Neuroprotective effect of AAV-mediated cell-specific expression of GDNF in Müller glial cells versus retinal ganglion cells in a murine model of glaucoma

Abstract

Purpose: To compare the neuroprotective effects of intravitreal AAV vector-mediated expression of GDNF in Müller glial cells (ShH10.GDNF) versus retinal ganglion cells (RGC) (AAV2.GDNF) in the murine model of microbead-induced ocular hypertension.

Methods: Polystyrene microbeads were injected into the anterior chamber of adult mice to elevate intraocular pressure (IOP) and induce glaucoma. An intravitreal injection containing 1x10¹⁰ vector genomes of either ShH10.GDNF or AAV2.GDNF was administered concomitantly. IOP was measured by TonoLab tonometry at weekly intervals. Mice were sacrificed at four weeks and RGC loss and optic nerve axon (ONA) loss was quantified. Enzyme-linked immunosorbent assay (ELISA) was performed to determine GDNF expression in the retina and optic nerve.

Results: Increased IOP was confirmed in microbead-injected eyes by tonometer. Neither Sh10.GDNF nor AAV2.GDNF had any effect on IOP. At four weeks, uninjected control eyes and microbead-injected eyes had a mean RGC count of 3065 ± 410 cells/mm² (n=15) and 1573 ± 75 cells/mm² (n=12). Microbead-injected eyes that received either ShH10.GDNF or AAV2.GDNF demonstrated 2185 ± 78 cells/mm² (n=13) and 1689± 77 cells/mm² (n=12) respectively. At four weeks, uninjected control eyes and microbead-injected eyes had a mean ONA count of 538842 ± 88924 axons/mm² (n=15) and 220716 ± 76253 axons/mm² (n=15). Microbead-injected eyes that received either ShH10.GDNF or AAV2.GDNF demonstrated 367415 ± 19701 axons/mm² (n=8) and 281800 ± 16044 axons/mm² (n=12) respectively. Thus, microbead-injected eyes treated with ShH10.GDNF contained ~130% increase in viable RGCs and axons at four weeks compared to eyes treated with AAV2.GDNF (p<0.05). Intravitreous ShH10.GDNF and AAV2.GDNF resulted in increased GDNF levels (pg GDNF/mg total protein) in the retina compared to uninjected eyes (5433 and 10874 vs 5.5) and optic nerve (2474 and 4310 vs 40) (n=10).

Conclusions: Intravitreal ShH10.GDNF and AAV2.GDNF result in GDNF expression in the retina and optic nerve with no effect on IOP. Although ELISA results showed relatively higher expression in retina and optic nerve tissues with AAV2 compared to ShH10, the ShH10 vector had a stronger neuroprotective effect on RGC and axon survival indicating that specific cellular targeting or indirect pathways may be an important consideration in the treatment of glaucoma.