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 having a 5′ end and a 3′ end, wherein the first strand is complementary to at least 15 nucleotides of a targeted gene of interest, and
a second strand of RNA having a 5′ end and a 3′ end,
wherein
(i) the first strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 49 and the second strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 50;
(ii) the first strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 51 and the second strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 52; or
(iii) the first strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 53 and the second strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 54; and
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, and a second strand of RNA,
wherein at least 12 nucleotides of the first and second strands are complementary to each other; and wherein
(i) the first strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 49 and the second strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 50;
(ii) the first strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 51 and the second strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 52; or
(iii) the first strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 53 and the second strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 54; and
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.
5. The RNA duplex of claim 4 , wherein the loop structure contains from 4 to 10 nucleotides.
6. An expression cassette comprising a nucleic acid encoding at least one strand of an isolated RNA duplex comprising a first strand of RNA, and a second strand of RNA,
wherein at least 12 nucleotides of the first and second strands are complementary to each other; and wherein
(i) the first strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 49 and the second strand is 18 to 30 nucleotides in length and comprises SEQ ID NO: 50;
(ii) the first strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 51 and the second strand is 20 to 30 nucleotides in length and comprises SEQ ID NO: 52; or
(iii) the first strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 53 and the second strand is 23 to 30 nucleotides in length and comprises SEQ ID NO: 54.
7. A vector comprising the expression cassette of claim 6 .
8. A vector comprising two expression cassettes, a first expression cassette comprising a nucleic acid encoding the first strand of the RNA duplex of claim 6 and a second expression cassette comprising a nucleic acid encoding the second strand of the RNA duplex of claim 6 .
9. A cell comprising the expression cassette of claim 6 .