New Reagents for Synthesis of High Potency siRNA

DESCRIPTION (provided by applicant): New Reagents for Synthesis of High Potency siRNAs Abstract Since the discovery of short interfering RNA (siRNA) molecules in the late 1990s, siRNA technology has developed rapidly as a powerful tool for functional genomic analysis, target validation and therapeutic purposes. However, the potency and persistence of unmodified siRNAs is dramatically limited by their sensitivity to nuclease degradation. Developing chemically modified siRNA duplexes with higher potency and improved nuclease resistance is essential, especially for therapeutics applications in vivo. In Phase I of this project, we demonstrated proof of principle for a new approach using phosphorodithioate siRNAs (PS2- siRNAs), prepared using novel ribonucleoside thiophosphoramidite reagents (R-thioamidites). We successfully synthesized the four ribonucleoside thiophosphoramidites (A, C, G, and U) at small scale and used them to synthesize a variety of PS2-siRNAs. Importantly, we showed that PS2-siRNAs had increased gene silencing activity against multiple different gene targets in cultured cells. These results demonstrate that PS2-siRNAs have the potential to greatly improve siRNA-based research and drug development. To achieve that potential, Phase II of this project will achieve the following aims: (1) increase the scale of R-thioamidite production; (2) optimize protocols for solid-phase synthesis of PS2-siRNA for in vitro and in vivo applications; (3) synthesize and screen several PS2-siRNA libraries to identify design rules that maximize the gene silencing activity of PS2-siRNAs; (4) demonstrate increased potency and antitumor efficacy of unformulated PS2-siRNAs in a murine model of metastatic colorectal cancer; (5) develop formulated PS2-siRNAs that provide increased potency and antitumor efficacy in a murine model of metastatic ovarian cancer. Successful completion of this project will demonstrate the value of PS2-siRNAs in vivo, and enable AM and its commercial partners to proceed with full commercialization of the R-thioamidite reagents and contribute toward the realization of effective siRNA-based therapeutics. PUBLIC HEALTH RELEVANCE: New Reagents for Synthesis of High Potency siRNAs Narrative The possible therapeutic applications of small interfering RNA (siRNA) are broad and far reaching for many diseases. However, chemically unmodified siRNA molecules are rapidly degraded by nucleases, and often have inadequate potency, especially in animals. The need to develop nuclease resistant siRNAs with improved activity in vivo is critical to the development of novel siRNA-based therapies, as well as for many research applications of siRNA. AM is developing a novel type of chemically-modified siRNA that is expected to significantly improve siRNA potency and stability, which may lead to improved treatments for multiple human diseases.