siRNA-mediated gene silencing

Patent metadata

Field	Value
Publication number	US-9487779-B2
Application number	US-201414297378-A
Country	US
Kind code	B2
Filing date	Jun 5, 2014
Priority date	Aug 5, 2002
Publication date	Nov 8, 2016
Grant date	Nov 8, 2016

Abstract

The present invention is directed to small interfering RNA molecules (siRNA) targeted against an allele of interest, and methods of using these siRNA molecules.

First claim

What is claimed is:

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.

Assignees

• Univ Iowa Res Found

Inventors

• Davidson Beverly L
• Paulson Henry
• Miller Victor
• Gouvion Cynthia

Classifications

• C12Y302/01031

  Beta-glucuronidase (3.2.1.31) · CPC title

• A01K2217/05

  Animals comprising random inserted nucleic acids (transgenic) · CPC title

• C12N2799/021

  for the expression of a heterologous nucleic acid · CPC title

• C12N2310/111

  spanning the whole gene, or a large part of it · CPC title

• C12N15/1137

  against enzymes (viral enzymes C12N15/1131; receptors C12N15/1138) · CPC title