Targeting white matter astrocytes with a Gfap-disruptive Cas9 nuclease benefits a murine model of Alexander disease.

Astrocytes are intriguing therapeutic targets due to their crucial role in initiating and promoting demyelination in the central nervous system. By efficient on-target editing, we can permanently disrupt detrimental genes and restore normal physiological processes. Here, we have optimized an AAV platform to deliver the CRISPR/Cas9 system to white matter astrocytes. A single neonatal intra-CSF administration of an all-in-one AAV-DJ8 vector expressing the Staphylococcus aureus Cas9 enzyme under an astrocyte-specific promoter resulted in effective downregulation of Glial Fibrillary Acidic Protein (GFAP), which is responsible for the primary astrocytic dysfunctions in Alexander disease (AxD), a fatal leukodystrophy. This treatment achieved an editing rate of up to 32% in Gfap+/R76H mice, leading to decreased gliosis and formation of Rosenthal fibers, an AxD pathological hallmark. The integration of the AAV vector occurred at low abundance in the host genome, showed a positive correlation with transduction and gene editing efficiencies, and did not cause tumorigenic events. Overall, this study demonstrates the feasibility of gene editing strategies and paves the way for therapies treating AxD and other demyelinating disorders.