A Novel Plant-Produced Asialo-rhuEPO Protects Brain from Ischemic Damage Without Erythropoietic Action

Mammalian cell-produced recombinant human erythropoietin (rhuEPO^M) has been shown to be a multimodal neuroprotectant targeting an array of key pathological mechanisms in experimental stroke models. However, the rhuEPO^M clinical trials were terminated due to increased risk of thrombosis, largely ascribed to its erythropoietic function. We recently took advantage of a plant-based expression system lacking sialylation capacity to produce asialo-rhuEPO^P, a rhuEPO derivative without sialic acid residues. In the present study, we proved that asialo-rhuEPO^P is non-erythropoietic by repeated intravenous injection (44 μg/kg bw) in mice showing no increase in hemoglobin levels and red blood cell counts, and confirmed that it is non-immunogenic by measuring humoral response after immunizing the mice. We demonstrate that it is neuroprotective in a cerebral ischemia and reperfusion (I/R) mouse model, exhibiting ~ 50% reduction in cerebral infarct volume and edema, and significant improvement in neurological deficits and histopathological outcome. Our studies further revealed that asialo-rhuEPO^P, like rhuEPO^M, displays pleiotropic neuroprotective effects, including restoring I/R-interrupted mitochondrial fission and fusion proteins, preventing I/R injury-induced increase in mitophagy and autophagy markers, and inhibiting apoptosis to benefit nerve cell survival. Most importantly, asialo-rhuEPO^P lacking erythropoietic activity and immunogenicity holds great translational potential as a multimodal neuroprotectant for stroke treatment.