Identification of a Therapeutic Dose of Continuously Delivered Erythropoietin in the Eye Using An Inducible Promoter System

Abstract

Erythropoietin (EPO) can protect the retina from acute damage, but long-term systemic treatment induces polycythemia. Intraocular gene delivery of EPO is not protective despite producing high levels of EPO likely due to its bell-shaped dose curve. The goal of this study was to identify a therapeutic dose of continuously produced EPO in the eye. We packaged a mutated form of EPO (EPOR76E) that has equivalent neuroprotective activity as wild-type EPO and attenuated erythropoietic activity into a recombinant adeno-associated viral vector under the control of the tetracycline inducible promoter. This vector was injected into the subretinal space of homozygous postnatal 5-7 day retinal degeneration slow mice, that express the tetracycline transactivators from a retinal pigment epithelium specific promoter. At weaning, mice received a single intraperitoneal injection of doxycycline and were then maintained on water with or without doxycycline until postnatal day 60. Intraocular EPO levels and outer nuclear layer thickness were quantified and correlated. Control eyes contained 6.1 ± 0.1 (SEM) mU/ml EPO. The eyes of mice that received an intraperitoneal injection of doxycycline contained 11.8 ± 2.0 (SEM) mU/ml EPO-R76E. Treatment with doxycycline water induced production of 35.9 ± 2.4 (SEM) mU/ml EPO-R76E in the eye. The outer nuclear layer was approximately 8μm thicker in eyes of mice that received doxycycline water as compared to the control groups. Our data indicates that drug delivery systems should be optimized to deliver at least 36mU/ml EPO into the eye since this dose was effective for the treatment of a progressive retinal degeneration.

Hypoxia-Inducible Factor-1α Target Genes Contribute to Retinal Neuroprotection

“…This suggests that hormone systemic effects contribute to progressive neuroprotection (Colella et al, 2011 ). Furthermore, high levels of EPO is not protective due to its bell-shaped dose curve (Hines-Beard et al, 2013 ), and the neuroprotective dose is higher than that required for erythropoiesis (Coleman and Brines, 2004 ). Early retinal EPO replenishment improves retinal vascular stability, but elevated EPO levels during the proliferation stage contribute to neovascularization and ocular diseases (Coleman and Brines, 2004 ; Chen et al, 2008 ).…”

“…An earlier report revealed that by either transgenic or systemic applications, EPO protected against apoptotic cell death during acute, light-induced photoreceptor cell death but not in genetically based retinal degeneration such as in retinal degeneration 1 (rd1) or dominant retinitis pigmentosa mouse models (Grimm et al, 2004 ). Findings from a study demonstrated that subretinal injection of EPO AAVs resulted in approximately an 8 μm thicker outer nuclear layer (ONL) in retinas of retinal degeneration slow (rds) mice compared to the control group (Hines-Beard et al, 2013 ). Nevertheless, one may argue that the neuroprotective effect of EPO is short-lived and the ONL may degenerate without a repeated hypoxia preconditioning (Grimm et al, 2005 ).…”

Antioxidant and Cytoprotective Potential of Erythropoietin in Mitigating Oxidative Stress-Induced Changes in the Retinal Pigment Epithelium

“…Intravenous EPO delivery improved visual acuity and color vision in patients following indirect traumatic neuropathy [8]. Studies show that systemic or retinal delivery of EPO or EPO-R76E, a modified form of EPO with reduced erythropoietic activity, can improve the function of retinal ganglion cells and photoreceptors cells [9][10][11][12][13][14][15][16][17][18][19].…”

Epobis is a Nonerythropoietic and Neuroprotective Agonist of the Erythropoietin Receptor with Anti-Inflammatory and Memory Enhancing Effects

“…Moreover, EPO has beneficial effects on memory and mood of animals and humans with depression-like symptoms [11]. Animal studies using EPO or EPO derivatives have demonstrated an improved memory and a reduced endothelial and neuronal degeneration in models of Alzheimer's disease [12–14], a protection against experimental cerebral malaria and pneumococcal meningitis [15, 16], downregulation of proinflammatory cytokines and upregulation of anti-inflammatory cytokines in a model of amyotrophic lateral sclerosis [17], a reduction of progressive retinal degeneration [18], and neuroprotective effects in relation to status epilepticus [19] and cervical subacute spinal cord compression [20]. Importantly, most of these beneficial effects of EPO are believed to be unrelated to the erythropoietic effects of EPO but not necessarily unrelated to signaling through the EPO receptor [1–3].…”