprise 2′-O-methyl nucleotides, one or more of the nucleotides of the oligonucleotide comprise ENA nucleotide analogues, and/or one or more of the nucleotides of the oligonucleotide comprise LNA nucleotides.
15 . The method of claim 1 , wherein the nucleotides of the oligonucleotide comprise comprising phosphorothioate internucleotide linkages between at least two nucleotides or between all nucleotides.
16 . An inhibitory oligonucleotide that is complementary or identical to at least 8 consecutive nucleotides of a CTCF binding site nucleotide sequence as set forth in Tables 1-2.
17 . The oligonucleotide of claim 16 , wherein the oligonucleotide does not comprise three or more consecutive guanosine nucleotides.
18 . The oligonucleotide of claim 16 , wherein the oligonucleotide is 8 to 30 nucleotides in length.
19 . The oligonucleotide of claim 16 , wherein at least one nucleotide of the oligonucleotide is a nucleotide analogue or at least one nucleotide of the oligonucleotide comprises a 2′ O-methyl.
20 . The oligonucleotide of claim 16 , wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, or at least one bridged nucleotide.
21 . The oligonucleotide of claim 20 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
22 . The oligonucleotide of claim 16 , wherein one or more of the nucleotides of the oligonucleotide comprise 2′-fluoro-deoxyribonucleotides or 2′-O-methyl nucleotides.
23 . The oligonucleotide of claim 16 , wherein one or more of the nucleotides of the oligonucleotide comprise ENA nucleotide analogues, and/or one or more of the nucleotides of the oligonucleotide comprise LNA nucleotides.
24 . The method of claim 1 , wherein the nucleotides of the oligonucleotide comprise phosphorothioate internucleotide linkages between at least two nucleotides.
25 . The method of claim 1 , wherein the nucleotides of the oligonucleotide comprise phosphorothioate internucleotide linkages between all nucleotides.
26 . The method of claim 1 , wherein the oligonucleotide is a gapmer or a mixmer.
27 . The oligonucleotide of claim 16 , which is a gapmer or a mixmer.
28 . A method of increasing expression of a selected gene listed in Tables 1 or 2 in a cell, the method comprising contacting the cell with a nucleic acid triplex-forming oligonucleotide (TFO) that binds specifically to a CTCF localization sequence or binding site associated with the selected gene.
29 . The method of claim 28 , wherein the TFO comprises one or more of DNA, RNA, PNA, HNA, MNA, ANA, LNA, CAN, INA, CeNA, TNA, (2′-NH)-TNA, (3′-NH)-TNA, alpha-L-Ribo-LNA, alpha-L-Xylo-LNA, beta-D-Ribo-LNA, beta-D-Xylo-LNA, [3.2.1]-LNA, Bicyclo-DNA, 6-Amino-Bicyclo-DNA, 5-epi-Bicyclo-DNA, alpha-Bicyclo-DNA, Tricyclo-DNA, Bicyclo[4.3.0]-DNA, Bicyclo[3.2.1]-DNA, Bicyclo[4.3.0]amide-DNA, beta-D-Ribopyranosyl-NA, alpha-L-Lyxopyranosyl-NA, 2′-R-RNA, 2′-OR-RNA, 2′-AE-RNA, alpha-L-RNA, and beta-D-RNA.
30 . The method of claim 29 , wherein the TFO includes one or more modifications described herein.