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bioRxiv · 10.1101/2021.06.24.449789

Rational engineering of an erythropoietin fusion protein to treat hypoxia

Lee, J.·Vernet, A.·Gruber, N. G.·Kready, K. M.·Burrill, D. R.·Way, J. C.·Silver, P. A.
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Abstract

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its pro-thrombotic activity is problematic for use of erythropoietin in treating hypoxia. We constructed a fusion protein that stimulates red blood cell production and neuroprotection without triggering platelet production, a marker for thrombosis. The protein consists of an anti-glycophorin A nanobody and an erythropoietin mutant (L108A). The mutation reduces activation of erythropoietin receptor homodimers that induce erythropoiesis and thrombosis, but maintains the tissue-protective signaling. The binding of the nanobody element to glycophorin A rescues homodimeric erythropoietin receptor activation on red blood cell precursors. In a cell proliferation assay, the fusion protein is active at 10-14 M, allowing an estimate of the number of receptor-ligand complexes needed for signaling. This fusion protein stimulates erythroid cell proliferation in vitro and in mice, and shows neuroprotective activity in vitro. Our erythropoietin fusion protein presents a novel molecule for treating hypoxia.

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Lee, J., Vernet, A., Gruber, N. G., Kready, K. M., Burrill, D. R., Way, J. C., Silver, P. A.. 2021-06-24. Rational engineering of an erythropoietin fusion protein to treat hypoxia. https://doi.org/10.1101/2021.06.24.449789

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