AAV-mediated Shrna and Mirna Silencing of Murine Peripherin-2 in vivo

Abstract

Purpose: : RNA interference (RNAi) is a promising tool for the treatment of dominant eye disorders. We aim to assess the efficiency of peripherin-2 (Prph2) silencing using either a standard short hairpin RNA (shRNA) or a microRNA (miRNA)-based hairpin. The impact of silencing on retinal morphology is measured in wild-type mice after delivery of the RNAi expression cassettes using an AAV2/8 vector. Our aim is to establish a viral RNAi platform that could be adapted for the treatment of dominant eye disorders.

Methods: : Short hairpins targeting Prph2 were constructed using the standard shRNA design (shRDS6) and an alternate design based on the endogenous miR30 sequence (miRDS6). A non-targeting control (miRCON) was also constructed and all three hairpins were cloned into the AAV backbone driven by a U6 promoter. In vitro evaluation of silencing was performed using quantitative real-time PCR (qPCR) on stable cell lines expressing either wild-type Prph2 or a non-targeted functional Prph2 cDNA. AAV2/8 preparations were subretinally injected into wild-type mice and eyes were analysed after 3 and 5 weeks using immunohistochemistry and electron microscopy.

Results: : Both hairpin designs silenced Prph2 in vitro and in vivo with the miRDS6 hairpin being more efficient by comparison to the standard shRDS6. The miRDS6 hairpin was more potent in silencing endogenous Prph2 in vivo at 3 and 5 weeks after injection. The shRDS6 hairpin reduced Prph2 levels in vivo by 20 % leading to photoreceptor outer segment (POS) disorganisation. The miRDS6 hairpin reduced Prph2 levels in vivo by 80 % leading to severe POS reduction and retinal degeneration. At 5 weeks after injection, miRDS6-mediated silencing resulted in almost 45 % reduction of photoreceptor cells.

Conclusions: : AAV-mediated RNAi leads to in vivo silencing of Prph2 in the murine retina. While establishing robust knockdown of Prph2, we also demonstrate evasion of silencing by an engineered Prph2 cDNA. These data could lead to the development of an RNAi therapeutic platform for the treatment of dominant RP.

Keywords: RNAi • gene transfer/gene therapy • photoreceptors