New Reagents for RNA-based Therapeutic Technologies Abstract Functional RNA molecules such as aptamers, siRNAs, miRNAs, and related compounds have enormous potential as human therapeutics and as tools for elucidating gene regulation in vivo. To reach this potential, such molecules must be highly potent and highly stable. Unmodified RNAs typically do not come close to meeting these requirements. Some success has been achieved in vitro and in vivo by using 2'-O- methyl-ribose (2'-OMe) and phosphorothioate (PS) backbone modifications, alone or in combination. However, both 2'-OMe and PS modified RNAs have limited in vivo stability and activity, which can be problematic. In addition, RNA containing PS modification(s) are chiral at phosphorus, resulting in two distinct isomers at each PS substitution. Therefore, there is a need for further improvements. In Phase I of this project, we demonstrated proof of principle for a new approach using RNA containing 2'-OMe-phosphorodithioate (MS2) modifications, prepared by using novel 2'-OMe-thiophosphoramidite (2'- OMe-thioamidite) reagents. We successfully synthesized the four 2'-OMe-thioamidites (A, C, G, and U) at small scale and used them to synthesize a variety of RNAs containing MS2 modifications. Significantly, we showed that incorporating MS2 modifications remarkably improved binding affinity toward the targeted VEGF protein more than 1000-fold, from 2 nM to <1 pM. In addition, we showed that siRNAs containing MS2 modifications had increased gene silencing activity against multiple gene targets in cultured cells. To realize the high potential o these new reagents, Phase II of this project will focus on the following aims: (1) increase the scale of 2'-OMe-thioamidite production;(2) optimize protocols for solid-phase synthesis of MS2-RNA for in vitro and in vivo applications;(3) determine the thermal stability and structure of MS2-RNA duplexes;(4) validate the cellular binding affinity and specificity of the selected VEGF MS2-aptamers;(5) develop formulated MS2-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 MS2-siRNAs in vitro and in vivo, and will enable AM and its commercial partners to proceed with full commercialization of the 2'-OMe- thioamidite reagents and contribute toward the realization of effective MS2 modified RNA-based therapeutics.

Public Health Relevance

Functional RNA molecules such as aptamers, miRNAs, and siRNAs have exciting potential as therapeutics in areas such as viral infections, cancer, genetic disorders, and neurological diseases. However, these potential RNA drugs require chemical modifications to achieve the necessary potency and stability. AM Biotechnologies (AM) will develop 2'-O-methyl-ribonucleoside thiophosphoramidite reagents that will allow the life science community to produce highly potent, highly stable phosphorodithioate 2'-O-methyl-RNA-based therapeutics. The unique reagents that AM will develop under this project could ultimately have a profound impact on public health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Small Business Innovation Research Grants (SBIR) - Phase II (R44)
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Special Emphasis Panel (ZRG1)
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Fabian, Miles
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Am Biotechnologies, LLC
United States
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