Methods of preventing off-target gene silencing
Aspects featured in the invention relate to compositions and methods for inhibiting off-target gene silencing by iRNA agents.
1 . A method of preventing off-target gene silencing in a cell comprising contacting a duplex RNA with the cell, wherein the duplex RNA comprises a modification on the sense strand, and
(a) the sense strand of said duplex RNA has a region of at least 70% complementarity to at least 10 nucleotides of a preselected gene; or
(b) the modified or unmodified sense strand has been tested for the ability to silence the off-target gene.
2 . The method of claim 1 , wherein the off-target gene is expressed in said cell.
3 . The method of claim 1 , wherein the off-target gene is expressed in a different cell type.
4 . The method of claim 1 , wherein the modification is on the 5′ terminus of the sense strand.
5 . The method of claim 4 , wherein the modification prevents hydrolysis of a 5′ terminal phosphate group.
6 . The method of claim 4 , wherein the modification comprises an L-sugar at the 5′ terminus of the sense strand.
7 . The method of claim 4 , wherein the modification comprises an alpha-nucleotide at the 5′ terminus of the sense strand.
8 . The method of claim 2 , wherein the modification comprises a 2′-5′ linkage.
9 . The method of claim 1 , wherein the modification is on the 3′ terminus of the sense strand.
10 . The method of claim 9 , wherein the modification is a steroidal compound conjugated to the 3′ terminal nucleotide of the sense strand.
11 . The method of claim 10 , wherein the steroidal compound is cholesterol.
12 . The method of claim 10 , wherein the steroidal compound is conjugated to the 3′ terminal nucleotide by a cationic linker.
13 . The method of claim 1 , wherein the modification is on a nucleotide that is not a terminal nucleotide.
14 . The method of claim 13 , wherein the modification causes the sense strand to have a DNA-like conformation.
15 . The method of claim 13 , wherein the modification is replacement of a ribonucleotide with a deoxyribonucleotide.
16 . The method of claim 13 , wherein the modification is replacement of a uridine with 2′-arabino-fluorodeoxyuridine.
17 . The method of claim 16 , wherein the 2′-arabino-fluorodeoxyuridine is methylated.
18 . A method of evaluating a modification of a sense strand of a duplex RNA for the ability to inhibit silencing of an off-target gene, the method comprising:
(a) modifying the sense strand of the duplex RNA, wherein the sense strand of said duplex RNA has a region of at least 70% complementarity for at least 10 nucleotides of the off-target gene,
(b) contacting the modified sense strand to a cell expressing the off-target gene;
(c) comparing expression of the off-target gene to expression of the off-target gene following contact with an unmodified sense strand of the duplex RNA.