Turning an old medicine into a new therapeutical candidate: from erythropoietin to a novel hyperglycosylated, non-erythropoietic but neuroprotective analogue

MARÍA DE LOS MILAGROS BÜRGI; VALENTINA WANDEL-PETERSEN; MATÍAS A. DEPETRIS; SOFÍA GIORGETTI; MARINA ETCHEVERRIGARAY; GABRIELA APARICIO; CAMILA SCORTICATI; SAHAB ARINRAD, ; ANJA RONNENBERG; HANNELORE EHRENREICH; RICARDO B. KRATJE; MARCOS OGGERO EBERHARDT

Lisboa

Congreso; 27th ESACT Meeting; 2022

European Society for Animal Cell Technology (ESACT)

Background and novelty: Neuropsychiatric disorders affect millions of people worldwide. They affect the central nervous system and are characterized by their chronicity and progressive evolution. In 2019, 1.5 billion people were diagnosed with some neurological disorder around the globe. Despite their exponential increment, the pharmaceutical market mainly offers medicines to relieve symptoms. Thus, it is necessary to develop new therapeutics which can produce a perceptible improvement in the patients. Human erythropoietin (hEPO) has been used in clinical trials due to its neurotrophic and cytoprotective properties. However, erythropoietic activity (EA) should be considered as a side effect. Some analogues like asialoEPO, carbamylated-EPO, or EPO-peptides have been developed showing different weaknesses: EA preservation, low stability, potential immunogenicity, or fast clearance.Experimental approach: Glycoengineering by hyperglycosylation was used as an innovative technology to block the EA of hEPO while preserving the neurological activity and conferring long lasting actions. A new consensus sequences for Nglycosylation was introduced by site directed mutagenesis within the hEPO sequence responsible for its EA. hEPOderivatives were then produced by CHO.K1 cell cultures, affinity-purified, and functionally analyzed studying their in vitro and in vivo EA and neurobiological actions. The in vitro neuroactivity was evaluated in neuron´s primary cultures by assessing neuritogenesis, filopodia density, synapses formation neuronal rescue from staurosporine-induced apoptosis. The in vivo neurobiology was evaluated by the voluntary running performance of young male C57Bl/6N mice during an overnight exposure to Complex Running Wheels (CRW) in their home cages.Results and discussion: Mut 45_47 (K45 > N45 + N47 > T47), Mut 104 (S104 > N104), and Mut 151_153 (G151 > N151 + K153 > T153) completely lost their EA both in vitro and in vivo, but preserved their in vitro neuroprotective activity.Particularly, Mut 45_47 and Mut 104 were more efficient to stimulate synapses formation in vitro than Mut 151_153 that showed a comparable activity respect to hEPO. When analyzing the in vivo potency to improve motor-cognitive performance in mice using overnight complex running wheels, only Mut 151-153 was comparable to recombinant hEPO (p>0.9 applying Friedman´s test and post hoc Dunn´s corrected multiple comparisons). Finally, the additional Nglycosylation site also improved the pharmacokinetic properties of Mut 45_47 and Mut 151_153 by reducing their clearance in plasma and increasing their half-life in blood.