Claims for Patent: 8,895,721

Summary for Patent: 8,895,721

Title:	RNA interference mediating small RNA molecules
Abstract:	Double-stranded RNA (dsRNA) induces sequence-specific post-transcriptional gene silencing in many organisms by a process known as RNA interference (RNAi). Using a Drosophila in vitro system, we demonstrate that 19-23 nt short RNA fragments are the sequence-specific mediators of RNAi. The short interfering RNAs (siRNAs) are generated by an RNase III-like processing reaction from long dsRNA. Chemically synthesized siRNA duplexes with overhanging 3' ends mediate efficient target RNA cleavage in the lysate, and the cleavage site is located near the center of the region spanned by the guiding siRNA. Furthermore, we provide evidence that the direction of dsRNA processing determines whether sense or antisense target RNA can be cleaved by the produced siRNP complex.
Inventor(s):	Tuschl; Thomas (Brooklyn, NY), Elbashir; Sayda Mahgoub (Cambridge, MA), Lendeckel; Winfried (Hohengandern, DE)
Assignee:	Max-Planck-Gesellschaft zur Forderung der Wissenschaften E.V. (Munich, DE) Massachusetts Institute of Technology (Cambridge, MA) Whitehead Institute for Biomedical Research (Cambridge, MA) University of Massachusetts (Boston, MA)
Application Number:	13/725,262
Patent Claims:	1. An isolated double-stranded RNA molecule, comprising: (i) a sense strand and an antisense strand that form a double-stranded region consisting of 14-24 base pairs; (ii) at least one strand having a single-stranded 3'-overhang; and (iii) at least one nucleotide analogue, wherein said RNA molecule is non-enzymatically processed and is capable of target-specific RNA interference, and said sense strand has an identity in the double-stranded region of at least 85 percent to a target RNA molecule. 2. The RNA molecule of claim 1, wherein the double-stranded region consists of 16-22 base pairs. 3. The RNA molecule of claim 1, wherein the double-stranded region consists of 20-23 base pairs. 4. The RNA molecule of claim 1, wherein the double-stranded region consists of 19 base pairs. 5. The RNA molecule of claim 1, wherein one end of the RNA molecule is blunt-ended. 6. The RNA molecule of claim 1, wherein the length of the sense strand and the antisense strand is the same. 7. The RNA molecule of claim 1, wherein the length of the sense strand and the antisense strand is different. 8. The RNA molecule of claim 1, wherein each strand independently consists of up to 25 nucleotides in length. 9. The RNA molecule of claim 1, wherein each strand independently consists of 19-25 nucleotides in length. 10. The RNA molecule of claim 2, wherein each strand independently consists of 19-23 nucleotides in length. 11. The RNA molecule of claim 2, wherein one or both strands consist of 21 nucleotides in length. 12. The RNA molecule of claim 1, which comprises two 3'-overhangs on each of the ends of the RNA molecule. 13. The RNA molecule of claim 12, wherein the length of the 3'-overhang is the same for each strand. 14. The RNA molecule of claim 12, wherein the length of the 3'-overhang is different for each strand. 15. The RNA molecule of claim 1, wherein the 3'-overhang is stabilized against degradation. 16. The RNA molecule of claim 1, wherein the 3'-overhang is of 1-5 nucleotides in length. 17. The RNA molecule of claim 1, wherein the 3'-overhang is of 1-3 nucleotides in length. 18. The double-stranded RNA molecule of claim 1, wherein the 3'-overhang is 2 nucleotides in length. 19. The RNA molecule of claim 1, wherein the nucleotide analogue is located at a position where the target-specific RNA interference is not substantially affected. 20. The RNA molecule of claim 1, wherein the nucleotide analogue is located at the 5'-end, the 3'-end, or both, of the RNA molecule. 21. The RNA molecule of claim 1, wherein the 3'-overhang comprises at least one nucleotide analogue. 22. The RNA molecule of claim 1, wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof. 23. The RNA molecule of claim 1, wherein the nucleotide analogue is a sugar-modified ribonucleotide. 24. The RNA molecule of claim 1, wherein the nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, N(R).sub.2 or CN, wherein R is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6 alkynyl and halo is F, Cl, Br or I. 25. The RNA molecule of claim 1, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphorothioate group. 26. The RNA molecule of claim 21, wherein the nucleotide analogue of the 3'-overhang is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof. 27. The RNA molecule of claim 21, wherein the nucleotide analogue of the 3'-overhang is a sugar-modified ribonucleotide. 28. The RNA molecule of claim 21, wherein the nucleotide analogue of the 3'-overhang is a backbone-modified ribonucleotide containing a phosphorothioate group. 29. The RNA molecule of claim 1, wherein the nucleotide analogue is a nucleobase-modified ribonucleotide. 30. The RNA molecule of claim 1, wherein the sense strand is at least 85 percent identical to the target RNA molecule. 31. The RNA molecule of claim 1, wherein the sense strand has 100% identity in the double-stranded region to a target RNA molecule. 32. The RNA molecule of claim 1, wherein the antisense strand is complementary to a target RNA molecule. 33. An isolated double-stranded RNA molecule, comprising: (i) a sense strand and an antisense strand that form a double-stranded region of up to 25 base pairs, said sense strand having an identity in the double-stranded region of at least 85 percent to a target RNA molecule; and (ii) at least one strand having a single-stranded 3'-overhang, wherein said 3'-overhang has been stabilized against degradation; and (iii) at least one nucleotide analogue, wherein said RNA molecule is capable of target-specific RNA interference. 34. The RNA molecule of claim 33, wherein the double-stranded regions consists of 20-23 base pairs, or 20-25 base pairs. 35. The RNA molecule of claim 33, wherein the double-stranded regions consists of 19 base pairs. 36. The RNA molecule of claim 33, wherein one end of the RNA molecule is blunt-ended. 37. The RNA molecule of claim 33, wherein the length of the sense strand and the antisense strand is the same. 38. The RNA molecule of claim 33, wherein the length of the sense strand and the antisense strand is different. 39. The RNA molecule of claim 33, wherein each strand independently consists of up to 25 nucleotides in length. 40. The RNA molecule of claim 33, wherein each strand independently consists of 19-25 nucleotides in length. 41. The RNA molecule of claim 33, wherein each strand independently consists of 19-23 nucleotides in length. 42. The RNA molecule of claim 33, which comprises two 3'-overhangs on each of the ends of the RNA molecule. 43. The RNA molecule of claim 42, wherein the length of the 3'-overhang is the same for each strand. 44. The RNA molecule of claim 42, wherein the length of the 3'-overhang is different for each strand. 45. The RNA molecule of claim 33, wherein the 3'-overhang is of 1-5 nucleotides in length. 46. The RNA molecule of claim 33, wherein the 3'-overhang is of 1-3 nucleotides in length. 47. The RNA molecule of claim 33, wherein the 3'-overhang is 2 nucleotides in length. 48. The RNA molecule of claim 33, wherein the nucleotide analogue is located at a position where the target-specific RNA interference is not substantially affected. 49. The RNA molecule of claim 33, wherein the nucleotide analogue is located at the 5'-end, the 3'-end, or both, of the RNA molecule. 50. The RNA molecule of claim 33, wherein the 3'-overhang comprises at least one nucleotide analogue. 51. The RNA molecule of claim 33, wherein the nucleotide analogue is selected from a sugar- or a backbone-modified ribonucleotide, or a combination thereof. 52. The RNA molecule of claim 33, wherein the nucleotide analogue is a sugar-modified ribonucleotide. 53. The RNA molecule of claim 33, wherein the modified nucleotide analogue is a sugar-modified ribonucleotide, wherein the 2'-OH group is replaced by a group selected from H, OR, R, halo, SH, SR, NH.sub.2, NHR, N(R).sub.2 or CN, wherein R is C.sub.1-C.sub.6 alkyl, C.sub.2-C.sub.6 alkenyl or C.sub.2-C.sub.6 alkynyl and halo is F, Cl, Br or I. 54. The RNA molecule of claim 33, wherein the nucleotide analogue is a backbone-modified ribonucleotide containing a phosphothioate group. 55. The RNA molecule of claim 33, wherein the sense strand is at least 85 percent identical to the target RNA molecule. 56. The RNA molecule of claim 33, wherein the sense strand has 100% identity in the double-stranded region to the target RNA molecule. 57. The RNA molecule of claim 33, wherein the antisense strand is complementary to the target RNA molecule. 58. A pharmaceutical composition comprising at least one RNA molecule of claim 1 and a pharmaceutical carrier. 59. A pharmaceutical composition comprising at least one RNA molecule of claim 33 and a pharmaceutical carrier. 60. The RNA molecule of claim 1, which is chemically synthesized. 61. The RNA molecule of claim 33, which is chemically synthesized. 62. The RNA molecule of claim 1, which mediates RNA interference of a mammalian RNA. 63. The RNA molecule of claim 33, which mediates RNA interference of a mammalian RNA. 64. The RNA molecule of claim 1, which mediates RNA interference of a human RNA. 65. The RNA molecule of claim 33, which mediates RNA interference of a human RNA. 66. The RNA molecule of claim 1, which mediates RNA interference of a plant RNA. 67. The RNA molecule of claim 33, which mediates RNA interference of a plant RNA. 68. The RNA molecule of claim 1, which mediates RNA interference of a target gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene. 69. The RNA molecule of claim 33, which mediates RNA interference of a target gene chosen from a pathogen-associated gene, a viral gene, a tumor-associated gene, or an autoimmune disease-associated gene. 70. The RNA molecule of claim 1, wherein the nucleotide analogue comprises a modified nucleobase or a non-naturally-occurring nucleobase. 71. The RNA molecule of claim 33, wherein the nucleotide analogue comprises a modified nucleobase or a non-naturally-occurring nucleobase. 72. The RNA molecule of claim 1, wherein the nucleotide analogue comprises a modified sugar or a non-naturally occurring sugar. 73. The RNA molecule of claim 33, wherein the nucleotide analogue comprises a modified sugar or a non-naturally occurring sugar. 74. The RNA molecule of claim 1, wherein the nucleotide analogue comprises a modified or a non-naturally occurring nucleoside. 75. The RNA molecule of claim 33, wherein the nucleotide analogue comprises a modified or a non-naturally occurring nucleoside. 76. An isolated double-stranded RNA molecule, comprising: (i) a sense strand and an antisense strand that form a double-stranded region consisting of 14-24 base pairs; (ii) at least one strand having a single-stranded 3'-overhang; and (iii) at least one nucleobase analogue, wherein said RNA molecule is non-enzymatically processed and is capable of target-specific RNA interference, and said sense strand is at least 85 percent to a target RNA molecule. 77. The RNA molecule of claim 1, wherein the sense strand has 100% identity in the double-stranded region to the target RNA molecule and the 3'-overhang is 1-5 nucleotides in length. 78. The RNA molecule of claim 33, wherein the sense strand has 100% identity in the double-stranded region to the target RNA molecule and the 3'-overhang is 1-5 nucleotides in length. 79. The RNA molecule of claim 76, wherein the sense strand has 100% identity in the double-stranded region to the target RNA molecule and the 3'-overhang is 1-5 nucleotides in length.