TARGETING OLIGONUCLEOTIDES AND RELATED METHODS FOR MODULATING FXN RNA
Aspects of the invention relate to methods for increasing gene expression in a targeted manner. In some embodiments, methods and compositions are provided that are useful for posttranscriptionally altering protein and/or RNA levels in a targeted manner. Aspects of the invention disclosed herein provide methods and compositions that are useful for protecting RNAs from degradation (e.g., exonuclease mediated degradation).
1 . A method of increasing FXN gene expression in a cell, the method comprising:
delivering to a cell an oligonucleotide comprising the general formula 5′-X 1 -X 2 -3′, wherein X 1 comprises 5 to 20 nucleotides that have a region of complementarity that is complementary with at least 5 contiguous nucleotides of an RNA transcript encoded by the FXN gene, wherein the nucleotide at the 3′-end of the region of complementary of X 1 is complementary with the nucleotide at the transcription start site of the RNA transcript; and X 2 comprises 1 to 20 nucleotides.
2 - 3 . (canceled)
4 . The method of claim 1 , wherein at least the first nucleotide at the 5′-end of X 2 is a pyrimidine complementary with guanine.
5 . (canceled)
6 . The method of claim 1 , wherein X 2 comprises the formula 5′-Y 1 -Y 2 -Y 3 -3′, wherein X 2 forms a stem-loop structure having a loop region comprising the nucleotides of Y 2 and a stem region comprising at least two contiguous nucleotides of Y 1 hybridized with at least two contiguous nucleotides of Y 3 .
7 . The method of claim 6 , wherein Y 1 , Y 2 and Y 3 independently comprise 1 to 10 nucleotides.
8 . The method of claim 6 or 7 , wherein Y 3 comprises, at a position immediately following the 3′-end of the stem region, a pyrimidine complementary with guanine.
9 . (canceled)
10 . The method of claim 1 , wherein X 2 comprises a region of complementarity that is complementary with at least 5 contiguous nucleotides of the RNA transcript that do not overlap the region of the RNA transcript that is complementary with the region of complementarity of X 1 .
11 . The method of claim 10 , wherein the region of complementarity of X 2 is within 100 nucleotides of a polyadenylation junction of the RNA transcript.
12 . The method of claim 11 , wherein the region of complementarity of X 2 is complementary with the RNA transcript immediately adjacent to or overlapping the polyadenylation junction of the RNA transcript.
13 . The method of claim 11 , wherein X 2 further comprises at least 2 consecutive pyrimidine nucleotides complementary with adenine nucleotides of the poly(A) tail of the RNA transcript.
14 . (canceled)
15 . The method of claim 1 , wherein the RNA transcript is an mRNA transcript, and wherein X 2 comprises a region of complementarity that is complementary with at least 5 contiguous nucleotides in the 3′-UTR of the transcript.
16 . The method of claim 1 , wherein the RNA transcript is an mRNA and the delivery results in an increase in the level of a protein encoded by the mRNA.
17 - 31 . (canceled)
31 . A method of increasing FXN gene expression in a cell, the method comprising delivering to a cell an oligonucleotide comprising the general formula 5′-X 1 -X 2 -3′, wherein X 1 comprises 2 to 20 pyrimidine nucleotides that form base pairs with adenine; and X 2 comprises a region of complementarity that is complementary with at least 3 contiguous nucleotides of a poly-adenylated RNA transcript encoded by the FXN gene, wherein the nucleotide at the 5′-end of the region of complementary of X 2 is complementary with the nucleotide of the RNA transcript that is immediately internal to the poly-adenylation junction of the RNA transcript.
32 . The method of claim 31 , wherein X 1 comprises 2 to 20 thymidines or uridines.
33 . The method of claim 31 , wherein the oligonucleotide comprises at least one modified internucleoside linkage.
34 . The method of claim 31 , wherein the oligonucleotide comprises at least one modified nucleotide.
35 . The method of claim 31 , wherein at least one nucleotide comprises a 2′ O-methyl.
36 . The method of claim 31 , wherein the oligonucleotide comprises at least one ribonucleotide, at least one deoxyribonucleotide, at least one 2′-fluoro-deoxyribonucleotides or at least one bridged nucleotide.
37 . The method of claim 36 , wherein the bridged nucleotide is a LNA nucleotide, a cEt nucleotide or a ENA modified nucleotide.
38 - 39 . (canceled)
40 . The method of claim 31 , wherein the oligonucleotide is mixmer.
41 - 42 . (canceled)
64 . An oligonucleotide comprising the general formula 5′-X 1 -X 2 -3′, wherein
X 1 comprises 5 to 20 nucleotides that have a region of complementarity that is complementary with at least 5 contiguous nucleotides of an RNA transcript of an FXN gene, wherein the nucleotide at the 3′-end of the region of complementary of X 1 is complementary with the nucleotide at the transcription start site of the RNA transcript; and X 2 comprises 1 to 20 nucleotides.
65 . (canceled)
66 . The oligonucleotide of claim 64 , wherein the RNA transcript has a 7-methylguanosine cap, and wherein the nucleotide at the 3′-end of the region of complementary of X 1 is complementary with the nucleotide of the RNA transcript that is immediately internal to the 7-methylguanosine cap.
67 . The oligonucleotide of claim 64 , wherein at least the first nucleotide at the 5′-end of X 2 is a pyrimidine complementary with guanine.
68 . (canceled)
69 . The oligonucleotide of claim 64 , wherein X 2 comprises the formula 5′-Y 1 -Y 2 -Y 3 -3′, wherein X 2 forms a stem-loop structure having a loop region comprising the nucleotides of Y 2 and a stem region comprising at least two contiguous nucleotides of Y 1 hybridized with at least two contiguous nucleotides of Y 3 .
70 . (canceled)
71 . The oligonucleotide of claim 69 , wherein Y 3 comprises, at a position immediately following the 3′-end of the stem region, a pyrimidine complementary with guanine.
72 . (canceled)
73 . The oligonucleotide of claim 64 , wherein X 2 comprises a region of complementarity that is complementary with at least 5 contiguous nucleotides of the RNA transcript that do not overlap the region of the RNA transcript that is complementary with the region of complementarity of X 1 .
74 . The oligonucleotide of claim 73 , wherein the region of complementarity of X 2 is within 100 nucleotides of a polyadenylation junction of the RNA transcript.
75 . The oligonucleotide of claim 74 , wherein the region of complementarity of X 2 is complementary with the RNA transcript immediately adjacent to or overlapping the polyadenylation junction of the RNA transcript.
76 - 77 . (canceled)
78 . The oligonucleotide of claim 64 , wherein the RNA transcript is an mRNA transcript, and wherein X 2 comprises a region of complementarity that is complementary with at least 5 contiguous nucleotides in the 3′-UTR of the transcript.
79 - 91 . (canceled)
92 . An oligonucleotide comprising the general formula 5′-X 1 -X 2 -3′, wherein
X 1 comprises 2 to 20 pyrimidine nucleotides that form base pairs with adenine; and X 2 comprises a region of complementarity that is complementary with at least 3 contiguous nucleotides of a poly-adenylated RNA transcript of an FXN gene, wherein the nucleotide at the 5′-end of the region of complementary of X 2 is complementary with the nucleotide of the RNA transcript that is immediately internal to the poly-adenylation junction of the RNA transcript.
93 . The oligonucleotide of claim 92 , wherein X 1 comprises 2 to 20 thymidines or uridines.
94 - 155 . (canceled)