Novel shRNA molecules and methods of use thereof

A method for reducing the expression level of a target gene, which comprises providing a cell with one or more precursor nucleic acid sequences that encode two or more RNA molecules, wherein:

• (a) a first RNA molecule comprises a first double stranded sequence, which comprises a first guide strand sequence that is complementary to a portion of an mRNA transcript encoded by the target gene;
• and
• (b) a second RNA molecule comprises a second double stranded sequence, which comprises a second guide strand sequence that is partially complementary to a portion of the mRNA transcript encoded by the target gene, wherein the second guide strand sequence comprises one or more bases that are mismatched with the nucleic acid sequence of the mRNA transcript encoded by the target gene, wherein:
• (i) the first and second double stranded sequences reside within a stem portion of a stem loop structure, wherein the stem portion comprises about 19-45 nucleotides, a loop portion of the stem loop structure comprises about 4-25 nucleotides, and the stem portion comprises a nucleic acid sequence that is substantially similar to a sequence of a naturally occurring miRNA molecule;
• (ii) the first double stranded sequence comprises a sequence that is presented to a cleavage dependent pathway and is capable of binding to the mRNA transcript encoded by the target gene, which causes degradation of the mRNA transcript;
• and
• (iii) the second double stranded sequence comprises a sequence that is presented to a cleavage independent pathway and is capable of sequestering the mRNA transcript encoded by the target gene and repressing the translation thereof.

A nucleic acid sequence, which may comprise a single contiguous sequence or multiple distinct sequences that, individually or collectively, encode two or more RNA molecules, wherein:

• (a) a first RNA molecule comprises a first double stranded sequence, which comprises a first guide strand sequence that is complementary to a portion of an mRNA transcript encoded by the target gene;
• and
• (b) a second RNA molecule comprises a second double stranded sequence, which comprises a second guide strand sequence that is partially complementary to a portion of the mRNA transcript encoded by the target gene, wherein the second guide strand sequence comprises one or more bases that are mismatched with the nucleic acid sequence of the mRNA transcript encoded by the target gene, wherein:
• (i) the first and second double stranded sequences reside within a stem portion of a stem loop structure, wherein the stem portion comprises about 19-45 nucleotides, a loop portion of the stem loop structure comprises about 4-25 nucleotides, and the stem portion comprises a nucleic acid sequence that is substantially similar to a sequence of a naturally occurring miRNA molecule;
• (ii) the first double stranded sequence comprises a sequence that is presented to a cleavage dependent pathway and is capable of binding to the mRNA transcript encoded by the target gene, which causes degradation of the mRNA transcript;
• and
• (iii) the second double stranded sequence comprises a sequence that is presented to a cleavage independent pathway and is capable of sequestering the mRNA transcript encoded by the target gene and repressing the translation thereof.