Methods and compositions for silencing genes without inducing toxicity
AssigneeAlnylam Pharmaceuticals, Inc.
InventorCatherine J. Pachuk
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Abstract
The present invention provides methods of post-transcriptional gene silencing which involve the use of a first dsRNA having substantial sequence identity to a target nucleic acid and a short, second dsRNA which inhibits dsRNA-mediated toxicity. These methods can be used to prevent or treat a disease or infection by silencing a gene associated with the disease or infection. The invention also provides methods for identifying nucleic acid sequences that modulate a detectable phenotype, including the function of a cell, the expression of a gene, or the biological activity of a target polypeptide.
Claims43
A method of for identifying a nucleic acid that modulates a detectable phenotype in a cell, said method comprising said steps of:
- (a) transforming a population of cells with a dsRNA expression library comprising long dsRNAs, wherein at least two cells of said population of cells are each transformed with a different nucleic acid from said dsRNA expression library;
- (b) transforming said population of cells with a short dsRNA or a nucleic acid encoding a short dsRNA;
- and
- (c) assaying for a modulation in said detectable phenotype, wherein said modulation identifies a nucleic acid that is associated with said phenotype,
- wherein the ratio between the long dsRNA and short dsRNA is 1:5 to 5:1.
A method for treating a disease, disorder, or an infection in an animal, said method comprising introducing into said animal a first agent that provides to said animal a first long dsRNA and a second agent that provides to said animal a short, second dsRNA, wherein said first long dsRNA has substantial sequence identity to a region of a target nucleic acid associated with said disease, disorder, or infection and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA does not decrease expression of an endogenous nucleic acid in the animal and inhibits dsRNA-mediated toxicity by inhibiting PKR dimerization and activation, and wherein the ratio between the long dsRNA and short dsRNA is 1:5 to 5:1.
A method for inhibiting the expression of a target nucleic acid in an animal, said method comprising introducing into said animal a first agent that provides to said animal a first long dsRNA and a second agent that provides to said animal a short, second dsRNA, wherein said first dsRNA has substantial sequence identity to a region of said target nucleic acid and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA does not decrease expression of an endogenous nucleic acid in the animal and inhibits dsRNA-mediated toxicity by inhibiting PKR dimerization and activation, and wherein the ratio between the long dsRNA and short dsRNA is 1:5 to 5:1.
A method for inhibiting the expression of a target nucleic acid in a cell, said method comprising introducing into said cell a first agent that provides to said cell a first long double stranded RNA (dsRNA) and a second agent that provides to said cell a short, second dsRNA, wherein said first dsRNA has substantial sequence identity to a region of said target nucleic acid and specifically inhibits said expression of said target nucleic acid, and wherein said short, second dsRNA does not decrease expression of a nucleic acid in the cell and inhibits dsRNA-mediated toxicity by inhibiting PKR dimerization and activation, and wherein the ratio between the long dsRNA and short dsRNA is 1:5 to 5:1.
Dependencies are inferred from the claim text.