Our goal is to investigate the neuroprotective efficacy of systemic rAAV gene delivery of erythropoiesis-attenuated erythropoietin-R76E (EPO-R76E) in a mouse model of experimental glaucoma. We investigated the efficacy of EPO-R76E gene therapy commenced before and after induction of ocular hypertension. C57BL/6 mice were injected intramuscularly with 109 vgc rAAV delivering eGFP or Epo-R76E behind a CMV promoter. To induce ocular hypertension, anterior chambers were injected with 15-μm diameter microbeads or saline, and elevation of intraocular pressure (IOP) was verified by tonometry. For pre-treatment, gene delivery was performed using rAAV2/8 serotype one month prior to IOP elevation. For delayed treatment, gene therapy was administered using rAAV2/1 serotype concurrently with IOP elevation. At 1 month post-IOP elevation, tissue was collected and retinal ganglion cell (RGC) axon function was assessed by optic nerve histology and fluorescent cholera toxin B anterograde transport. Hematocrit was also assessed at collection. Hematocrit was significantly elevated in mice pre-treated with rAAV2/8.CMV.Epo-R76E (59.6±9.1%) and in mice given rAAV2/1.CMV.Epo-R76E delayed (55.0±3.6%) compared to eGFP controls (42.7±3.2%). Mean IOP of 20.3±2.9 mmHg was observed in microbead-injected eyes 1 week post-induction compared to 15.4±2.1 mmHg in saline-injected controls. At 1 month post-induction, microbead-injected eGFP controls developed 20% reduction of RGC axons and 22% impairment of RGC anterograde transport compared to saline-injected controls. Microbead-injected mice pre-treated with rAAV2/8.CMV.Epo-R76E showed 4% reduction of RGC axons and 5% impairment in RGC anterograde transport. Microbead-injected mice given delayed treatment with rAAV2/1.CMV.Epo-R76E showed 18% reduction of RGC axons and 6% impairment of RGC anterograde transport. Gene delivery of modified EPO attenuates glaucomatous pathology of retinal ganglion cells in the induced model of ocular hypertension. Pre-treatment with EPO gene therapy provides more robust preservation of RGC axons than EPO gene therapy commenced at onset of hypertension. Delayed gene therapy preserves axon transport significantly, however, which may be due to delayed therapy preserving normal function of surviving RGC axons while failing to attenuate early axon loss.
