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.
Claims35
An adeno-associated viral (AAV) vector comprising 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 consisting of a sequence of about 18 to about 40 nucleotides that is complementary to a sequence of the of the mRNA of the target gene;
- (ii) a second stem portion consisting of 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;
- and
- (iii) a loop portion consisting of 2 to 9 nucleotides that connects the two stem portions.
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 consisting of a sequence of about 18 to about 40 nucleotides that is complementary to a sequence of the mRNA of the target gene;
- (ii) a second stem portion consisting of 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;
- and
- (iii) a loop portion consisting of 2 to 9 nucleotides that connects the two stem portions.
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 consisting of a sequence of about 18 to about 40 nucleotides that is complementary to a sequence of the of the mRNA of the target gene;
- (ii) a second stem portion consisting of 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;
- 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.