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Publication Number
US-9290763-B2
Publication Date
2016-03-22
Expiration Date
Abstract
A method for designing a bi-shRNA expression cassette encoding a bi-shRNA comprising: selecting one or more target site sequences; providing a backbone sequence comprising a first and a second stem-loop structure, inserting a first passenger strand and a second passenger strand and providing for synthesis of the bi-shRNA expression cassette.
Core Innovation
The disclosed invention relates to a vector-driven bifunctional bi-shRNA for enhanced RNA interference potency and durability by harnessing both cleavage-dependent and cleavage-independent RISC loading pathways. The invention is directed to an expression cassette that encodes a bi-shRNA having two linked stem-loop structures to generate guide strands fully complementary to target mRNA sequences while routing their activity to different Ago-containing RISCs.
The first stem-loop structure and the second stem-loop structure are linked by a sequence longer than 5 nucleotides within a backbone sequence that includes a first and a second stem-loop structure. Each stem-loop includes insertion sites within a stem connected by a loop sequence, and the cassette inserts passenger strands and guide strands to form the stems.
Target selection is constrained by selecting one or more first target site sequences and one or more second target site sequences having low homology with other mRNAs of the targeted species, with low homology selected from less than 75%, less than 80%, less than 90%, less than 95%, and less than 98% homology. The second passenger strand and the second guide strand are partially complementary, where partially complementary is defined by having allowed nucleotide mismatches when paired.
Claims Coverage
The partial content identifies two independent claims covering a bifunctional shRNA expression cassette and a method for designing such an expression cassette. Across these independent claims, the core inventive features combine low-homology target-site selection, a specific linked dual stem-loop backbone architecture with passenger/guide insertion, and defined passenger/guide partial complementarity based on allowed nucleotide mismatches.
Low-homology first and second target site selection
Selecting one or more first target site sequences and one or more second target site sequences of a targeted species having low homology with other mRNAs of the targeted species, where low homology is less than 75%, less than 80%, less than 90%, less than 95%, or less than 98% homology.
Linked dual stem-loop backbone architecture with defined insertion and loop linkage
Providing a backbone sequence comprising a first and a second stem-loop structure, where the first stem-loop structure includes two first insertion sites linked by a first loop sequence within the first stem and the second stem-loop structure includes two second insertion sites linked by a second loop sequence within the second stem, and where the first and the second stem-loop structures are linked by a sequence longer than 5 nucleotides.
First stem passenger/guide complementarity for first target sites
Inserting a first passenger strand and a first guide strand into the two first insertion sites to form the first stem, where the first passenger strand is homologous to the one or more first target site sequences and the first guide strand is complementary to the first passenger strand, or where the first passenger strand is identical to the reverse orientation of the first guide strand.
Second stem partial complementarity defined by allowed mismatch counts
Inserting a second passenger strand and a second guide strand into the two second insertion sites to form the second stem-loop structure, where the second passenger strand and the second guide strand are partially complementary, with partially complementary defined as having 1, 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, or greater nucleotide mismatches when paired.
Across the independent claims, the coverage is centered on a bi-shRNA expression cassette with low-homology selection of first and second target site sequences, a linked dual stem-loop backbone with insertion sites and loop linkage longer than 5 nucleotides, first-stem passenger/guide complementarity for first target sites, and second-stem passenger/guide partial complementarity defined by allowed nucleotide mismatch ranges.
Stated Advantages
Enhanced RNA interference potency and durability by harnessing both cleavage-dependent and cleavage-independent RISC loading pathways.
Documented Applications
Therapeutic and research applications, including cancer-related targets and pathogen targets.
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