Stable miRNA overexpression in human CAP cells: Engineering alternative production systems for advanced manufacturing of biologics using miR-136 and miR-3074.

Chinese hamster ovary (CHO) cells still represent the major production host for therapeutic proteins. However, multiple limitations have been acknowledged leading to the search for alternative expression systems. CEVEC's amniocyte production (CAP) cells are human production cells demonstrated to enable efficient overexpression of recombinant proteins with human glycosylation pattern. However, CAP cells have not yet undergone any engineering approaches to optimize process parameters for a cheaper and more sustainable production of biopharmaceuticals. Thus, we assessed the possibility to enhance CAP cell production capacity via cell engineering using miRNA technology. Based on a previous high-content miRNA screen in CHO-SEAP cells, selected pro-productive miRNAs including, miR-99b-3p, 30a-5p, 329-3p, 483-3p, 370-3p, 219-1-3p, 3074-5p, 136-3p, 30e-5p, 1a-3p, and 484-5p, were shown to act pro-productive and product independent upon transient transfection in CAP and CHO antibody expressing cell lines. Stable expression of miRNAs established seven CAP cell pools with an overexpression of the pro-productive miRNA strand. Subsequent small-scale screening as well as upscaling batch experiments identified miR-136 and miR-3074 to significantly increase final mAb concentration in CAP-mAb cells. Transcriptomic changes analyzed by microarrays identified several lncRNAs as well as growth and apoptosis-related miRNAs to be differentially regulated in CAP-mAb-miR-136 and -miR-3074. This study presents the first engineering approach to optimize the alternative human expression system of CAP-cells.