Reduced size self-delivering RNAi compounds
The present invention relates to RNAi constructs with minimal double-stranded regions, and their use in gene silencing. RNAi constructs associated with the invention include a double stranded region of 8-14 nucleotides and a variety of chemical modifications, and are highly effective in gene silencing. The RNAi constructs may be, for instance, miRNA constructs that are miRNA modulators.
1. An isolated nucleic acid molecule comprising
a guide strand of 18-23 nucleotides in length that has complementarity to a miRNA sequence, and
a passenger strand of 8-16 nucleotides in length,
wherein the guide strand and the passenger strand form the nucleic acid molecule such that the nucleic acid molecule has a double stranded region of 10-15 nucleotides in length and a single stranded region, wherein the single stranded region is at the 3′ end of the guide strand and is 2-13 nucleotides in length and comprises at least two phosphorothioate modifications, wherein at least 50% of the pyrimidines in the nucleic acid molecule are modified, and wherein the isolated nucleic acid molecule modulates miRNA-mediated gene expression in a mammalian cell.
2. The nucleic acid molecule of claim 1 , wherein the nucleotide in position one of the guide strand has a 2′-O-methyl modification.
3. The nucleic acid molecule of claim 2 , wherein at least 60%, at least 80%, at least 90% or wherein 100% of the pyrimidines in the nucleic acid molecule are modified.
4. The nucleic acid molecule of claim 2 , wherein modified pyrimidines are 2′fluoro or 2′O methyl modified.
5. The nucleic acid molecule of claim 2 , wherein at least one U or C includes a hydrophobic modification.
6. The nucleic acid molecule of claim 5 , wherein the hydrophobic modification is a methyl or ethyl hydrophobic base modification.
7. The nucleic acid molecule of claim 2 , wherein the guide strand contains 6-8 phosphorothioate modifications.
8. The nucleic acid molecule of claim 2 , wherein the guide strand includes 4-14 phosphate modifications.
9. The nucleic acid molecule of claim 2 , wherein the single stranded region of the guide strand is 6 nucleotides long or 8 nucleotides long.
10. The nucleic acid molecule of claim 2 , wherein the double stranded region is 13 nucleotides long.
11. The nucleic acid molecule of claim 2 , wherein the double stranded nucleic acid molecule has one end that is blunt or includes a one nucleotide overhang.
12. The nucleic acid molecule of claim 2 , wherein the passenger strand is linked at the 3′ end to a sterol.
13. The nucleic acid molecule of claim 2 , wherein the isolated double stranded nucleic acid molecule is an miRNA mimic and wherein the miRNA sequence to which the guide strand is complementary is a miRNA recognition element.
14. The nucleic acid molecule of claim 2 , wherein the isolated double stranded nucleic acid molecule is an miRNA inhibitor and wherein the miRNA sequence to which the guide strand is complementary is an antisense strand of a mature miRNA.
15. The nucleic acid molecule of claim 14 , wherein the guide strand is at least 50% chemically modified.
16. The nucleic acid molecule of claim 14 , wherein the mature miRNA is miR 17-92.
17. A method for modulating miRNA-mediated gene expression in a mammalian cell, comprising contacting the mammalian cell with an isolated double stranded nucleic acid molecule of claim 1 in an effective amount to modulate miRNA-mediated gene expression.
18. The method of claim 17 , wherein the mammalian cell is contacted with the isolated nucleic acid in vivo or ex vivo.
19. A method for modulating miRNA-mediated gene expression in a stem cell, comprising contacting the stem cell with an isolated double stranded nucleic acid molecule of claim 1 in an effective amount to modulate miRNA-mediated gene expression in the stem cell.
20. The nucleic acid molecule of claim 2 , wherein the 2′-O-methyl modification is a 5P-2′O-methyl U modification.
21. The nucleic acid molecule of claim 2 , wherein a plurality of U's and/or C's include a hydrophobic modification.
22. The nucleic acid molecule of claim 2 , wherein the 3′ terminal 10 nucleotides of the guide strand include at least eight phosphorothioate modifications.
23. The nucleic acid molecule of claim 12 , wherein the sterol is cholesterol.
24. The nucleic acid molecule of claim 13 , wherein the miRNA mimic is a mimic of a miRNA selected from the group consisting of miR21, miR 139, miR 7, miR29, miR 122, miR 302-367 cluster, miR 221, miR-96, miR 126, miR 225 and miR 206.