Modified siRNA molecules and uses thereof
View Patent ↗The present invention provides chemically modified siRNA molecules and methods of using such siRNA molecules to silence target gene expression. Advantageously, the modified siRNA of the present invention is less immunostimulatory than its corresponding unmodified siRNA sequence and retains RNAi activity against the target sequence. The present invention also provides nucleic acid-lipid particles comprising a modified siRNA, a cationic lipid, and a non-cationic lipid, which can further comprise a conjugated lipid that inhibits aggregation of particles. The present invention further provides methods of silencing gene expression by administering a modified siRNA to a mammalian subject. Methods for identifying and/or modifying an siRNA having immunostimulatory properties are also provided.
1. A modified siRNA comprising a double-stranded region of about 15 to about 30 nucleotides in length, wherein from 15% to about 30% of the nucleotides in the double-stranded region comprise 2′-O-methyl (2′OMe) nucleotides,
wherein the modified siRNA comprises 2′OMe nucleotides in both strands of the modified siRNA,
wherein the modified siRNA comprises at least one 2′OMe-guanosine nucleotide and at least one 2′OMe-uridine nucleotide in the double-stranded region,
wherein the modified siRNA does not comprise 2′OMe-cytosine nucleotides in the double-stranded region,
wherein the modified siRNA is less immunostimulatory than a corresponding unmodified siRNA sequence, and
wherein the modified siRNA is capable of silencing expression of a target sequence.
2. The modified siRNA of claim 1 , wherein 2′OMe nucleotides are the only modified nucleotides present in the double-stranded region.
3. The modified siRNA of claim 1 , wherein the modified siRNA comprises a double-stranded region of about 19 to about 25 nucleotides in length.
4. The modified siRNA of claim 1 , wherein the modified siRNA is chemically synthesized.
5. The modified siRNA of claim 1 , wherein the modified siRNA comprises 3′ overhangs in one or both strands of the modified siRNA.
6. The modified siRNA of claim 5 , wherein the 3′ overhangs in one or both strands of the modified siRNA comprise unmodified nucleotides, modified nucleotides, or combinations thereof.
7. The modified siRNA of claim 1 , wherein from about 20% to about 30% of the nucleotides in the double-stranded region comprise 2′OMe nucleotides.
8. The modified siRNA of claim 1 , wherein from about 25% to about 30% of the nucleotides in the double-stranded region comprise 2′OMe nucleotides.
9. The modified siRNA of claim 1 , further comprising a carrier system.
10. The modified siRNA of claim 9 , wherein the carrier system is selected from the group consisting of a nucleic acid-lipid particle, a liposome, a micelle, a virosome, a nucleic acid complex, and mixtures thereof.
11. A nucleic acid-lipid particle comprising:
a modified siRNA of claim 1 ;
a cationic lipid; and
a non-cationic lipid.
12. The nucleic acid-lipid particle of claim 11 , further comprising a conjugated lipid that inhibits aggregation of particles.
13. The nucleic acid-lipid particle of claim 12 , wherein the conjugated lipid that inhibits aggregation of particles is a member selected from the group consisting of a polyethyleneglycol (PEG)-lipid conjugate, a polyamide (ATTA)-lipid conjugate, and a mixture thereof.
14. The nucleic acid-lipid particle of claim 13 , wherein the PEG-lipid conjugate is a member selected from the group consisting of a PEG-diacylglycerol conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide conjugate, and a mixture thereof.
15. The nucleic acid-lipid particle of claim 14 , wherein the PEG-DAA conjugate is a member selected from the group consisting of a PEG-dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG-dipalmityloxypropyl (C16) conjugate, and a PEG-distearyloxypropyl (C18) conjugate.
16. The nucleic acid-lipid particle of claim 14 , wherein the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (C14) conjugate.
17. The nucleic acid-lipid particle of claim 11 , wherein the cationic lipid comprises from about 2 mol % to about 60 mol % of the total lipid present in the particle.
18. The nucleic acid-lipid particle of claim 11 , wherein the non-cationic lipid comprises from about 5 mol % to about 90 mol % of the total lipid present in the particle.
19. The nucleic acid-lipid particle of claim 12 , wherein the conjugated lipid that inhibits aggregation of particles comprises from about 0.5 mol % to about 20 mol % of the total lipid present in the particle.
20. The nucleic acid-lipid particle of claim 11 , wherein the non-cationic lipid comprises a phospholipid and cholesterol.
21. The nucleic acid-lipid particle of claim 11 , wherein the modified siRNA is fully encapsulated in the nucleic acid-lipid particle.
22. A pharmaceutical composition comprising a modified siRNA of claim 1 and a pharmaceutically acceptable carrier.
23. A pharmaceutical composition comprising a nucleic acid-lipid particle of claim 11 and a pharmaceutically acceptable carrier.
24. A method for introducing an siRNA that silences expression of a target sequence into a cell, the method comprising:
contacting the cell with a modified siRNA of claim 1 .
25. A method for introducing an siRNA that silences expression of a target sequence into a cell, the method comprising:
contacting the cell with a nucleic acid-lipid particle of claim 11 .
26. A method for in vivo delivery of an siRNA that silences expression of a target sequence, the method comprising:
administering to a mammalian subject a modified siRNA of claim 1 .
27. A method for in vivo delivery of an siRNA that silences expression of a target sequence, the method comprising:
administering to a mammalian subject a nucleic acid-lipid particle of claim 11 .