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Publication Number
US-8735058-B2
Publication Date
2014-05-27
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 bifunctional short hairpin RNA (shRNA) molecules that engage RNA-induced silencing complex (RISC) pathways to reduce the expression level of a target gene. A bifunctional RNA molecule activates both cleavage-dependent and cleavage-independent RNA-induced silencing complex mechanisms.
The bifunctional RNA molecule includes a first double stranded sequence and a second double stranded sequence. The first double stranded sequence comprises a guide strand sequence fully complementary to a passenger strand and an mRNA transcript encoded by the target gene, while the second double stranded sequence comprises a partially complementary guide strand sequence with mismatches to route to cleavage-independent RISC for mRNA sequestration and/or degradation and/or translation repression.
The description also supports bifunctional designs arranged as precursor encoding two RNA molecules and/or within stem-loop structures that include sequences substantially similar to naturally occurring miRNA motifs, including miR-30. Functional examples described for target genes such as STMN1 and Acid Ceramidase (AC) indicate longer-lasting knockdown and enhanced anti-tumor outcomes in colon cancer and breast cancer models, with apoptotic outcomes described for caspase 3 and Annexin V measurements.
Claims Coverage
The partial claim set provides two independent claims. Each independent claim requires a bifunctional RNA molecule that activates both cleavage-dependent and cleavage-independent RISC to reduce expression of a target gene, while differing in how the first and second portions and structural constraints are defined.
Bifunctional RNA activation of cleavage-dependent and cleavage-independent RISC using complementary and mismatched sequences
A bifunctional RNA molecule comprising a first double stranded sequence with a guide strand sequence that is fully complementary to a passenger strand and an mRNA transcript encoded by the target gene, and a second double stranded sequence with a guide strand sequence that is 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.
Stem-loop bifunctional RNA with miRNA-substantially similar stem portion and complementary/mismatched guide sequences for dual-pathway RISC activation
A bifunctional RNA molecule comprising a first portion with a first double stranded sequence including a first guide strand sequence complementary to a portion of an mRNA transcript encoded by the target gene and a second portion with a second double stranded sequence including a second guide strand sequence partially complementary to the mRNA transcript encoded by the target gene, wherein the second guide strand sequence comprises one or more bases mismatched with the nucleic acid sequence of the passenger strand and the mRNA transcript encoded by the target gene; wherein the first and second double stranded sequences reside within a stem portion of a stem loop structure having a stem portion comprising about 19-45 nucleotides and a loop portion comprising about 4-25 nucleotides and wherein the stem portion further comprises a nucleic acid sequence substantially similar to a sequence of a naturally occurring miRNA molecule; 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.
Both independent claims are directed to bifunctional RNA molecules that reduce target gene expression by activating cleavage-dependent and cleavage-independent RNA-induced silencing complex using a fully complementary guide sequence for the cleavage-dependent activity and a partially complementary, mismatched guide sequence for the cleavage-independent activity. One independent claim further requires that the double stranded sequences reside in stem portions of stem loop structures with specified stem and loop nucleotide ranges and a stem nucleic acid sequence substantially similar to a naturally occurring miRNA sequence.
Stated Advantages
Longer-lasting knockdown compared with siRNA is described for STMN1-targeting constructs.
Enhanced knockdown is described for combinatorial or single-vector bifunctional designs with improved apoptotic outcomes.
Stronger anti-tumor effects are described in colon cancer and breast cancer models.
Improved cell-kill is described for Acid Ceramidase (AC) targeting.
Documented Applications
Reducing STMN1 expression by using STMN1-targeting bifunctional shRNA designs.
Anti-tumor activity in colon cancer models using bifunctional shRNA designs.
Anti-tumor activity in breast cancer models using bifunctional shRNA designs.
Targeting Acid Ceramidase (AC) to produce increased cell-kill using bifunctional shRNA designs.
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