Predicting favorable landing pads for targeted integrations in Chinese hamster ovary cell lines by learning stability characteristics from random transgene integrations

Chinese Hamster Ovary (CHO) cell lines are considered to be the preferred platform for the production of biotherapeutics, but issues related to expression instability remain unresolved. In this study, we investigated potential causes for an unstable phenotype by comparing cell lines that express stably to ones that undergo loss in titer across 10 passages. Factors related to transgene integrity and copy number as well as the genomic profile around the integration sites were analyzed. Horizon Discovery CHO-K1 (HD-BIOP3) derived production cell lines selected for phenotypes with low, medium or high copy number, each with stable and unstable transgene expression, were sequenced for capturing changes at genomic and transcriptomic levels. The exact sites of the random integration events in all the cell lines were also identified, followed by profiling genomic, transcriptomic and epigenetic patterns around them. Based on the information deduced from these random integration events, genomic loci that potentially favor reliable and stable transgene expression were reported for targeted transgene integration. By comparing stable vs unstable phenotype across the journey of the transgene from integration to expression, we could establish that expression stability can be controlled at three levels: 1) Good choice of integration site, 2) Ensuring integrity of transgene and observing concatemerization pattern after integration, and 3) Checking for potential stress related cellular processes. Genome wide favorable and unfavorable genomic loci for targeted transgene integration can be browsed at https://www.borthlabchoresources.boku.ac.at/cgr-stablelandingpads/jbrowse/