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Publication Number
US-9556431-B2
Publication Date
2017-01-31
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 a bifunctional RNA molecule that comprises a first RNA molecule and a second RNA molecule, where each RNA molecule includes a double-stranded sequence with a guide strand sequence. The first guide strand sequence is complementary to a portion of an mRNA transcript encoded by a target gene, and the second guide strand sequence is partially complementary to a portion of the same mRNA transcript, with one or more bases mismatched with a nucleic acid sequence of the mRNA transcript.
The bifunctional RNA molecule activates cleavage-dependent RNA-induced silencing complex and also activates cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene. The disclosed architecture uses both cleavage-dependent and cleavage-independent RNA silencing pathways in the same bifunctional RNA molecule to improve target gene reduction outcomes.
The disclosed nucleotide expression and processing architectures include use of a promoter and an expression vector to encode the bifunctional RNA molecule, including architectures where the first and second RNA molecules initially reside within a single primary transcript. The disclosure further describes structural placement of the first and second double-stranded sequences within separate stem-loop stem portions and incorporates miRNA-derived loop elements through pri-miRNA and related Drosha and Dicer processing concepts.
The disclosed examples compare target knockdown and silencing performance, including experiments directed to Stathmin-1 (STMN1) and Acid Ceramidase (AC), and report enhanced and more durable knockdown and silencing relative to conventional cleavage-dependent or cleavage-independent shRNAs and siRNA. The document also describes improved functional outcomes in colon and breast cancer cell models and includes combinatorial and paired construct efficacy.
Claims Coverage
One independent claim is identified. It covers a bifunctional RNA molecule that includes two double-stranded sequences encoding two RNA molecules that target an mRNA transcript of a target gene, while activating both cleavage-dependent and cleavage-independent RISC using a mismatched second guide strand.
Bifunctional RNA molecule with first complementary and second partially complementary mismatched guide strands
A bifunctional RNA molecule comprises first and second RNA molecules with double-stranded sequences; the first double-stranded sequence includes a first guide strand sequence complementary to a portion of an mRNA transcript encoded by the target gene, and the second double-stranded sequence includes a second guide strand sequence partially complementary to a portion of the mRNA transcript and comprising one or more bases mismatched with a nucleic acid sequence of the mRNA transcript.
Activating cleavage-dependent and cleavage-independent RISC for reducing target gene expression
The bifunctional RNA molecule activates cleavage-dependent RNA-induced silencing complex and cleavage-independent RNA-induced silencing complex for reducing the expression level of the target gene.
The claim set centers on a bifunctional RNA molecule combining an mRNA-cleavage-activating first guide strand with a mismatched partially complementary second guide strand to activate both cleavage-dependent and cleavage-independent RISC, with dependent claims further specifying optional structural, transcriptional, promoter, and stem and loop sizing refinements.
Stated Advantages
Enhanced and more durable knockdown and silencing versus conventional cleavage-dependent or cleavage-independent shRNAs and siRNA.
Improved functional outcomes in colon and breast cancer cell models, including reported effects such as growth arrest and apoptosis.
Documented Applications
Knockdown and silencing of Stathmin-1 (STMN1) and Acid Ceramidase (AC) in cancer cell models, with reported functional outcomes in colon and breast cancer cell models.
Use of bifunctional shRNA constructs in a paired and combinatorial format to achieve efficacy for target knockdown and silencing in the disclosed experimental context.
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