Glycoengineering of CHO-DG44 cells for the production of recombinant human R-Spondin 1 (rhRSPO1) with high content of terminally sialylated N-linked glycan chains

Abstract

R-Spondins (RSPOs) comprise a family of secreted glycoproteins known for their important roles in cell proliferation, differentiation and death by inducing the Wnt pathway. Studies have demonstrated the importance of RSPOs in the regulation of several tissue-specific processes, in particular, the proliferation of intestinal stem cells. Aiming at its application in bowel regeneration in short bowel syndrome (SBS), which causes serious nutrition and development problems in affected patients, our group established platforms for recombinant expression of RSPO1 protein and evaluated the activity of the recombinant protein (rhRSPO1). It was concluded that treatment with rhRSPO1 and the consequent activation of the Wnt canonical pathway proved to be very promising with regard to the generation of a functional engineered murine intestine and improvement of SBS symptoms. However, in purified rhRSPO1 preparations, produced from a CHO-DG44 animal cell line, reduced protein stability was observed, making its long-term storage impossible and making its purification process difficult. It is believed that the main reason for this reduction in stability is due to changes in the glycosylation profile of the molecule, particularly in the absence of terminal sialylation in CHO-DG44 cells, which can significantly alter the protein folding and secretion process, aside from altering the pharmacokinetics and biological activity of the molecule. The enzyme ST6GAL1 (ST6 ²-galactoside ±-2,6-sialyltransferase), responsible for adding a sialic acid molecule to the ends of N-linked oligosaccharide chains during the glycosylation process, is not expressed in CHO-DG44 cells. With this, this project proposes the establishment of a CHO-DG44 cell line capable of producing glycoproteins with sialylated N-linked glycan chains for the production of the rhRSPO1 protein, through the promotion of the ectopic expression of the ST6GAL1 enzyme and the RSPO1 protein in cGMP conditions, in order to increase the stability of the purified molecule and its biological activity, aiming at its future application in clinical trials. (AU)