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

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Abstract

Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been shown to be a multimodal neuroprotectant targeting an array of key pathological mechanisms in experimental stroke models. However, the rhuEPOM 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-rhuEPOP, a rhuEPO derivative without sialic acid residues. In the present study, we proved that asialo-rhuEPOP 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-rhuEPOP, like rhuEPOM, 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-rhuEPOP lacking erythropoietic activity and immunogenicity holds great translational potential as a multimodal neuroprotectant for stroke treatment.

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Acknowledgements

We thank Qingping He for the help with immunofluorescence assay.

Funding

This work was supported by the National Institute of General Medical Sciences grant SC1GM111178 to Jiahua Xie.

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Author notes

  1. Maotao He and Farooqahmed S. Kittur contributed equally to this work

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Biomanufacturing Research Institute & Technology Enterprise, North Carolina Central University, Durham, NC, 27707, USA

    Maotao He, Farooqahmed S. Kittur, Chiu-Yueh Hung, Jianhui Zhang, P. Andy Li & Jiahua Xie

  2. Department of Pathology, Weifang Medical University, Weifang, 261000, China

    Maotao He

  3. Department of Pathology, Ningxia Medical University, Yinchuan, 750004, China

    Li Jing

  4. Carilion Clinic and Virginia Tech Carilion School of Medicine, Roanoke, VA, 24014, USA

    David C. Sane

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  1. Maotao He
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Contributions

F.S.K, D.C.S, P.A.L, and J.X. designed research; M.H, F.S.K, C.-Y.H, and J.Z. performed the experiments; M.H, F.S.K, P.A.L., J.Z. L.J., D.C.S, and J.X. analyzed the data. M.H, F.S.K., and J.X. drafted the manuscript with input from all authors.

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Correspondence to P. Andy Li or Jiahua Xie.

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This study was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the Association and Accreditation of Laboratory Animal Care International guidelines and under the authorization of the Institutional Animal Care and Use Committee at the North Carolina Central University, Durham, NC, USA.

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Conflict of Interest

JX, FK, and C-Y H are inventors of filed patent “Methods for the production of cytoprotective asialo-erythropoietin in plants and its purification from plant tissues” (PCT NUMBER: US2013031382, pending).

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He, M., Kittur, F.S., Hung, CY. et al. A Novel Plant-Produced Asialo-rhuEPO Protects Brain from Ischemic Damage Without Erythropoietic Action. Transl. Stroke Res. 13, 338–354 (2022). https://doi.org/10.1007/s12975-021-00943-z

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