In vivo production of small interfering RNAS that mediate gene silencing
First Claim
1. An isolated nucleic acid molecule capable of inducing RNA interference (RNAi) in a mammalian cell in vivo, comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor, wherein the precursor comprises a stem from a naturally-occurring stRNA or miRNA precursor, the stem comprising a first stem portion of about 18 to about 40 nucleotides and a second stem portion of about 18 to about 40 nucleotides, wherein the first stem portion comprises an stRNA or miRNA sequence within additional stem sequences of the first stem portion, and wherein the second stem portion comprises a sequence complementary to the stRNA or miRNA sequence within additional stem sequences of the second stem portion, wherein:
- (i) the stRNA or miRNA sequence within the first stem portion and the sequence within the second stem portion that is complementary to the stRNA or miRNA sequence are replaced with an siRNA antisense strand sequence that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell and an siRNA sense strand sequence that is complementary to the siRNA antisense strand sequence;
(ii) the second stem portion is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem;
(iii) a loop portion connects the two stem portions; and
(iv) the engineered RNA precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA.
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Accused Products
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.
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Citations
68 Claims
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1. An isolated nucleic acid molecule capable of inducing RNA interference (RNAi) in a mammalian cell in vivo, comprising a regulatory sequence operably linked to a nucleic acid sequence that encodes an engineered ribonucleic acid (RNA) precursor, wherein the precursor comprises a stem from a naturally-occurring stRNA or miRNA precursor, the stem comprising a first stem portion of about 18 to about 40 nucleotides and a second stem portion of about 18 to about 40 nucleotides, wherein the first stem portion comprises an stRNA or miRNA sequence within additional stem sequences of the first stem portion, and wherein the second stem portion comprises a sequence complementary to the stRNA or miRNA sequence within additional stem sequences of the second stem portion, wherein:
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(i) the stRNA or miRNA sequence within the first stem portion and the sequence within the second stem portion that is complementary to the stRNA or miRNA sequence are replaced with an siRNA antisense strand sequence that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell and an siRNA sense strand sequence that is complementary to the siRNA antisense strand sequence; (ii) the second stem portion is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem; (iii) a loop portion connects the two stem portions; and (iv) the engineered RNA precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40)
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41. An engineered RNA precursor capable of inducing RNA interference (RNAi) in a mammalian cell in vivo, comprising a stem from a naturally-occurring stRNA or miRNA precursor, the stem comprising a first stem portion of about 18 to about 40 nucleotides and a second stem portion of about 18 to about 40 nucleotides, wherein the first stem portion comprises an stRNA or miRNA sequence within additional stem sequences of the first stem portion, and wherein the second stem portion comprises a sequence complementary to the stRNA or miRNA sequence within additional stem sequences of the second stem portion, wherein:
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(i) the stRNA or miRNA sequence within the first stem portion and the sequence within the second stem portion that is complementary to the stRNA or miRNA sequence are replaced with an siRNA antisense strand sequence that is complementary to a sequence of a messenger RNA (mRNA) of a target gene in the mammalian cell and an siRNA sense strand sequence that is complementary to the siRNA antisense strand sequence; (ii) the second stem portion is sufficiently complementary to the first stem portion to hybridize with the first stem portion to form a duplex stem; (iii) a loop portion connects the two stem portions; and (iv) the engineered RNA precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68)
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Specification