Abstract
Purpose :
Glaucoma is a progressive optic neuropathy characterized by high intraocular pressure (IOP) and apoptotic degeneration of retinal ganglion cell (RGC) death and is the leading cause of irreversible vision loss throughout the world. Approaches targeting the ciliary body to lower IOP are not only limited but also have adverse systemic effects like metabolic acidosis and kidney stones. In addition, low patient adherence rates for long-term self-administration of IOP-lowering eye drops is a major problem contributing to disease progression despite therapy. In this study, we aim to develop a long-lasting gene therapy for glaucoma by regulating aquaporin 1 (AQP1) and carbonic anhydrase (CA2) using CRISPR/Cas13d.
Methods :
Cas13, a family of RNA-targeting CRISPR effectors, is utilized to gene edit two water-transporting transmembrane protein APQ1 and CA2 in vitro, wild-type mice and a steroid-induced glaucoma mouse model. Editing efficiency of hfCas13x and pfxCas13d were compared in HEK293T and N2a cells. pfxCas13d has better editing efficiency than hfCas13x, so ShH10 AAV-Cas13d simultaneously targeting AQP1 and CA2 were produced. 2x1010 genome copies of ShH10 AAV-Cas13d was delivered into each eye of 16 wild-type mice by intravitreal injection; 16 wild-type mice were intravitreally injected with ShH10 AAV-GFP as control. IOP was measured before and after intravitreal treatment. In addition, the ciliary body of those mice were dissected and utilized for qPCR test or western blotting. The same methodology was applied to steroid-induced glaucoma model with 32 treated animals and 16 controls; first group were treated with ShH10 AAV-Cas13 and the other 16 were treated with ShH10 AAV-GFP.
Results :
IOP of ShH10 AAV-Cas13d treated WT mice significantly reduced by AQP1 and CA2 knockdown compared with those treated with ShH10 AAV-GFP (paired t test, n=16. P=0.0276). qPCR and western blotting further confirmed a decrease in AQP1 and CA2 expression after ShH10 AAV-Cas13 injection. However, OCT, PERG and OKR results exhibited no difference in both groups. IOP of steroid-induced glaucoma models treated with ShH10 AAV-Cas13d was significantly lower than that of glaucoma models (paired t test, n=16. P=0.0008).
Conclusions :
CRISPR/13d-mediated knockdown of AQP1 and CA2 lowers IOP in wild-type mice and a steroid-induced glaucoma mouse model, holding great potential for being a novel therapy for glaucoma.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.