Glycoengineering by hyperglycosylation: an innovative strategy to block the undesired effects of human erythropoietin as a neurotherapeutic candidate

BURGI, MM.; APARICIO, G.; WANDEL-PETERSEN, V.; DEPETRIS, M.; KRATJE, R.; SCORTICATI, C.; OGGERO EBERHARDT, M.

Buenos Aires

Congreso; Congreso conjunto SAIB-SAMIGE 2021: LVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB) y de la Asociación Civil de Microbiología General (SAMIGE); 2021

SAIB-SAGIME

Neurological disorders affect millions of people worldwide causing behavior-cognitive disorders. 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, nowadays there is no effective treatment for them. The pharmaceutical market only offers medicines to relieve symptoms. Thus, it is necessary to develop new therapeutics which can produce a perceptible improvement in the patient. 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 analogs like asialoEPO, carbamylated-EPO, or EPO-peptides have been developed showing different weaknesses: EA preservation, low stability, potential immunogenicity, or fast clearance. This work is based on the hypothesis that glycoengineering by hyperglycosylation would be an appropriate technology to block the EA of hEPO while preserving the neurological activity and conferring long-lasting actions. N-glycoengineering was carried out to add a new glycosylation site within the hEPO sequence responsible for its EA. Thus, one or two amino acids were changed by site-directed mutagenesis to create the N-X-S consensus sequence required to incorporate a N-oligosaccharide. hEPO-derivatives were produced by CHO.K1 cell cultures, affinity-purified, and functionally analyzed studying their in vitro and in vivo EA. The neurobiological activities were evaluated by assessing neuritogenesis, filopodia density, and synapses formation in neuron´s primary cultures. We also accomplished the analysis of neuronal rescue from estaurosporine-apoptotosis induction. 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 neuroprotective activity more efficiently than hEPO. Furthermore, they enhanced neuritogenesis and induced filopodia formation more competently than hEPO. In particular, Mut 45_47 and Mut 104 were more efficient to stimulate synapses formation than Mut 151_153 that showed a comparable activity respect to hEPO.Finally, this modification 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.In conclusion, the use of glycoengineering by hyper-N-glycosylation was a proper procedure to differentiate the hEPO activities by blocking the hematopoietic action, and consequently its undesirable effects, while preserving its neurobiological function. Each mutein encompasses distinct particularities that will guide this research to a proof-of-concept trial in wild type mice to explore their potentiality as biotherapeutics for neurological disorders.