siRNA-mediated gene silencing
The present invention is directed to small interfering RNA molecules (siRNA) targeted against an allele of interest, and methods of using these siRNA molecules.
1. A mammalian cell comprising
a first strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end, wherein the first strand is complementary to at least 15 nucleotides of a mutant Ataxin-3 transcript encoded by siC7 (5′-CTGTCCTGATAGGTCCCGCTGC-3′ SEQ ID NO: 16), siG10 (5′-CTGATAGGTCCCCCTGCTGC-3′ SEQ ID NO:18), siC7/8 (5′-CTGTCCTGATAGGTCCGGCTGC-3′ SEQ ID NO:20), or siC10 (5′-CTGATAGGTCCCGCTGCTGC-3′ SEQ ID NO:22), and wherein the 5′ end of the first strand of RNA is operably linked to a G nucleotide to form a first segment of RNA, and
a second strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end,
wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences only one allele of the targeted gene in the cell.
2. The cell of claim 1 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure.
3. The cell of claim 2 , wherein the loop structure contains from 4 to 10 nucleotides.
4. An isolated RNA duplex comprising a first strand of RNA having a 5′ end and a 3′ end, and a second strand of RNA, wherein the first strand comprises 22 nucleotides complementary to mutant Ataxin-3 transcript encoded by siC7 (5′-CTGTCCTGATAGGTCCCGCTGC-3′ SEQ ID NO: 16), siG10 (5′-CTGATAGGTCCCCCTGCTGC-3′ SEQ ID NO:18), siC7/8 (5′-CTGTCCTGATAGGTCCGGCTGC-3′ SEQ ID NO:20), or siC10 (5′-CTGATAGGTCCCGCTGCTGC-3′ SEQ ID NO:22), wherein the 5′ end of the first strand of RNA is operably linked to a G nucleotide to form a first segment of RNA, and wherein the second strand is complementary to all the nucleotides of the first strand.
5. The RNA duplex of claim 4 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure.
6. The RNA duplex of claim 4 , wherein the loop structure contains from 4 to 10 nucleotides.
7. An expression cassette comprising a nucleic acid encoding at least one strand of the RNA duplex of claim 4 .
8. A vector comprising the expression cassette of claim 7 .
9. A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding the first strand of the RNA duplex of claim 4 and a second expression cassette comprising a nucleic acid encoding the second strand of the RNA duplex of claim 4 .
10. A cell comprising the expression cassette of claim 7 .
11. An isolated RNA duplex comprising a first strand of RNA having a 5′ end and a 3′ end, and a second strand of RNA, wherein the first strand comprises 22 nucleotides complementary to mutant APP transcript encoded by siT8/C9 (5′-ATTCTGCATCCAGATTCACT-3′ SEQ ID NO: 83), siT9/C10 (5′-TTCTGCATCCAGATTCACTT-3′ SEQ ID NO:85), siT10/C11 (5′-TCTGCATCCAGATTCACTTC-3′ SEQ ID NO:86), siT11/C12 (5′-TGCATCCAGATTCACTTCA-3′ SEQ ID NO:88), or siT12/C13 (5′-TGCATCCAGATTCACTTCAG-3′ SEQ ID NO: 90), wherein the 5′ end of the first strand of RNA is operably linked to a G nucleotide to form a first segment of RNA, and wherein the second strand is complementary to all the nucleotides of the first strand.
12. The RNA duplex of claim 11 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure.
13. The RNA duplex of claim 11 , wherein the loop structure contains from 4 to 10 nucleotides.
14. An expression cassette comprising a nucleic acid encoding at least one strand of the RNA duplex of claim 11 .
15. A vector comprising the expression cassette of claim 14 .
16. A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding the first strand of the RNA duplex of claim 14 and a second expression cassette comprising a nucleic acid encoding the second strand of the RNA duplex of claim 14 .
17. A cell comprising the expression cassette of claim 14 .
18. A mammalian cell comprising
an isolated first strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end, wherein the first strand is complementary to at least 15 nucleotides of mutant APP transcript encoded by siT8/C9 (5′-ATTCTGCATCCAGATTCACT-3′ SEQ ID NO: 83), siT9/C10 (5′-TTCTGCATCCAGATTCACTT-3′ SEQ ID NO:85), siT10/C11 (5′-TCTGCATCCAGATTCACTTC-3′ SEQ ID NO:86), siT11/C12 (5′-TGCATCCAGATTCACTTCA-3′ SEQ ID NO:88), or siT12/C13 (5′-TGCATCCAGATTCACTTCAG-3′ SEQ ID NO: 90), and wherein the 5′ end of the first strand of RNA is operably linked to a G nucleotide to form a first segment of RNA, and
an isolated second strand of RNA of 15 to 30 nucleotides in length having a 5′ end and a 3′ end,
wherein at least 12 nucleotides of the first and second strands are complementary to each other and form a small interfering RNA (siRNA) duplex under physiological conditions, and wherein the siRNA silences only one allele of the targeted gene in the cell.
19. The cell of claim 18 , wherein the first strand and the second strand are operably linked by means of an RNA loop strand to form a hairpin structure comprising a duplex structure and a loop structure.
20. The cell of claim 19 , wherein the loop structure contains from 4 to 10 nucleotides.