A high-fidelity RNA-targeting Cas13X downregulates connexin43 in astrocytes: A novel neuroprotective strategy for glaucoma
Glaucoma is a neurodegenerative disease characterized by the progressive degeneration of retinal ganglion cells (RGCs) and their axons, ultimately leading to irreversible vision loss. Elevated intraocular pressure (IOP) is the primary risk factor; however, neurodegeneration continues even after effective IOP management, underscoring the need for neuroprotective therapies. This study investigates the role of connexin43 (Cx43), which is extensively expressed in retinal macroglia, in regulating microglial activation and optic nerve degeneration in glaucoma. We employed a high-fidelity CRISPR-Cas13 (hfCas13X) system to selectively target and knock down Cx43 expression in astrocytes. Our findings reveal that Cx43-mediated ATP release through hemichannels exacerbates microglial activation and neuroinflammation, thereby contributing to RGC loss. Notably, in a mouse model of chronic ocular hypertension (COH) glaucoma, knocking down Cx43 in astrocytes using the hfCas13X system significantly promoted the survival of RGCs and the integrity of the optic nerve, and improved visual function. The hfCas13X system, which offers high-fidelity RNA editing with minimal off-target effects, represents a novel and promising therapeutic strategy for glaucoma, highlighting the potential of gene editing technologies in the management of neurodegenerative diseases. Overall design: RNA-seq from retinal cells of control and WT-G3w and Cx43ko-G3w mice to investigate the neuroprotective mechanisms of conditional astrocyte Cx43 knockout in glaucoma.