Lentiviral Vector-Mediated Gene Delivery for the Generation of Chinese Hamster Ovary Cell Lines with Improved Productivity and Stability

Recombinant cell line generation by standard transfection techniques is a time-consuming and labor-intensive process often leading to an unpredictable outcome as transgene integration is a rare, random event. Consequently, the population of cells obtained after standard gene delivery is heterogeneous in terms of specific growth and recombinant protein productivity. Therefore, genetic selection is usually applied to the population for up to 3 weeks to eliminate non-recombinant cells. A large number of the surviving cell lines must be screened to find a few which stably produce the recombinant protein at the desired level. These must be maintained in culture for 2-4 months in the absence of selection to assess the stability of protein production. In contrast, lentiviral vectors (LVs) deliver the recombinant gene to target cells through infection and a controlled integration process, which allows the inserted transgene to become a permanent genetic element of the transduced cell. Moreover, lentiviruses tend to integrate into transcriptionally active sites of the host cell genome. Hence, LV-mediated gene transfer was expected to increase the probability of generating a stable high-producing clone in comparison to the standard methods based on plasmid transfection. Therefore, the objective of this PhD thesis was to improve the efficiency of stable cell line generation with the use of LVs. For this purpose, LVs carrying the enhanced green fluorescent protein (eGFP) gene under the control of either the cytomegalovirus immediate early promoter/enhancer (CMV) or the elongation factor-1 alpha promoter (EF-1α) were generated and used to infect Chinese hamster ovary (CHO) cells. Two days after gene delivery at a multiplicity of infection (MOI) of 2, up to 98% of infected cells were eGFP-positive. Following isolation by fluorescence-activated cell sorting (FACS), CHO cell pools and clonal cell lines were analyzed for the stability of eGFP expression over time. In these studies, the EF-1α promoter was found to yield more stable and homogeneous protein expression compared to the CMV promoter. For comparison, cell pools and cell lines were generated by transfection with the LV transfer plasmid bearing the eGFP gene. The level and stability of eGFP expression was greater in LV-generated pools and cell lines than in those established by transfection. Subsequently, an LV expressing the tumor necrosis factor receptor-Fc (TNFR-Fc) fusion gene and the eGFP gene from a bicistronic mRNA under the control of the EF-1α promoter (LV_EF-TNFR) was generated and used to infect CHO cells. Based on selective FACS gating for high eGFP expression, stable CHO clones showing volumetric productivities of up to 100 mg/L of TNFR-Fc in a non-optimized batch process could be isolated within 2 days of infection at an MOI of 2. The level of expression of the recombinant protein remained constant for 3 months in serum-free suspension culture and in the absence of chemical selective agents.