90. miRNA Shuttles Improve Therapeutic RNAi

Abstract

Top of pageAbstract Huntington's disease (HD) and Spinocerebellar ataxia type I (SCA1) are dominant neurodegenerative diseases caused by polyglutamine (polyQ) expansion. The mutations produce toxic proteins, with polyQ length predictive of disease onset and severity. There is no therapy for HD or SCA1, supporting the investigation of novel strategies. In other work, we show that gene silencing by RNA interference (RNAi) can be achieved in vitro and in vivo by expressing short hairpin RNAs (shRNAs) which are subsequently processed into small interfering RNAs (siRNAs) specific for ataxin-1 or huntingtin mRNA. Viral delivery of RNAi to disease mouse models resulted in partial recovery of morphological and behavioral phenotypes, suggesting improvements could be explored. To improve our RNAi strategy, we have previously developed an inducible RNAi expression system which exploits microRNAs (miRNAs) as siRNA shuttles. Although previous studies suggest that miRNA shuttles are processed by RNAi machinery more efficiently than shRNAs, the silencing efficiencies of miRNA shuttles and shRNAs have not been adequately compared. To address this, we generated RNAi constructs which express siRNAs specific for unique targets (ataxin-1, huntingtin or eGFP) in the context of either miRNA shuttles or shRNAs. shRNAs were driven by the polIII-based U6 or H1 promoters, while miRNA shuttles were driven by U6 or the polII-based CMV promoter. We assessed the ability of the RNAi constructs to silence Renilla luciferase with 3'UTRs containing target sequences corresponding to either ataxin-1, huntingtin or eGFP. Furthermore, sense and antisense sequences were targeted to determine which RNAi strands (guide or passenger) enter RISC. RNAi and luciferase target constructs were co-transfected into 293 cells and luciferase assays performed 48h later. Preliminary results show that the RNAi constructs direct sequence-specific silencing of the appropriate luciferase target. Furthermore, silencing was more efficient with miRNA shuttles than shRNAs, independent of the targeted sequence. These results suggest that miRNA shuttles may be used to increase the therapeutic efficacy of RNAi in SCA1 and HD mouse models.