Allele selective inhibition of mutant C9orf72 foci expression by duplex RNAS targeting the expanded hexanucleotide repeat
View Patent ↗Provided herein are compositions and methods for reducing expression of C9orf72 transcripts in cells containing expanded intronic GGGGCC regions, including those in subjects having or at risk of developing amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Provided herein are a double-stranded oligonucleotides of 13 to 22 nucleobases in length targeting a GGGGCC expanded repeat region in an intron of C9orf72, comprises (a) 3-5 central mismatches (within bases 9-14) within a target sequence comprising the expanded repeat sequence, or (h) 3-5 mismatches outside of the seed sequence (bases 2-8 within the guide strand complementary to the expanded repeat sequence).
1. A double-stranded oligonucleotide of 18 to 22 nucleobases in length targeting a GGGGCC expanded repeat region in an intron of C9orf72, comprises (a) 3-5 central mismatches (within bases 9-14) within a target sequence comprising said expanded repeat sequence, or (b) 3-5 mismatches outside of the seed sequence (bases 2-8 within the guide strand complementary to the expanded repeat sequence).
2. The double-stranded oligonucleotide of claim 1 , wherein said oligonucleotide comprises one or more chemically-modified nucleobases.
3. The double-stranded oligonucleotide of claim 2 , wherein said one or more chemically-modified nucleobases is a nuclease-resistant modification.
4. The double-stranded oligonucleotide of claim 3 , wherein said nuclease-resistant modification is a modified sugar moiety or a modified internucleoside linkage.
5. The double-stranded oligonucleotide of claim 4 , wherein said modified sugar moiety is a high-affinity sugar modification.
6. The double-stranded oligonucleotide of claim 5 , wherein the high-affinity sugar modification is a bicyclic sugar moiety, or a 2′-modified sugar moiety.
7. The double-stranded oligonucleotide of claim 4 , wherein the modified sugar moiety is a 4′ to 2′ bicyclic sugar moiety.
8. The double-stranded oligonucleotide of claim 1 , wherein said double-stranded oligonucleotide comprises terminal dT residues.
9. The double-stranded oligonucleotide of claim 1 , wherein said double-stranded oligonucleotide comprises 3′ and/or ‘5 2’-O-methyl modifications.
10. The double-stranded oligonucleotide of claim 1 , wherein the nucleobases are linked by phosphate internucleoside linkages.
11. The double-stranded oligonucleotide of claim 1 , wherein said double-stranded oligonucleotide comprises DNA nucleobases, RNA nucleobases or a mixture of DNA and RNA nucleobases.
12. The double-stranded oligonucleotide of claim 1 , wherein said double-stranded oligonucleotide is selected from the following RNAs, or a DNA cognate thereof:
CGGCCCCGAAACCGGCCCCdTdT (AS)
SEQ ID NO: 1
dTdTUCCGGGGCUUUGGCCGGGG (S)
SEQ ID NO: 2
CGGCCCCGAAAACGGCCCCdTdT (AS)
SEQ ID NO: 3
dTdTUCCGGGGCUUUUGCCGGGG (S)
SEQ ID NO: 4
CGGCCCCAAAAACGGCCCCdTdT (AS)
SEQ ID NO: 5
dTdTUCCGGGGCUUUUGCCGGGG (S)
SEQ ID NO: 6
CGGCCCCGAAAACGACCCCdTdT (AS)
SEQ ID NO: 7
dTdTUCCGGGGCUUUUGCCGGGG (S)
SEQ ID NO: 8
CGGCCCCGAAAACGACCACdTdT (AS)
SEQ ID NO: 9
dTdTUCCGGGGCUUUUGCCGGGG (S)
SEQ ID NO: 10
CGGCCCCGAACCAGGACCCdTdT (AS)
SEQ ID NO: 11
dTdTUCCGGGGCUUGGUCCUGGG (S)
SEQ ID NO: 12
CGGCCCCGAACCAGACCCCdTdT (AS)
SEQ ID NO: 13
dTdTUCCGGGGCUUGGUCCUGGG (S)
SEQ ID NO: 14
CGGCCCCGAAACCGACCCCdTdT (AS)
SEQ ID NO: 15
dTdTUCCGGGGCUUUGGCCGGGG (S)
SEQ ID NO: 16
CGGCCCCGAACCCGACCCCdTdT (AS)
SEQ ID NO: 17
dTdTUCCGGGGCUUGGGCUGGGG (S)
SEQ ID NO: 18
CGGCCCCGAAACCGACCCCdTdT (AS)
SEQ ID NO: 19
dTdTUCCGAGGCUUUGGCCGGGG (S)
SEQ ID NO: 20
CGGCCCCGAAACCGGCCCUdTdT (AS)
SEQ ID NO: 21
dTdTGCCGAGGCUUUGGCCGGGG (S)
SEQ ID NO: 22
CGGCCCCGAAACCGGCCCUdTdT (AS)
SEQ ID NO: 23
dTdTGCCGAGACCUUGGCCGGGG (S)
SEQ ID NO: 24.
13. The double-stranded oligonucleotide of claim 1 , wherein said central mismatches comprise one or more abasic or unlocked nucleotides.
14. A method of selectively decreasing the expression of C9orf72 transcripts in a cell having an expanded GGGGCC repeat in an intron of C9orf72 comprising contacting the cell with a double-stranded oligonucleotide of 13 to 22 nucleobases in length targeting a GGGGCC expanded repeat region in an intron of C9orf72, comprises (a) 3-5 central mismatches (within bases 9-14) within a target sequence comprising said expanded repeat sequence, or (b) 3-5 mismatches outside of the seed sequence (bases 2-8 within the guide strand complementary to the expanded repeat sequence).
15. The method of claim 14 , wherein the expanded GGGGCC repeat region contains 500 or more repeats.
16. The method of claim 14 , wherein the expanded GAA repeat region contains about 700 to 1600 repeats.
17. The method of claim 14 , where said cell is contacted with said double-stranded oligonucleotide at about 5-75 nM.
18. The method of claim 14 , wherein the cell is located in a subject suffering from a GGGGCC repeat disease.
19. The method of claim 18 , wherein contacting comprises administering said double-stranded oligonucleotide by direct administration into the central nervous system, cerebrospinal fluid, or mediated uptake across the blood brain barriers, and/or administering said double-stranded oligonucleotide more than once.
20. The method of claim 18 , further comprising administering a second therapeutic agent to said subject.
21. The method of claim 18 , wherein said subject has or exhibits a symptom of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
22. The method of claim 21 , wherein ALS/FTD foci in the brain tissue of said subject are reduced in number or size.