CRISPR ACTIVATION AS A NEUROPROTECTIVE STRATEGY FOR SPINOCEREBELLAR ATAXIA TYPE 3

Machado-Joseph disease, also known as Spinocerebellar ataxia type 3 (MJD/SCA3), is a polyglutamine (polyQ) neurodegenerative disorder caused by abnormal expansion of the CAG tract in the ATXN3/MJD1 gene, resulting in accumulation of mutant ataxin-3. This results in progressive neurodegeneration in different brain regions, such as the cerebellum and brainstem, ultimately causing debilitating motor symptoms. Currently, there is no effective treatments.
Previously, our group identified sirtuin-1 as a neuroprotective factor in MJD/SCA3-associated neuropathology. Both MJD/SCA3 mouse models and patient-derived fibroblasts showed decreased sirtuin-1 levels, while its overexpression reduced ataxin-3 aggregation and blocked neurodegeneration, supporting Sirt1 upregulation as a potential therapy for MJD/SCA3.
In this study, we explored CRISPR activation (CRISPRa)-based gene therapy approach to selectively enhance endogenous sirtuin-1. Compared to conventional overexpression strategies, CRISPRa offers a physiologically relevant modulation of gene expression, minimizing side effects associated with excessive protein levels. We developed an adeno-associated virus (AAV)-delivered CRISPRa system to upregulate Sirt1 in vitro and in vivo. This strategy led to a significant reduction of mutant ataxin-3 protein levels, suggesting a neuroprotective effect.
Furthermore, we leveraged the CRISPRa approach to co-activate a second neuroprotective gene, which is involved in stress response and RNA metabolism. This co-activation led to a further reduction in mutant ataxin-3 levels. Overall, our findings support AAV-delivered CRISPRa as a promising gene therapy platform for polyQ disorders and highlight the potential of multigene activation in targeting neurodegeneration mechanisms.