Identification of Loci with High Transgene Expression in CHO Cells

Chinese Hamster Ovary (CHO) cells are commonly used for producing therapeutic proteins in the biopharmaceutical industry. Targeted integration (TI) of therapeutic protein-encoding transgenes into predetermined high and stably expressing genomic loci can simplify the cell line development processes for biologics production. Establishing a successful TI system requires identifying genomic loci that allow a high expression of the integrated transgenes. In this work, we demonstrated that the Thousands of Reporters Integrated in Parallel (TRIP) technology can identify such transcriptional hotspots in CHO cells. TRIP simplifies screening for transcriptional hotspots since it utilizes randomly integrated barcoded reporters and uses each barcode as a unique identifier to track the genomic location and activity of the corresponding reporter by next-generation sequencing. Transcriptional hotspots identified by TRIP resulted in up to a 9.4-fold increase in mRNA levels and a 5.6-fold increase in fed-batch titers of a test molecule compared with a medium-expressing control locus. Moreover, single copy expression from one of the identified transcriptional hotspots resulted in up to a 1.6-fold higher titer in comparison to the piggyBac-mediated stable expression of the same molecule. Reporter expression levels from TRIP loci showed positive correlations with active chromatin marks; however, the proximity to active marks was not consistently deterministic. These results suggest that TRIP is a powerful functional screening method for identifying transcriptional hotspots in CHO cells without the need for more complex epigenomic analyses.