Inhibiting complement C3 activation by gene therapy reduces glaucoma progression

Abstract

Purpose : Complement activation is associated with glaucoma, and precedes neurodegeneration in animal models. Knockout of the classic pathway initiator C1q delays disease in DBA/2J (D2) mice. All three complement activation pathways converge at C3 cleavage. Here, we test the therapeutic effect of limiting C3 activation during glaucoma progression in D2 mice using ocular gene therapy.

Methods : We utilized CR2-Crry, which is the soluble rodent-specific complement inhibitor (sCrry) linked to a complement receptor 2 (CR2) targeting moiety that directs sCrry to sites of C3b-fragment (iC3b/C3dg/C3d) deposition. CR2-sCrry was packaged using quadruple YF mutant capsid AAV2 vector with CBA promoter. AAV2-CR2-Crry or control AAV2-GFP was delivered by bilateral intravitreal injection in 7-month old (mo) D2 female mice. At 10 and 12mo, RGC and axonal density were quantified in confocal images of retinal wholemounts immunostained for RGC and axonal markers (Brn3b, pNF), and optic nerve damage was scored as mild, moderate or severe by proportion of degenerative/lost axons (< 10%, 10-50% or > 50%, respectively). Naive 5-7mo D2 retinal wholemounts were immunostained for C3d, Brn3 and pNF. We conducted one-way ANOVA for Brn3b/pNF counts by group/age, followed by Student’s t-test.

Results : Preceding therapy, 5-7mo D2 showed C3d-stained RGC somata, dendrites and axons. At 10 and 12mo, AAV-CR2-Crry-treated D2 mice maintained 38% more healthy optic nerves, and had 37% and 52% less severe nerves at each age, compared with naïve D2 (FIG.1). AAV-GFP had a minimal effect, with 8% more healthy and 17% less severe nerves relative to 10mo naïve D2. AAV-CR2-Crry-treated retinas showed uniformly intact and bundled intraretinal axons at 750-µm eccentricity, with 3.3 axons/fascicle at 10mo (p=0.014) and 2.6 at 12mo (p<0.001), compared to variably dystrophic and depleted fascicles in age-matched naive mice (2.6 and 1.5 axons), and in 10mo AAV-GFP controls (2.4 axons). AAV2-CR2-Crry suppressed RGCs loss, showing 84% higher Brn3b-nuclei density at 10mo (p=0.001) and 168% at 12mo (p=0.001), whereas 10mo AAV-GFP-treated eyes only increased RGC density by 27% (FIG.2).

Conclusions : Viral gene therapy targeted to block C3 activation provides a high-precision and potent neuroprotective strategy to reduce the advance of chronic glaucoma. Ongoing studies test this in acute models, and define molecular changes in retina, nerve and their innate immune cells.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.