Rational engineering of an erythropoietin fusion protein to treat hypoxia

Erythropoietin enhances oxygen delivery and reduces hypoxia-induced cell death, but its prothrombotic 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 tissueprotective 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.

Automated summary

The fusion protein developed in this study stimulates red blood cell production and neuroprotection, while avoiding thrombosis. It is composed of an anti-glycophorin A nanobody and an erythropoietin mutant. The protein exhibited promising activity in vitro and in mouse models, highlighting its potential as a novel therapeutic molecule for hypoxia.