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 mu g/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 similar to 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.

Automated summary

The study demonstrates that non-sialylated rhuEPO(P) is a non-erythropoietic and non-immunogenic neuroprotectant in a cerebral ischemia and reperfusion mouse model, showing significant reduction in cerebral infarct volume and edema, as well as improvement in neurological deficits and histopathological outcome. Non-sialylated rhuEPO(P) displays pleiotropic neuroprotective effects, similar to rhuEPO(M), making it a promising candidate for stroke treatment.