RNAi therapy for Machado-Joseph disease: long-term safety profile of lentiviral vectors encoding shRNAs targeting mutant ataxin-3.

Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 (SCA3) is an autosomal dominantly-inherited neurodegenerative disorder caused by the over-repetition of a CAG codon in the MJD1 gene. This expansion translates into a long polyglutamine tract that confers a toxic gain-of-function to the mutant protein - ataxin-3, leading to neurodegeneration in specific brain regions. No treatment able to modify the disease progression is available. However, we and others have previously shown that specific silencing mutant ataxin-3 by RNA interference with viral vectors is a promising therapeutic strategy for MJD. Nevertheless, reports of cytotoxic effects of this technology led us to investigate the safety profile of the previously tested lentiviral vectors encoding shRNAs (LV-shmutatx3) targeting mutant ataxin-3 upon brain injection. For this purpose, we injected the vectors in the mouse striata and evaluated neuronal dysfunction, degeneration, gliosis, off-target effects and saturation of the RNAi machinery. We found that LV-shmutatx3 mediated: a) stable and long-term expression of the shRNA in neurons of the mouse striatum, b) neuronal dysfunction evaluated by darpp-32, NeuN and cresyl violet staining's comparable to PBS injection that resolved within 20 weeks, c) astrocytic activation that resolved within 8 weeks, d) microglial activity and proinflammatory cytokines release, which resolved and normalized within 20 weeks, e) no off-target effects or saturation of the endogenous RNAi processing machinery in the mouse striatum. Our data show that injection of lentiviral vectors encoding a shRNA targeting mutant ataxin-3 in the mouse brain induce transient dysfunctions, which resolve within 20 weeks. Importantly, the long-term expression (up to 20 weeks post-injection) of this shRNA (driven by H1 promoter) led to no toxic effect in vivo. This study constitutes thus an additional step in a future translation of gene silencing as a therapy for MJD.