GENOMIC UNDERSTANDING OF CLONAL VARIATION IN RECOMBINANT CHO CELLS

摘要

In the current manufacturing platform for large-scale production of therapeutic proteins, recombinant Chinese hamster ovary (rCHO) cell line generation is probably the most time-consuming step. The high degree of phenotypic heterogeneity in the absolute transgene expression level and variable responses to culture conditions among the selected clones, which has been referred to as 'clonal variation', makes the cell line generation process laborious and time-consuming. Upon transfection, a transgene is randomly integrated into the chromosomes of the host cells, and the integration site of the transgene is believed to be responsible for this clonal variation. However, the lack of understanding the molecular basis involved in clonal variation has hindered rapid cell line generation with a predicted culture performance. With the availability of CHO genome sequences and targeted genome sequencing/editing technologies, we investigated 'clonal variation' with an emphasis on the transgene integration sites and configuration of the integrated transgenes. rCHO cell clones expressing GFP, which were constructed by random integration of the GFP expression vector into CHOK1 host cells, revealed a remarkable variability in GFP expression at 37℃. Furthermore, they showed different responses to culture temperature shifts from 37℃ to 33℃ confirming the existence of clonal variation. Application of targeted sequencing by the proximity ligation to the transgenes enabled the mapping of the integrated transgenes, and thereby the genomic integration sites of the transgene in the representative rCHO cell clones showing different responses to hypothermia were identified. To determine whether the different responses of the rCHO clones to hypothermia were due to the different integration sites of the transgenes, rCHO cell clones expressing GFP were also constructed by CRISPR/Cas9-mediated targeted integration of an intact transgene into CHOK1 host cells at the integration sites that were identified in the rCHO cells exhibiting different responses to hypothermia. Surprisingly, the rCHO clones constructed with targeted integration, regardless of the integration site of the gene, had similar expression patterns in terms of the absolute expression level and responses to hypothermia. They all exhibited enhanced GFP expression with hypothermia. Evidence of several rearrangements in the integrated transgene was detected in the rCHO clones constructed with random integration, which may interrupt the normal function of regulatory elements, particularly promoters. Promoter replacement and dissection results support the crucial role of promoter elements in the differential transgene expression patterns at the identical genomic site. Taken together, we demonstrate the complex nature of 'clonal variation' in rCHO cells which encompasses the concept of vector elements and their rearrangement upon random integration besides just the genomic integration sites. Further characterization of the interactions between the integration sites and vector regulatory elements together with controlled integration of transgenes could lead to the tailored control of recombinant gene expression in rCHO cells while minimizing clonal variation.