COMPOSITIONS AND METHODS FOR RNA INTERFERENCE
The disclosure provides inhibitory RNA polynucleotides that have partial complementarity to a target gene. The inhibitory RNA polynucleotides have at least one mismatched nucleotide and can be designed to increase or decrease the cleavage rate when loaded onto the RNA-induced silencing complex (RISC).
1 . An inhibitory RNA polynucleotide comprising between 15 and 30 nucleotides,
wherein the inhibitory RNA polynucleotide is partially complementary to an equal length portion of a target gene,
wherein the inhibitory RNA polynucleotide comprises at least one mismatched nucleotide at position 5, 7, 8, 12, 16, 17, 18, 19, 20, or 21, and
wherein the inhibitory RNA polynucleotide guides an RNA-induced silencing complex (RISC) to cleave the target gene.
2 . The inhibitory RNA polynucleotide of claim 1 , where the inhibitory RNA polynucleotide comprises at least one mismatched nucleotide at position 12, 16, 17, 18, 19, 20, or 21.
3 . The inhibitory RNA polynucleotide of claim 1 , where the inhibitory RNA polynucleotide comprises one mismatched nucleotide at position 12 and/or at position 18.
4 . (canceled)
5 . The inhibitory RNA polynucleotide of claim 1 , wherein the inhibitory RNA polynucleotide comprises at least two mismatched nucleotides at positions selected from 5, 7, 8, 12, 15, 16, 17, 18, 19, 20, and 21.
6 . The inhibitory RNA polynucleotide of claim 5 , wherein the inhibitory RNA polynucleotide comprises two mismatched nucleotides, and wherein:
(a) a first mismatched nucleotide is at position 12 and a second mismatched nucleotide is at position 5, 7, 8, 15, 16, 17, 18, 19, 20, or 21; or
(b) a first mismatched nucleotide is at position 18 and a second mismatched nucleotide is at position 5, 7, 8, 12, 15, 16, 17, 19, 20, or 21; or
(c) a first mismatched nucleotide is at position 12 and a second mismatched nucleotide is at position 18.
7 - 8 . (canceled)
9 . The inhibitory RNA polynucleotide of claim 1 , wherein the inhibitory RNA polynucleotide comprises at least two mismatched nucleotides at positions selected from positions 15, 16, 17, 18, 19, 20, and 21.
10 . The inhibitory RNA polynucleotide of claim 9 , wherein:
(a) the inhibitory RNA polynucleotide comprises mismatched nucleotides at positions 15, 16, 17, 18, 19, 20, and 21; or
(b) the inhibitory RNA polynucleotide comprises mismatched nucleotides at positions 17, 18, 19, 20, and 21.
11 . (canceled)
12 . The inhibitory RNA polynucleotide of claim 1 , wherein the inhibitory RNA polynucleotide guides the RISC to cleave the target gene at a faster cleavage rate than the corresponding cleavage rate of RISC when RISC is guided by a corresponding inhibitory RNA polynucleotide having complete complementarity to the equal length portion of the target gene.
13 . The inhibitory RNA polynucleotide of claim 1 , wherein the inhibitory RNA polynucleotide is single-stranded or double-stranded.
14 . (canceled)
15 . A pharmaceutical composition comprising an inhibitory RNA polynucleotide of claim 1 and a pharmaceutically acceptable carrier.
16 . The pharmaceutical composition of claim 15 , wherein the inhibitory RNA polynucleotide is encapsulated in a nanoparticle.
17 . The pharmaceutical composition of claim 16 , wherein the nanoparticle is a liposome.
18 . The pharmaceutical composition of claim 17 , wherein the liposome is a polyethylene glycol (PEG) liposome.
19 . A method of increasing the cleavage rate of an RNA-induced silencing complex (RISC) in cleaving a target gene, comprising introducing at least one mismatched nucleotide to an inhibitory RNA polynucleotide comprising between 15 and 30 nucleotides,
wherein the inhibitory RNA polynucleotide is partially complementary to an equal length portion of a target gene,
wherein the inhibitory RNA polynucleotide comprises at least one mismatched nucleotide at position 5, 7, 8, 12, 16, 17, 18, 19, 20, or 21, and
wherein the RISC is guided by the inhibitory RNA polynucleotide to bind and cleave the target gene at a faster cleavage rate than the corresponding cleavage rate of RISC when RISC is guided by a corresponding inhibitory RNA polynucleotide having complete complementarity to the equal length portion of the target gene.
20 . The method of claim 19 , wherein the inhibitory RNA polynucleotide comprises at least two mismatched nucleotides at positions selected from positions 5, 7, 8, 12, 15, 16, 17, 18, 19, 20, and 21.
21 . A method of decreasing the cleavage rate of an RNA-induced silencing complex (RISC) in cleaving a target gene, comprising introducing at least two mismatched nucleotides to an inhibitory RNA polynucleotide comprising between 15 and 30 nucleotides,
wherein the inhibitory RNA polynucleotide is partially complementary to an equal length portion of a target gene,
wherein the inhibitory RNA polynucleotide comprises at least two mismatched nucleotides at positions selected from positions 9, 10, 11, and 13, and
wherein the RISC is guided by the inhibitory RNA polynucleotide to bind and cleave the target gene at a slower cleavage rate than the corresponding cleavage rate of RISC when RISC is guided by a corresponding inhibitory RNA polynucleotide having complete complementarity to the equal length portion of the target gene.
22 . A method of decreasing the expression level of a target gene in a cell, comprising contacting the cell with an inhibitory RNA polynucleotide of claim 1 .
23 . A method of treating a disease in a subject in need thereof, comprising administered to the subject an inhibitory RNA polynucleotide of claim 1 ,
wherein the inhibitory RNA polynucleotide decreases the expression level of the target gene.
24 . The method of claim 23 , wherein the inhibitory RNA polynucleotide comprises at least two mismatched nucleotides at positions selected from positions 5, 7, 8, 9, 10, 12, 13, 15, 16, 17, 18, 19, 20, and 21.
25 . A method of synthesizing an inhibitory RNA polynucleotide of claim 1 , comprising:
(a) providing a sequence of the target gene;
(b) selecting a portion of the sequence of the target gene where the inhibitory RNA polynucleotide binds;
(c) selecting at least one position from positions 5, 7, 8, 12, 16, 17, 18, 19, 20, and 21 of the inhibitory RNA polynucleotide to introduce a mismatched nucleotide at the position; and
(c) introducing the mismatched nucleotide at the selected position of the inhibitory RNA polynucleotide during synthesis of the inhibitory RNA polynucleotide,
wherein the inhibitory RNA polynucleotide is partially complementary to an equal length portion of the target gene, and
wherein an RNA-induced silencing complex (RISC) is guided by the inhibitory RNA polynucleotide to bind and cleave the target gene at a faster cleavage rate than the corresponding cleavage rate of RISC when RISC is guided by a corresponding inhibitory RNA polynucleotide having complete complementarity to the equal length portion of the target gene.