Development of an AAV-based microRNA gene therapy to treat Machado-Joseph disease

Spinocerebellar ataxia type 3 (SCA3), or Machado-Joseph disease (MJD), is a progressive neurodegenerative disorder caused by a CAG expansion in the gene. The expanded CAG repeat is translated into a prolonged polyglutamine repeat in the ataxin-3 protein and accumulates within inclusions, acquiring toxic properties, which results in degeneration of the cerebellum and brain stem. In the current study, a non-allele-specific silencing approach was investigated using artificial microRNAs engineered to target various regions of the gene (miATXN3). The mi candidates were screened based on their silencing efficacy on a luciferase (Luc) reporter co-expressing . The three best miATXN3 candidates were further tested for target engagement and potential off-target activity in induced pluripotent stem cells (iPSCs) differentiated into frontal brain-like neurons and in a SCA3 knockin mouse model. Besides a strong reduction of mRNA and protein, small RNA sequencing revealed efficient guide strand processing without passenger strands being produced. We used different methods to predict alteration of off-target genes upon AAV5-mi treatment and found no evidence for unwanted effects. Furthermore, we demonstrated in a large animal model, the minipig, that intrathecal delivery of AAV5 can transduce the main areas affected in SCA3 patients. These results proved a strong basis to move forward to investigate distribution, efficacy, and safety of AAV5-mi in large animals.