shRNA library of 3 million sequences for identification of small-RNA therapeutic candidates, new targets and pathways, as well as conventional chemical-compound drugs in cell-culture disease models.
Problem:
ShRNA Drug Discovery: RNA interference (RNAi) using short hairpin RNA (shRNA) is commonly used to inhibit gene expression. shRNA-expressing libraries may have important applications in identifying RNA molecules/sequences with specific biological activity and thus therapeutic implications. Typically, shRNA libraries are limited to sequences that target single mRNAs. Because 7-nucleotide “seed” sequences within shRNAs are sufficient for partial inhibition of target mRNAs, shRNAs are inherently promiscuous. Thus, the single-gene-targeting approach is complicated by off-target effects, which diminish therapeutic indices, and fails to take advantage of multi-gene targeting, which enhances potency. Discovery of Drugs, Targets, and Pathways: Traditional drug discovery process involving high-throughput screening is labor-intensive, expensive, often ineffective, and infeasible when cell-culture disease models are unsuitable for microtiter-plate formats.
Solution:
ShRNA Drug Discovery: To challenge the single-gene-targeting paradigm of small-RNA-based therapeutic initiatives, Dr. Wilson’s lab designed, synthesized, validated, and now further optimized shRNA-expressing libraries that are completely random at the nucleotide level. Three million shRNAs can be screened in a single tissue-culture flask, selecting for the desired phenotype with “hit” sequences retrieved by PCR. Because the libraries are completely random, the screens are unbiased: favorable cell phenotypes reveal which shRNAs are most effective and least toxic. This approach allows identification of sequences that target multiple genes and/or act through non-canonical mechanisms. Discovery of Drugs, Targets, and Pathways: Random shRNA library screening can be combined with bioinformatic pattern analyses of hit sequences to identify targets, pathways, and conventional chemical-compound therapeutic candidates, bypassing in vivo delivery issues. Thus, each phenotypic screen has the potential to identify, (i) small-RNA therapeutic candidates, (ii) conventional, chemical-compound therapeutic candidates, (iii) target candidates for conventional drug development, and (iv) information on pathways relevant to disease mechanisms.
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Docket # Y6140, R3854