Gene therapy for Glaucoma by CRISPR-Cas9 mediated disruption of Aquaporin 1 in the Ciliary Body

Abstract

Purpose : Glaucoma is the commonest cause of irreversible vision loss worldwide, however reducing intraocular pressure (IOP) invariably delays or halts disease progression. IOP lowering eye drops have frequent side-effects and compliance is poor, whilst surgery risks catastrophic complications and often fails over time. Complete ciliary body destructive procedures reduce aqueous production and are effective but hypotony, loss of neurotrophic factors and phthisis bulbi have precluded wider use. We trialled an approach targeting the ciliary body to selectively lower IOP alone following a single intravitreal injection, using CRISPR-Cas9 to disrupt Aquaporin 1 (Aqp1) – a key gene involved in aqueous humour production.

Methods : Staphylococcus aureus derived Cas9 and two short-guide RNA sequences targeting exon 1 of mouse Aqp1 were produced as ShH10 serotype adeno-associated virus (AAV) by the triple transfection method and AVB column purification. 2x10¹¹ genome copies were injected into the vitreous cavity of adult C57BL/6J mice with ShH10 expressing GFP vector and non-injected eyes as controls. Disruptive indel formation by CRISPR was assessed using T7 endonuclease 1 assay. IOP was measured by rebound tonometry (iCare TonoLab) and retinal and corneal OCT performed with the Micron IV platform. The paramagnetic microbead model of ocular hypertension was used with injection of 3x10⁶ beads into the anterior chamber of each eye and assessed by Brn3a ganglion cell counts using retinal flat-mount immunohistochemistry.

Results : The ShH10 serotype of AAV could transduce non-pigmented ciliary body epithelium after intravitreal injection, unlike AAV 1, 2, 5 and 6 serotypes. IOP was reduced in CRISPR injected eyes (10.4+/-2.4 mmHg) compared to GFP control (13.2+/-2.0 mmHg) and non-injected eyes (13.1+/-2.8 mmHg, P<0.001, n=12) three weeks after injection. Indel formation in CRISPR treated ciliary body was identified and no off-target increases in corneal or retinal thickness were detected by OCT imaging. Injected seven days after the induction of experimental glaucoma, ganglion cell counts were higher in CRISPR (202+/-30 per field) than untreated eyes (156+/-25, P<0.01, n=8) six weeks later. Mean & SD shown.

Conclusions : CRISPR-Cas9 disruption of Aqp1 in the ciliary body following AAV delivery by intravitreal injection results in reduced IOP in the mouse that prevented ganglion cell loss in an experimental Glaucoma model.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.