Development of human cell-based production platform for therapeutic proteins using site-specific integration

Abstract

Human cell lines, such as human embryonic kidney (HEK) 293, HT-1080, and PER.C6 have been used for the production of therapeutic proteins because of human-like post-translational modifications (PTMs), thereby ensuring proper function of recombinant proteins and eliminating the risk of immunogenic reactions. A traditional strategy for cell line development has been dependent on the random integration of gene-of-interest (GOI), which is often leading to instability of genetic and production profiles of recombinant cell lines. To overcome the drawbacks of random integration, site-specific integration of GOIs has been attempted in mammalian cell lines, including Chinese hamster ovary (CHO) and HEK293 cells. Most notably, a platform using recombinase-mediated cassette exchange (RMCE) coupled with the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) allows for the facile generation of recombinant cell lines producing therapeutic proteins. In this study, human genomic safe harbors (GSHs) were assessed and compared for the establishment of human cell-based RMCE platform. Then, various therapeutic proteins, erythropoietin, Fc-fusion proteins, monoclonal antibodies (mAbs), and coagulation factor Ⅷ, were produced in recombinant cells using this platform. Dual-landing pad cell lines were generated and RMCE system was optimized to facilitate the development of recombinant cells stably expressing multigene cassettes at safe harbors. Finally, RMCE-based CRISPR/Cas9 library screening platform was established in HEK293 cells, and novel target genes were identified from the osmotic stress screening, enabling the generation of stress-resistant human cells for improved productivity of therapeutic proteins. Comprehensive analysis of target sites for transgene expression is necessary for the establishment of cell line development platform based on site-specific integration. Human cell lines are being increasingly used as host cells to produce therapeutic glycoproteins, due to their human glycosylation machinery. In an attempt to develop a platform for generating isogenic human cell lines producing therapeutic proteins based on targeted integration, three well-known human GSHs – AAVS1, CCR5, and human ROSA26 loci – were evaluated with respect to the transgene expression level and stability in HEK293 cells. Among the three GSHs, the AAVS1 locus showed the highest eGFP expression with the highest homogeneity. Transgene expression at the AAVS1 locus was sustained without selection for approximately 3 months. Furthermore, the CMV promoter showed the highest expression, followed by the EF1α, SV40, and TK promoters at the AAVS1 locus. Master cell lines were created using CRISPR/Cas9-mediated integration of the landing pad into the AAVS1 locus and were used for faster generation of recombinant cell lines that produce therapeutic proteins with RMCE. A platform, based on targeted integration of transgenes using RMCE coupled with CRISPR/Cas9, is increasingly being used for the development of mammalian cell lines that produce therapeutic proteins, because of reduced clonal variation and predictable transgene expression. However, low efficiency of the RMCE process has hampered its application in multicopy or multisite integration of transgenes. To improve RMCE efficiency, nuclear transport of RMCE components such as site-specific recombinase and donor plasmid was accelerated by incorporation of nuclear localization signal and DNA nuclear-targeting sequence, respectively. Consequently, the efficiency of RMCE in dual-landing pad HEK293 cell lines harboring identical or orthogonal pairs of recombination sites at two well-known human safe harbors (AAVS1 and ROSA26 loci), increased 6.7- and 8.1-fold, respectively. This platform with enhanced RMCE efficiency enabled the integration of large gene cassettes, and simultaneous integration of transgenes at the two sites using a single transfection without performing selection and enrichment processes. The use of a homotypic dual-landing pad HEK293 cell line capable of incorporating the same transgenes at two sites resulted in a 2-fold increase in the transgene expression level compared to a single-landing pad HEK293 cell line. In addition, the use of a heterotypic dual-landing pad HEK293 cell line, which can incorporate transgenes for a recombinant protein at one site and an effector transgene for cell engineering at another site, increased recombinant protein production. For the application of an optimized RMCE systems, we established the CRISPR/Cas9 library screening platform in HEK293 cells based on the guide RNA integration mediated by RMCE to interrogate gene functions in a high-throughput manner. During the mammalian cell culture, cellular stresses are induced by the exogenous and endogenous changes, such as temperature, pH, osmolality, metabolite concentration, and unfolded/misfolded proteins. These stresses often aggravate cell proliferation and eventually lead to cell death, thereby impeding the efficient production of recombinant proteins with proper quality in bioprocesses. Genetic engineering of endogenous genes related to stress responses has been attempted to minimize the negative effects of stress conditions

however, the elucidation of novel targets has been labor-intensive and limited to previous studies. Using RMCE-based CRISPR/Cas9 screening platform, we screened for genes whose perturbation confers the resistance to hyperosmotic stress, which inhibits cell growth and induces apoptosis. As a result, we identified human GSPT1 gene pertaining to the apoptosis, and knockout of this gene significantly increased cell growth and prolonged culture longevity, resulting in improved productivity of therapeutic proteins during the fed-batch culture. In conclusion, the cell line development platform based on RMCE coupled with CRISPR/Cas9 has facilitated the rapid and reliable generation of recombinant cells producing various therapeutic proteins in human cells. Furthermore, the optimized RMCE process allows for the multigene expression of transgenes, thereby extending the scope of human cell engineering. Lastly, CRISPR screening platform using RMCE-mediated library integration enables the identification of novel targets associated with cellular stress, and the generation of stress-resistant producers for recombinant proteins.