In vivo production of small interfering RNAS that mediate gene silencing
AssigneeUniversity of Massachusetts
InventorsPhillip D. Zamore, Juanita McLachlan, Gyorgy Hutvagner, Alla Grishok, Craig C. Mello
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Abstract
The invention provides engineered RNA precursors that when expressed in a cell are processed by the cell to produce targeted small interfering RNAs (siRNAs) that selectively silence targeted genes (by cleaving specific mRNAs) using the cell's own RNA interference (RNAi) pathway. By introducing nucleic acid molecules that encode these engineered RNA precursors into cells in vivo with appropriate regulatory sequences, expression of the engineered RNA precursors can be selectively controlled both temporally and spatially, i.e., at particular times and/or in particular tissues, organs, or cells.
Claims18
A method of inducing ribonucleic acid interference (RNAi) of a target gene in a cell, the method comprising introducing into a host cell in vitro an isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor;
- and culturing the cell under conditions sufficient for the cell to express the RNA precursor and to form a small interfering ribonucleic acid (siRNA) within the cell, thereby inducing RNAi of the target gene in the cell, wherein the precursor comprises
- (i) a first stem portion consisting of a sequence of 18 to about 30 nucleotides that is complementary to a sequence of a messenger RNA (mRNA) of the target gene;
- (ii) a second stem portion consisting of a sequence of 18 to about 30 nucleotides that is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem;
- and
- (iii) a loop portion that connects the two stem portions, wherein the loop portion consists of 2 to 9 nucleotides.
A method of inducing ribonucleic acid interference (RNAi) of a target gene in a cell, the method comprising introducing into a host cell in vitro an isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor;
- and culturing the cell under conditions sufficient for the cell to express the RNA precursor and to form a small interfering ribonucleic acid (siRNA) within the cell, thereby inducing RNAi of the target gene in the cell, wherein the precursor comprises
- (i) a first stem portion consisting of a sequence of 18, 19, 20, 21 or 22 nucleotides that is complementary to a sequence of a messenger RNA (mRNA) of the target gene;
- (ii) a second stem portion consisting of a sequence of 18, 19, 20, 21 or 22 nucleotides that is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem;
- and
- (iii) a loop portion that connects the two stem portions, wherein the loop portion consists of 2 to 9 nucleotides.
Dependencies are inferred from the claim text.