Interested in licensing this patent?
MTEC can help explore whether this patent might be available for licensing for your application.
Publication Number
US-8252526-B2
Publication Date
2012-08-28
Expiration Date
Abstract
The present invention relates to certain novel shRNA molecules and methods of use thereof. According to certain embodiments of the present invention, methods for reducing the expression level of a target gene are provided. Such methods generally comprise providing a cell with one or more precursor nucleic acid sequences that encode two or more RNA molecules. A first RNA molecule comprises a double stranded sequence, which includes a guide strand sequence that is complementary to a portion of an mRNA transcript encoded by the target gene. In addition, a second RNA molecule comprises a second double stranded sequence, which includes a second guide strand sequence that is partially complementary to a portion of the mRNA transcript encoded by the target gene. Preferably, the second guide strand sequence comprises one or more bases that are mismatched with a nucleic acid sequence of the mRNA transcript encoded by the target gene.
Core Innovation
The invention relates to short hairpin RNA (shRNA) bifunctional designs that reduce the expression level of a target gene by simultaneously activating cleavage-dependent and cleavage-independent RNA-induced silencing complex. The bifunctional RNA molecule includes a first fully complementary guide strand region and a second partially complementary mismatched region, and each region activates a different RNA-induced silencing complex activity for reducing target gene expression.
The disclosure distinguishes cleavage-dependent RNA-induced silencing complex activity associated with a guide strand that is fully complementary to a passenger strand and an mRNA transcript encoded by the target gene, and cleavage-independent RNA-induced silencing complex activity associated with a partially complementary sequence containing mismatches or bulges to a passenger strand and complementary to the mRNA transcript. The bifunctional RNA molecule therefore engages both pathways to lower target gene expression.
The bifunctional RNA molecule is provided in a form that can include a single contiguous bifunctional RNA molecule or a bifunctional RNA molecule with first and second portions. The first and second double stranded sequences can be arranged within a single primary transcript or within stem-loop nucleic acid structures, and the disclosure also addresses bifunctional RNA precursor nucleic acid formats and promoter/transcription considerations, including RNA polymerase II promoter or RNA polymerase III promoter contexts.
Claims Coverage
The partial content identifies two independent methods. Each independent claim centers on providing a bifunctional RNA molecule that activates both cleavage-dependent and cleavage-independent RNA-induced silencing complex to reduce expression of a target gene.
Single contiguous bifunctional RNA molecule activates both RISC activities
Providing a cell with a single contiguous bifunctional RNA molecule comprising a first double stranded sequence with a guide strand sequence fully complementary to a passenger strand and an mRNA transcript encoded by the target gene, and a second double stranded sequence partially complementary to a mismatched passenger strand and complementary to the mRNA transcript encoded by the target gene, wherein the bifunctional RNA molecule activates cleavage-dependent and cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene.
Bifunctional RNA first portion presents cleavage-dependent guide and second portion presents cleavage-independent sequence
Providing a cell with a bifunctional RNA molecule comprising a first and a second portion, where the first portion comprises a first double stranded sequence with a guide strand sequence fully complementary to a passenger strand and a portion of an mRNA transcript encoded by the target gene and the second portion comprises a second double stranded sequence partially complementary to a mismatched passenger strand and to the mRNA transcript encoded by the target gene, wherein the bifunctional RNA molecule activates both cleavage-dependent and cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene.
Across the independent claims, the core claim coverage is the bifunctional RNA arrangement that uses a fully complementary guide region to activate cleavage-dependent RISC and a partially complementary mismatched region to activate cleavage-independent RISC, thereby reducing the expression level of a target gene.
Stated Advantages
Reduces the expression level of a target gene by activating cleavage-dependent and cleavage-independent RNA-induced silencing complex.
Documented Applications
Therapeutic use is described, including cancer.
Research use includes experiments for reducing expression of target genes such as STMN1 (Stathmin-1) and Acid Ceramidase (AC) via bifunctional shRNA designs.
Interested in licensing this patent?