WIDESPREAD CNS TARGETING BY ALLELE-SPECIFIC BICISTRONIC MICRORNAS IMPROVES MOTOR AND MOLECULAR OUTCOMES IN SPINOCEREBELLAR ATAXIA TYPE 3

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is an autosomal dominant neurodegenerative disorder caused by a CAG repeat expansion in the ATXN3 gene, leading to toxic gain-of-function of the mutant protein. Despite extensive research, no disease-modifying therapies are currently available. Here, we developed an allele-specific gene silencing strategy using recombinant adeno-associated virus (rAAV) to deliver bicistronic artificial microRNAs targeting mutant ATXN3. In vitro screening identified a lead construct, miATXN3-10x2, designed to selectively target a single nucleotide polymorphism linked to the expanded allele. Following rAAV9 packing, intra-cerebellar administration in two SCA3 mouse models resulted in robust and selective suppression of mutant ATXN3 in the cerebellum. To evaluate long-term therapeutic efficacy, rAAV9-miATXN3-10x2 was delivered via intra-cisterna magna injection in a severe transgenic SCA3 mouse model, achieving widespread CNS distribution and sustained transgene expression across disease-relevant brain regions. Treated animals exhibited significant and sustained improvements in motor performance at 5-, 8-, and 11-weeks post-injection. Histological analyses revealed reduced mutant ATXN3 aggregation and a trend toward preservation of cerebellar molecular layer thickness. These effects were supported by dose-dependent reductions in mutant ATXN3 mRNA, decreased expression of neuroinflammatory markers, and increased levels of the neuronal marker NeuN. Transcriptomic profiling further demonstrated partial normalization of cerebellar gene expression, with treated transgenic mice shifting toward a wild-type molecular signature. Together, these findings establish rAAV-mediated delivery of bicistronic artificial microRNAs as a promising and translationally relevant therapeutic strategy, providing a strong preclinical foundation for the advancement of allele-specific RNA interference toward clinical development for SCA3.