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.
Claims33
An isolated nucleic acid molecule comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor, wherein the precursor comprises
- (i) a first stem portion comprising a sequence of at least 18 nucleotides that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell;
- (ii) a second stem portion 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 that connects the two stem portions, wherein one or more of the following optional criteria are met:
- the first stem portion is fully complementary to the mRNA sequence,
- the second stem portion is fully complementary to the first stem portion,
- the first stem portion is located at a 5′ end of the RNA precursor,
- the first stem portion is located at a 3′ end of the RNA precursor, and
- the loop portion comprises at least 4 nucleotides, comprises at least 7 nucleotides or comprises 11 nucleotides.
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An engineered RNA precursor comprising
- wherein the precursor comprises
- (i) a first stem portion comprising a sequence of at least 18 nucleotides that is complementary to a portion of a messenger RNA (mRNA) of a target gene;
- (ii) a second stem portion 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 that connects the two stem portions, wherein one or more of the following optional criteria are met:
- the first stem portion is fully complementary to the mRNA sequence,
- the second stem portion is fully complementary to the first stem portion,
- the first stem portion is located at a 5′ end of the RNA precursor,
- the first stem portion is located at a 3′ end of the RNA precursor, and
- the loop portion comprises at least 4 nucleotides, comprises at least 4 nucleotides or comprises 11 nucleotides.
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Dependencies are inferred from the claim text.