CRISPR-Cas9 Disruption of Aquaporin 1: An Alternative to Glaucoma Eye Drop Therapy?

Main Text

Glaucoma is a group of optic neuropathies and a leading cause of irreversible blindness worldwide, affecting more than 70 million people, with a rising prevalence in aging populations.^1,2 The hallmark and final common pathway of all forms of glaucoma is progressive retinal ganglion cell (RGC) death and optic nerve degeneration.³ The pathogenic triggering mechanisms are largely unknown, but key risk factors include intraocular pressure (IOP)-related biomechanical stress.³ Both medical and surgical therapies are available to control IOP, either by reducing production or increasing drainage of aqueous humor. While these treatments are quite effective, there are shortfalls that may result in persistent progressive vision loss despite therapy.^3,4 In this issue of Molecular Therapy, Wu et al.⁵ describe a new treatment strategy that could result in effective, long-term control of IOP. They propose to disrupt aquaporin 1 (AQP1) expression within the ciliary epithelium by adeno-associated virus (AAV)-mediated delivery of a CRISPR-Cas9 system (Figure 1), and they provide proof-of-concept in experimental mouse glaucoma models and cultured human ciliary body.

Figure 1.

Aqueous Humor Dynamics and Disruption of Aquaporin 1 (AQP1) within the Ciliary Epithelium Using CRISPR-Cas9

Figure originally published in Wu et al.⁵

In healthy eyes, IOP is maintained in a physiologic range by the balance of aqueous humor production by the ciliary body and drainage through the iridocorneal angle (Figure 1). Most patients suffer from hypertensive glaucoma with elevated IOP due to increased aqueous humor outflow resistance.³ In the most common disease forms, such as primary open-angle glaucoma (POAG), the underlying pathogenesis is still largely unknown and a cure is not available.⁴ Current treatments are limited to lowering IOP in order to slow or prevent further RGC loss and damage to the optic nerve.^3,4 Reduction of IOP is the only proven method to treat glaucoma and slow progression of vision loss; this can be achieved by medical and surgical treatments, including daily eye drops as well as laser and incisional surgeries.^3,4 Low patient adherence rates for long-term self-administration of IOP-lowering eye drops is a major problem that contributes to disease progression despite therapy.^6,7

Strategies are being developed to address this problem, most importantly by use of sustained drug delivery technologies, with a number of drug implants in pre-clinical and clinical testing.⁶ By taking advantage of the latest gene therapy vector and gene editing advances, Wu et al.⁵ propose another alternative to eye drops by AAV-mediated intraocular delivery of CRISPR-Cas9 to disrupt the AQP1 gene within the non-pigmented ciliary epithelium, which encodes for a membrane protein channel important for aqueous humor production.8, 9, 10 Their strategy is viable in view of numerous ongoing clinical gene therapy trials for retinal and optic nerve diseases and the recent approval of Luxturna (Spark Therapeutics) by the United States Food and Drug Administration.^10,11 First, Wu et al.⁵ recognized the ability of the AAV ShH10 serotype to target transgene expression to the non-pigmented ciliary epithelium under the control of the constitutive cytomegalovirus (CMV) promoter following intravitreal injection. Subsequently, they designed two Staphylococcus aureus-derived Cas9 (SaCas9)-compatible short guide RNA (sgRNA) sequences within the exon 1 of AQP1 that, in combination, efficiently disrupted the gene. When the combined AAV vector mix with both sgRNAs was administered intravitreally, a significant reduction in IOP was achieved in wild-type C57BL/6J mice and in two mouse models of glaucoma based on corticosteroid- and microbead-induced ocular hypertension (Figure 1). During the 3–7-week observation period, reduction of AQP1 protein levels within the ciliary body and the prevention of RGC loss were observed in treated eyes. To investigate the potential translational value of this treatment, targeting of gene expression to the human non-pigmented ciliary epithelium by the AAV ShH10 serotype was demonstrated in tissue culture. Finally, detectable editing was observed in human cell and tissue culture with a sgRNA aligned to exon 1 of human AQP1.

The results presented by Wu et al.⁵ are very promising, but additional testing of safety and efficacy in larger animal species and disease models are necessary before clinical application in human patients can be considered. Such studies would have to include vector dose-related effects on aqueous humor dynamics and IOP. While this treatment does not address specific glaucoma disease mechanisms and does not result in a cure, it is potentially applicable to many forms of hypertensive glaucoma. The decrease in aqueous humor production resulting from the disruption of AQP1 expression is comparable to other currently available treatments targeting the ciliary body, including laser cyclophotocoagulation and topical eye drop administration, such as carbonic anhydrase inhibitors, beta-adrenergic receptor blockers, and alpha-adrenergic receptor agonists.^3,4 A major advantage of the proposed gene therapy approach would be an avoidance of any patient adherence issues with daily eye drop administration, a major concern in glaucoma therapy. Because of the negligible ciliary epithelium turnover,¹² the one-time vector injection may provide a long-term treatment effect; this is in contrast to sustained drug delivery implants, which have to be replaced once depleted.⁶ Even though the authors speculate that AQP4 could compensate for excessive IOP reduction, potential issues could arise from the inability to adjust the dosing and/or treatment effect of the AAV-delivered CRISPR-Cas9 system to the needs of the individual patient, including any changes in disease stage and severity. An adjustable AQP1 gene silencing approach may have advantages over gene editing.

While no off-target effects and no structural abnormalities were observed in mice, therapy-related uveitis with inflammatory cells present in the vitreous in some of the bilaterally treated mice was observed as an adverse effect. Even though the eye is considered immune privileged, the dose-related occurrence of inflammation following intravitreal AAV administration is being increasingly recognized and will have to be addressed before considering clinical application.¹³

In summary, Wu et al.⁵ show that IOP can be reduced by AAV-mediated delivery of a CRISPR-Cas9 system for disruption of the AQP1 gene expression within the ciliary epithelium. If proven safe in preclinical trials, this treatment has the potential to address the major problem of poor glaucoma eye drop adherence, because a one-time intravitreal injection could provide long-term IOP control.

Conflicts of Interest

There are no conflicts of interest.