5?-Vinylphosphonate improves tissue accumulation and efficacy of conjugated siRNAs in vivo.

ABSTRACT: 5?-Vinylphosphonate modification of siRNAs protects them from phosphatases, and improves silencing activity. Here, we show that 5?-vinylphosphonate confers novel properties to siRNAs. Specifically, 5?-vinylphosphonate (i) increases siRNA accumulation in tissues, (ii) extends duration of silencing in multiple organs and (iii) protects siRNAs from 5?-to-3? exonucleases. Delivery of conjugated siRNAs requires extensive chemical modifications to achieve stability in vivo. Because chemically modified siRNAs are poor substrates for phosphorylation by kinases, and 5?-phosphate is required for loading into RNA-induced silencing complex, the synthetic addition of a 5?-phosphate on a fully modified siRNA guide strand is expected to be beneficial. Here, we show that synthetic phosphorylation of fully modified cholesterol-conjugated siRNAs increases their potency and efficacy in vitro, but when delivered systemically to mice, the 5?-phosphate is removed within 2 hours. The 5?-phosphate mimic 5?-(E)-vinylphosphonate stabilizes the 5? end of the guide strand by protecting it from phosphatases and 5?-to-3? exonucleases. The improved stability increases guide strand accumulation and retention in tissues, which significantly enhances the efficacy of cholesterol-conjugated siRNAs and the duration of silencing in vivo. Moreover, we show that 5?-(E)-vinylphosphonate stabilizes 5? phosphate, thereby enabling systemic delivery to and silencing in kidney and heart.

SUBMITTER: Haraszti RA 

PROVIDER: S-EPMC5570069 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature