Patent ID: 8530438

Claim:
A method of inducing ribonucleic acid interference (RNAi) of a target gene in a mammalian cell in vivo, the method comprising (a) contacting the cell with an isolated nucleic acid molecule, said 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 the 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 target gene in the mammalian cell, 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, and (b) inducing the cell to express the engineered RNA precursor encoded by the nucleic acid molecule, wherein the precursor is processed by the cell to generate an siRNA that mediates cleavage of the mRNA and wherein the sequences flanking the antisense and sense strands in the stem portions facilitate processing of the precursor in the cell to form the siRNA, thereby inducing RNAi of the target gene in the mammalian cell in vivo.