ArticleBioprocess and biosystems engineering2025
Evaluation of precursor MicroRNA (pre-miRNA) as a powerful tool for robust CHO production cell line platform development.
Article in Bioprocess and biosystems engineering, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Chinese hamster ovary (CHO) cells are the most widely used host for the commercial production of recombinant therapeutic proteins. The rapidly growing demand for large quantities of biologics at controllable cost-of-goods requires continuous cell engineering and process optimization of the CHO host cells. MicroRNAs (miRNAs) have been shown to enhance recombinant protein production in CHO cells. While studies have demonstrated that transient overexpression of certain miRNAs can increase recombinant protein yields, systematic comparisons of different miRNA overexpression forms (primary, precursor, and mature) remain limited. Furthermore, their application in stable cell line development, particularly for difficult-to-express proteins, has yet to be thoroughly explored. This study evaluated three miRNA overexpression strategies: primary miRNAs (pri-miRNAs), precursor miRNAs (pre-miRNAs), and flanked mature miRNAs (incorporating the mature sequence plus reverse complementary and loop sequences), to enhance the expression of difficult-to-express proteins in stable CHO cell lines. Notably, these miRNA constructs were built-in with the gene of interest (GOI) on the same vector to simplify stable cell line generation. Our results indicate that the pre-miRNA overexpression strategy is the most effective. Overexpression of premiR-92a, premiR-200a, premiR-483, and premiR-106b significantly increased the expression level of a bispecific antibody (BsAb) and an Fc-fusion protein without compromising product quality. Further clone evaluation of the premiR-92a and premiR-483 overexpression groups revealed an improved proportion of high-productivity and stable clones. In conclusion, this study demonstrates that integrating pre-miRNA expression cassettes into therapeutic protein vectors for co-expression is a valuable and effective engineering strategy for developing a robust stable CHO expression platform.
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