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.
Claims29
1-54. (canceled)
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 precursor comprises
- (i) a first stem portion comprising a sequence of about 18 to about 40 nucleotides that is complementary to a sequence of the of the mRNA of the target gene, wherein the first stem portion consists essentially of the antisense strand of the siRNA, flanked by sequences not having complementarity to the sequence of the mRNA;
- (ii) a second stem portion comprising a sequence of about 18 to about 40 nucleotides that is sufficiently complementary to the about 18 to about 40 nucleotide sequence of the first stem portion to hybridize with the first stem portion to form a duplex stem, wherein the second stem portion consists essentially of the sense strand of the siRNA, flanked by sequences not sharing identity with the sequence of the mRNA;
- and
- (iii) a loop portion that connects the two stem portions, wherein the sequences flanking the antisense and sense strands in the stem portions facilitate processing of the precursor in a mammalian cell to form the siRNA.
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, said engineered RNA precursor comprising,
- (i) a first stem portion comprising a sequence of about 18 to about 40 nucleotides that is complementary to a sequence of the mRNA of the target gene, wherein the first stem portion consists essentially of the antisense strand of the siRNA, flanked by sequences not having complementarity to the sequence of the mRNA;
- (ii) a second stem portion comprising a sequence of about 18 to about 40 nucleotides that is sufficiently complementary to the about 18 to about 40 nucleotide sequence of the first stem portion to hybridize with the first stem portion to form a duplex stem, flanked by sequences not sharing identity with the sequence of the mRNA;
- and
- (iii) a loop portion that connects the two stem portions,
- wherein the sequences flanking the antisense and sense strands in the stem portions facilitate processing of the precursor in a mammalian cell to form the siRNA.
A method of inducing ribonucleic acid interference (RNAi) of a target gene in a mammalian cell in vitro, the method comprising
- (a) contacting the cell with the engineered ribonucleic acid (RNA) precursor of claim 2;
- and
- (b) maintaining the cell under conditions sufficient to permit processing of the precursor by the cell to generate the siRNA that mediates cleavage of the mRNA, thereby inducing RNAi of the target gene in the mammalian cell in vitro.
Dependencies are inferred from the claim text.