Novel erythropoietin-based therapeutic candidates with extra N-glycan sites that block hematopoiesis but preserve neuroplasticity.
Burgi, M., Aparicio, G. I., Dorella, A., Kratje, R., Scorticati, C. and Oggero, M.
UNL, CONICET, FBCB, Centro Biotecnologico del Litoral, Ciudad Universitaria -C.C. 242- (S3000ZAA) Santa Fe, Pcia, Santa Fe, Argentina.
IIB-UNSAM, IIBio-CONICET, Laboratorio de Neurobiologia Molecular y Celular. Campus Miguelete, San Martin, Buenos Aires, Argentina.
UNL, FBCB, Centro Biotecnologico del Litoral, Ciudad Universitaria -C.C. 242- (S3000ZAA) Santa Fe, Pcia, Santa Fe, Argentina.
Neurological disorders affect millions of people worldwide causing behavior-cognitive disabilities. Nowadays there is no effective treatment for them. Human erythropoietin (hEPO) has been used in clinical tests because of its neurotrophic and cytoprotective properties. However, the erythropoietic activity (EA) should be considered as a side effect. Some analogs like asialoEPO, carbamylated-EPO or EPO-peptides have been developed showing different weaknesses: erythropoiesis preservation, low stability, potential immunogenicity, or fast clearance. In this work, we used a novel strategy that blocks the EA but preserves hEPO neurobiological actions. N-glycoengineering was carried out to add a new glycosylation site within the hEPO sequence responsible for its EA. hEPO-derivatives were produced by CHO.K1 cell cultures, affinity-purified and functionally analyzed studying their in vitro and in vivo EA. Also, we studied the in vitro neuronal plasticity in hippocampal neurons and neuroprotective action by rescuing hippocampal neurons from apoptosis. The muteins: Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) completely lost their EA in vitro and in vivo but preserved their neuroprotection activity, which enhanced neuroplasticity more efficiently than hEPO. Interestingly, Mut 45_47 resulted in a promising candidate to be explored as a neurotherapeutic considering not only its biopotency but also its pharmacokinetic potential due to the hyperglycosylation. This article is protected by copyright. All rights reserved.
Biotechnology Journal : e2000455 (2021)