Allele selective inhibition of mutant c9orf72 foci expression by duplex rnas targeting the expanded hexanucleotide repeat

Abstract

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).

1 . 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). 
     
     
         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 . (canceled) 
     
     
         8 . The double-stranded oligonucleotide of  claim 4 , wherein the modified sugar moiety is a 4′ to 2′ bicyclic sugar moiety. 
     
     
         9 - 12 . (canceled) 
     
     
         13 . The double-stranded oligonucleotide of  claim 1 , wherein said double-stranded oligonucleotide comprises terminal dT residues. 
     
     
         14 . The double-stranded oligonucleotide of  claim 1 , wherein said double-stranded oligonucleotide comprises 3′ and/or ′5 2′-O-methyl modifications. 
     
     
         15 . The double-stranded oligonucleotide of  claim 1 , wherein the nucleobases are linked by phosphate internucleoside linkages. 
     
     
         16 - 17 . (canceled) 
     
     
         18 . 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. 
     
     
         19 . The double-stranded oligonucleotide of  claim 1 , wherein said double-stranded oligonucleotide is selected from the following RNAs, or a DNA cognate thereof: 
       
         
           
                 
                 
               
                     
                 
                   CGGCCCCG   AAA   CCGGCCCCdTdT (AS) 
                   SEQ ID NO: 1 
                 
                   dTdT   U   CCGGGGC   UUU   GGCCGGGG (S) 
                   SEQ ID NO: 2 
                 
                     
                 
                   CGGCCCCG   AAAA   CGGCCCCdTdT (AS) 
                   SEQ ID NO: 3 
                 
                   dTdT   U   CCGGGGC   UUUU   GCCGGGG (S) 
                   SEQ ID NO: 4 
                 
                     
                 
                   CGGCCCC   AAAAA   CGGCCCCdTdT (AS) 
                   SEQ ID NO: 5 
                 
                   dTdT   U   CCGGGGC   UUUU   GCCGGGG (S) 
                   SEQ ID NO: 6 
                 
                     
                 
                   CGGCCCCG   AAAA   CG   A   CCCCdTdT (AS) 
                   SEQ ID NO: 7 
                 
                   dTdT   U   CCGGGGC   UUUU   GCCGGGG (S) 
                   SEQ ID NO: 8 
                 
                     
                 
                   CGGCCCCG   AAAA   CG   A   CC   A   CdTdT (AS) 
                   SEQ ID NO: 9 
                 
                   dTdT   U   CCGGGGC   UUUU   GCCGGGG (S) 
                   SEQ ID NO: 10 
                 
                     
                 
                   CGGCCCCG   AA   CC   A   GG   A   CCCdTdT (AS) 
                   SEQ ID NO: 11 
                 
                   dTdT   U   CCGGGGC   UU   GG   U   CC   U   GGG (S) 
                   SEQ ID NO: 12 
                 
                     
                 
                   CGGCCCCG   AA   CC   A   G   A   CCCCdTdT (AS) 
                   SEQ ID NO: 13 
                 
                   dTdT   U   CCGGGGC   UU   GG   U   CC   U   GGG (S) 
                   SEQ ID NO: 14 
                 
                     
                 
                   CGGCCCCG   AAA   CCG   A   CCCCdTdT (AS) 
                   SEQ ID NO: 15 
                 
                   dTdT   U   CCGGGGC   UUU   GGCCGGGG (S) 
                   SEQ ID NO: 16 
                 
                     
                 
                   CGGCCCCG   AA   CCCG A CCCCdTdT (AS) 
                   SEQ ID NO: 17 
                 
                   dTdT   U   CCGGGGC   UU   GGGC   U   GGGG (S) 
                   SEQ ID NO: 18 
                 
                     
                 
                   CGGCCCCG   AAA   CCG   A   CCCCdTdT (AS) 
                   SEQ ID NO: 19 
                 
                   dTdT   U   CCG   A   GGC   UUU   GGCCGGGG (S) 
                   SEQ ID NO: 20 
                 
                     
                 
                   CGGCCCCG   AAA   CCGGCCC   U   dTdT (AS) 
                   SEQ ID NO: 21 
                 
                   dTdTGCCG   A   GGC   UUU   GGCCGGGG (S) 
                   SEQ ID NO: 22 
                 
                     
                 
                   CGGCCCCG   AAA   CCGGCCC   U   dTdT (AS) 
                   SEQ ID NO: 23 
                 
                   dTdTGCCG   A   G   A   CC   UU   GGCCGGGG (S) 
                   SEQ ID NO: 24 
                 
                     
                 
             
                
               
               
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         20 . The double-stranded oligonucleotide of  claim 1 , wherein said central mismatches comprise one or more abasic or unlocked nucleotides. 
     
     
         21 . 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). 
     
     
         22 . The method of  claim 21 , wherein the expanded GGGGCC repeat region contains 500 or more repeats. 
     
     
         23 . The method of  claim 21 , wherein the expanded GAA repeat region contains about 700 to 1600 repeats. 
     
     
         24 . The method of  claim 21 , where said cell is contacted with said double-stranded oligonucleotide at about 5-75 nM. 
     
     
         25 . The method of  claim 21 , wherein the cell is located in a subject suffering from a GGGGCC repeat disease. 
     
     
         26 . The method of  claim 25 , 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. 
     
     
         27 . (canceled) 
     
     
         28 . The method of  claim 25 , further comprising administering a second therapeutic agent to said subject. 
     
     
         29 . The method of  claim 25 , wherein said subject has or exhibits a symptom of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). 
     
     
         30 . The method of  claim 29 , wherein ALS/FTD foci in the brain tissue of said subject are reduced in number or size.