Methods and compositions for enhancing the efficacy and specificity of RNA silencing
The present invention provides methods of enhancing the efficacy and specificity of RNA silencing. The invention also provides compositions for mediating RNA silencing. In particular, the invention provides siRNAs, siRNA-like molecules, shRNAs, vectors and transgenes having improved specificity and efficacy in mediating silencing of a target gene. Therapeutic methods are also featured.
1. An asymmetrical substituted dsRNA silencing agent comprising one or more mismatched base pairs, the dsRNA silencing agent comprising a sense strand and an antisense strand, each strand having a 5′ end and a 3′ end, wherein the one or more mismatched base pairs are within 5 base pairs from the antisense strand 5′ (AS 5′) and the sense strand 3′ (S 3′) end, such that entry of the antisense strand into a RISC complex is promoted relative to the sense strand; such that the antisense strand preferentially guides cleavage of a desired target mRNA by the RISC complex, wherein the asymmetrical substituted dsRNA silencing agent is made by:
(a) selecting a first dsRNA silencing agent comprising a sense strand and an antisense strand, each strand having a 5′ and a 3′ end, wherein the first dsRNA silencing agent directs cleavage by a RISC complex at a phosphodiester bond within a desired target mRNA, the first dsRNA silencing agent having a base pairing strength within 5 base pairs from the antisense strand 5′ (AS 5′) and the sense strand 3′ end (S 3′) relative to a base pairing strength within 5 base pairs from the antisense strand 3′ (AS 3′) and the sense strand 5′ end (S 5′), and
(b) synthesizing the substituted dsRNA silencing agent comprising one or more substituted base pairs with respect to the first dsRNA silencing agent, wherein the substituted dsRNA silencing agent comprises a sense strand and an antisense strand, each strand having a 5′ and a 3′ end, and wherein the substituted dsRNA silencing agent directs cleavage by the RISC complex at the same phosphodiester bond within the desired target mRNA as the first dsRNA silencing agent, the substituted dsRNA silencing agent having a base pairing strength within 5 base pairs from the AS 5′ and the S 3′ end and a base pairing strength within 5 base pairs from the AS 3′ and the S 5′ end, wherein the one or more substituted base pairs comprise at least one mismatched base pair and are within 5 base pairs from the AS 5′ and the S 3′ end of the substituted dsRNA silencing agent,
such that the base pairing strength within 5 base pairs from the AS 5′ and the S 3′ end of the substituted dsRNA silencing agent relative to the base pairing strength within 5 base pairs from the AS 3′ and the S ′5 end of the substituted dsRNA silencing agent is lessened as compared to the base pairing strength within AS 5′ and the S 3′ end of the first dsRNA silencing agent relative to the base pairing strength between the AS3′ and the S 5′ end of the first dsRNA silencing agent.
2. The substituted dsRNA silencing agent of claim 1 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:U, G:G, A:A, C:C and U:U.
3. The substituted dsRNA silencing agent of claim 1 , wherein the mismatched base pair is selected from the group consisting of G:A, C:A, C:T, G:G, A:A, C:C and U:T.
4. A composition comprising the substituted dsRNA silencing agent of claim 1 , formulated to facilitate entry of the substituted dsRNA silencing agent into a cell.
5. A pharmaceutical composition comprising the substituted dsRNA silencing agent of claim 1 .
6. The substituted dsRNA silencing agent of claim 1 , wherein the substituted dsRNA silencing agent is derived from an engineered precursor.
7. The substituted dsRNA silencing agent of claim 1 , wherein the substituted dsRNA silencing agent is chemically synthesized.
8. The substituted dsRNA silencing agent of claim 1 , wherein the substituted dsRNA silencing agent is enzymatically synthesized.
9. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of a cellular protein.
10. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of an extracellular protein.
11. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of a protein associated with a pathological condition.
12. The substituted dsRNA silencing agent of claim 11 , wherein the protein is a pathogen-associated protein.
13. The substituted dsRNA silencing agent of claim 11 , wherein the protein is a host protein.
14. The substituted dsRNA silencing agent of claim 11 , wherein the protein is a tumor-associated protein.
15. The substituted dsRNA silencing agent of claim 11 , wherein the protein is an autoimmune disease-associated protein.
16. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of an endogenous protein.
17. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of a heterologous protein expressed in a recombinant cell or a genetically altered organism.
18. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA specifies the amino acid sequence of a protein encoded by a transgene.
19. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA is expressed in a plant cell.
20. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA is expressed in a mammalian cell.
21. The substituted dsRNA silencing agent of claim 1 , wherein the desired target mRNA is expressed in a human cell.
22. The substituted dsRNA silencing agent of claim 1 , wherein the substituted dsRNA silencing agent is a siRNA duplex.
23. A composition comprising the siRNA duplex of claim 22 , formulated to facilitate entry of the siRNA duplex into a cell.
24. A pharmaceutical composition comprising the siRNA duplex of claim 22 .
25. An engineered pre-miRNA comprising the siRNA duplex of claim 22 .
26. A vector encoding the pre-miRNA of claim 25 .
27. A cell comprising the vector of claim 26 .
28. A pri-miRNA comprising the pre-miRNA of claim 25 .
29. A vector encoding the pri-miRNA of claim 28 .
30. A cell comprising the vector of claim 29 .
31. A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the sense and antisense strand of the siRNA duplex of claim 22 .
32. The shRNA of claim 31 , wherein the nucleotide sequence identical to the sense strand is upstream of the nucleotide sequence identical to the antisense strand.
33. The shRNA of claim 31 , wherein the nucleotide sequence identical to the antisense strand is upstream of the nucleotide sequence identical to the sense strand.
34. A vector encoding the shRNA of claim 31 .
35. A cell comprising the vector of claim 34 .
36. A transgene encoding the shRNA of claim 31 .
37. The substituted dsRNA silencing agent of claim 1 , wherein the substituted dsRNA silencing agent is a siRNA-like duplex, wherein the antisense strand is a miRNA strand and the sense strand is a miRNA* strand.
38. A composition comprising the siRNA-like duplex of claim 37 , formulated to facilitate entry of the siRNA duplex into a cell.
39. A pharmaceutical composition comprising the siRNA-like duplex of claim 37 .
40. A small hairpin RNA (shRNA) comprising nucleotide sequence identical to the miRNA and miRNA* strand of the siRNA-like duplex of claim 37 .