Targeted integration of miR-17 using CRIS-PITCh/RMCE hybrid system in CHO cells is associated with increased recombinant ACE2 production

Chinese hamster ovary (CHO) cells are widely used for recombinant therapeutic protein production. MicroRNA engineering represents a potential strategy for improving CHO cell performance; however, conventional random integration can introduce variability in transgene expression. Here, we investigated targeted miR-17 integration using a combined CRIS-PITCh/recombinase-mediated cassette exchange (RMCE) strategy in an hrsACE2-producing CHO-K1 cell line. A Bxb1-compatible landing pad was targeted near the S100A locus using CRIS-PITCh, followed by RMCE-mediated integration of miR-17 or a scrambled control cassette. Engineered clones were characterized by junction PCR, flow cytometry, qPCR, and RT-qPCR. Cell growth, viability, and hrsACE2 production were subsequently evaluated. In the representative single-copy clones evaluated, miR-17 expression was associated with enhanced cell viability and extended culture duration. The miR-17-expressing clone showed an approximately 2.3-fold increase in volumetric productivity and an approximately 2.8-fold increase in estimated specific productivity compared with the scramble control clone. These proof-of-concept findings demonstrate the feasibility of combining CRIS-PITCh and RMCE for targeted miRNA engineering in recombinant CHO cells and suggest that targeted miR-17 expression may improve hrsACE2 production and culture longevity. Further evaluation across independently derived clones is required to establish the generalizability of these effects.Keywords: CRIS-PITCh, RMCE, miR-17, Productivity, Growth rate.