Identification and engineering of Chinese hamster ovary (CHO) cell genes whose expression is correlated with age-related growth rate acceleration

Generational age-related Chinese hamster ovary (CHO) cell growth changes are undesirable for large-scale manufacturing cell lines. An age-related high growth phenotype in CHO cells has previously been shown to be associated with a Chromosome 2 (Chr2) duplication event in late generation cells. We have evaluated the contribution of a subset of the 123 genes involved in the duplication event in relation to the high growth phenotype of late generation cells (> 90 generations post-cloning). We show that this age correlated high growth phenotype associated with a Chr2 duplication event in CHO cells can be reversed by single knockdown of several transcripts that are upregulated as a result of the duplication event. Further, one of these genes, Cxxc1, when overexpressed in low growth phenotype cells, results in the generation of high growth phenotype cell pools. Thus, Cxxc1 overexpression engineering in CHO cells can impart a high growth phenotype that correlates with those observed in Chr2 duplication event cell lines. Furthermore, this impact can be reversed by subsequent knockdown of Cxxc1 transcript levels such that the cells revert to a low growth phenotype. The high growth phenotype of Cxxc1 overexpressing engineered cell pools was shown to be stable for 100 generations, and although the high growth phenotype was associated with a reduced cell specific productivity, volumetric titre was maintained/compensated for by the increased cell number. We propose targeted cell engineering of CHO cells to overexpress Cxxc1 may be a potential strategy to overcome the issue of age-related genetic instability associated growth phenotype to generate new host cell lines with a stable, high growth phenotype.