Gene therapy for glaucoma using a combination targeting multiple genes with CRISPR-CasRx

Glaucoma affects approximately 80 million individuals worldwide, a condition for which current treatment options are inadequate. The primary risk factor for glaucoma is elevated intraocular pressure, which is currently the only controllable treatment approach. Elevated intraocular pressure is determined by the balance between the secretion and outflow of aqueous humor (AH). Utilizing CRISPR Cas9 to disrupt the expression of the AQP1 protein, which decreases the ciliary AH secretion, resulted in an approximate 22% reduction in intraocular pressure. Nevertheless, the effectiveness of single-gene therapies is limited. To address this limitation, we conducted experiments using two mouse models of ocular hypertension. Our findings indicate that reducing the expression of the genes Rock1 and Rock2, which are associated with the outflow of AH at the trabecular meshwork (TM), as well as the genes Aqp1 and Adrb2, which are related to the generation of AH at the ciliary body (CB), can significantly reduce intraocular pressure and provide protection to the retina ganglion cells (RGCs), ultimately delaying disease progression. In addition, we elucidated the mechanisms by which the knockdown of Rock1 and Rock2, or Aqp1 and Adrb2, reduces the IOP and secures RGCs by single-cell sequencing. Overall design: The glucocorticoid-induced ocular hypertension model was prepared for single-cell RNA sequence. 10 weeks after mold and virus injection, eyes were surgically removed from the mice and trimmed, then surgical scissors were used to take samples 1mm before and after the ora serrata immediately and kept in a special preservation solution. All operations were performed on ice. The samples were delivered to CapitalBio Technology (Beijing, China) and sequenced for analysis.