Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference
View Patent ↗The present invention relates to a multifunctional short interfering nucleic acid (siNA) having a structure of Formula MF-III: wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length about 15 nucleotides to about 50 nucleotides; X comprises a nucleotide sequence that is complementary to a nucleotide sequence present in region Y′; X′ comprises a nucleotide sequence that is complementary to a nucleotide sequence present in region Y; one or more of X, X′, Y, and Y′ is independently complementary to a first, second, third, or fourth target sequence, respectively, or a portion thereof; and W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y, wherein the siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference.
1. A chemically modified multifunctional short interfering nucleic acid (siNA) assembled from two separate double-stranded siNAs, each double-stranded siNA having a sense strand and an antisense strand of a length from about 18 to about 28 nucleotides,
wherein either (a) one end of each sense strand of the two double-stranded siNAs is covalently bonded via a tether, or (b) one end of each antisense strand of the two double-stranded siNAs is covalently bonded via a tether,
wherein the antisense strand of each double-stranded siNA comprises a sequence complementary to a target sequence or a portion thereof,
wherein the sense or antisense strand of each double-stranded siNA is annealed to its corresponding strand that has been tethered at one end to another strand of the other double-stranded siNA,
wherein the tether is a nucleotide linker or a non-nucleotide linker, and
wherein the chemically modified multifunctional siNA comprises one or more chemical modifications.
2. The chemically modified multifunctional siNA of claim 1 , wherein the antisense strand of each double-stranded siNA comprises a sequence complementary to a first and second target sequence, respectively, or a portion thereof, and wherein the first target sequence or a portion thereof is different than the second target sequence or a portion thereof.
3. The chemically modified multifunctional siNA of claim 2 , wherein the first and second target sequences are present in the same target nucleic acid molecule.
4. The chemically modified multifunctional siNA of claim 2 , wherein the first and second target sequences are present in different target nucleic acid molecules.
5. The chemically modified multifunctional siNA of claim 1 , wherein the tether covalently bonds the 3′-ends of the sense strands of the two double-stranded siNAs or bonds the 5′-ends of the sense strands of the two double-stranded siNAs.
6. The chemically modified multifunctional siNA of claim 1 , wherein the tether covalently bonds the 5′-end of one sense strand of one double-stranded siNA to the 3′-end of the sense strand of the other double-stranded siNA.
7. The chemically modified multifunctional siNA of claim 1 , wherein the tether covalently bonds the 3′-ends of the antisense strands of the two double-stranded siNAs or bonds the 5′-ends of the antisense strands of the two double-stranded siNAs.
8. The chemically modified multifunctional siNA of claim 1 , wherein the tether covalently bonds the 5′-end of one antisense strand of one double-stranded siNA to the 3′-end of the antisense strand of the other double-stranded siNA.
9. The chemically modified multifunctional siNA of claim 1 , further comprising one or more terminal phosphate groups at the end of the antisense strand(s) of one double-stranded siNA or both double-stranded siNAs.
10. The chemically modified multifunctional siNA of claim 1 , wherein the tether is a biodegradable linker.
11. The chemically modified multifunctional siNA of claim 10 , wherein the tether is a biodegradable nucleic acid linker molecule containing 2 to 20 nucleotides in length.
12. The chemically modified multifunctional siNA of claim 11 , wherein the tether contains ribonucleotides; deoxyribonucleotides; 2′-modified nucleotides selected from the group consisting of 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl, and 2′-O-allyl; or combination thereof.
13. The chemically modified multifunctional siNA of claim 1 , wherein the tether contains a non-nucleotide linker selected from the group consisting of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, and combinations thereof.
14. The chemically modified multifunctional siNA of claim 1 , wherein the sense strand(s) and antisense strand(s) of one double-stranded siNA or both double-stranded siNAs have a length from about 19 to about 23 nucleotides.
15. The chemically modified multifunctional siNA of claim 1 , further comprising a second tether covalently bonding either one end of each sense strand of the two double-stranded siNAs or one end of each antisense strand of the two double-stranded siNAs, so that the sense strands of the two double-stranded siNAs have one end covalently bonded by one tether and the antisense strands of the two double-stranded siNAs have one end covalently bonded by another tether, respectively.
16. The chemically modified multifunctional siNA of claim 15 , wherein the second tether is a non-nucleotide linker.
17. The chemically modified multifunctional siNA of claim 16 , wherein the non-nucleotide linker is selected from the group consisting of an abasic nucleotide, polyether, polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, and combinations thereof.
18. The chemically modified multifunctional siNA of claim 1 , wherein the one or more chemical modifications are selected from the group consisting of phosphorothioate internucleotide linkage, 2′-deoxy modification, 2′-O-methyl modification, 2′-deoxy-2′-fluoro modification, 4′-thio modification, 2′-O-trifluoromethyl modification, 2′-O-ethyl-trifluoromethoxy modification, 2′-O-difluoromethoxy-ethoxy modification, 2′-deoxy-2′-fluoroarabino modification, universal base modification, acyclic modification, 5-C-methyl modification, glyceryl modification, abasic modification, inverted deoxy abasic modification, and combinations thereof.
19. A pharmaceutical composition comprising the chemically modified multifunctional siNA of claim 1 , alone or in combination with a pharmaceutically acceptable carrier or excipient.
20. A method for inhibiting the expression of a target gene comprising the step of administering the chemically modified multifunctional siNA of claim 1 , in an amount sufficient to inhibit expression of the target gene.