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.
Claims36
An isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor which is processed to form a small interfering ribonucleic acid (siRNA) comprising an antisense strand complementary to a sequence of a messenger RNA (mRNA) of a target gene, and a sense strand having a sequence complementary to the antisense strand, wherein the engineered RNA precursor comprises:
- (i) a first stem portion of the engineered RNA precursor comprising a sequence of at least 18 nucleotides that is complementary to a sequence of the mRNA;
- (ii) a second stem portion of the engineered RNA precursor comprising a sequence of at least 18 nucleotides that is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex;
- and
- (iii) a loop portion of the engineered RNA precursor consisting of 2 to 9 nucleotides that connects said first and said second stem portions.
An engineered RNA precursor which is processed to form an siRNA comprising an antisense strand complementary to a sequence of an mRNA of a target gene, and a sense strand having a sequence complementary to the antisense strand, wherein the engineered RNA precursor comprises:
- (i) a first stem portion of the engineered RNA precursor comprising a sequence of at least 18 nucleotides that is complementary to a portion of the mRNA;
- (ii) a second stem portion of the engineered RNA precursor comprising a sequence of at least 18 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 of the engineered RNA precursor consisting of 2 to 9 nucleotides that connects the two stem portions.
Dependencies are inferred from the claim text.