Streamlined Human Cell-Based Recombinase-Mediated Cassette Exchange Platform Enables Multigene Expression for the Production of Therapeutic Proteins

Abstract

A platform, based on targeted integration of transgenes using recombinase-mediated cassette exchange (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 duallanding pad human embryonic kidney 293 (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 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. Overall, a streamlined RMCE platform can be a versatile tool for mammalian cell line development by facilitating multigene expression at genomic safe harbors.

Hybrid cell line development system utilizing site-specific integration and methotrexate-mediated gene amplification in Chinese hamster ovary cells

“…The HS4 insulator was amplified from #154834. The NLS-Cre-NLS vector has been described in a previous study ( Shin et al, 2021 ). An RMCE donor integrating the C12orf35 locus was constructed by combining CMVcore-DHFR-BGHpA and LoxP-CMV-EGFP-Lox2272-SV40pA.…”

CRISPR‐interceded CHO cell line development approaches

“…In both sLP and homotypic dLP, NP N‐terminal NLS and NF‐κB 5′/3′ DTS were the most effective, and the highest efficiency was reported when both NLS and DTS were utilized. In heterotypic dLP (harboring Bxb1/attP LP), the RMCE efficiency was the best when NP C‐terminal NLS and NF‐κB5′/3′ DTS were applied and were the highest in the presence of both NP C‐terminal NLS and NF‐κB 5′/3′ DTS (S. Shin, Kim, Lee & Lee, 2021).…”

“…Precise insertion of transgenes into the so‐called genomic safe harbors ensures production consistency in long‐term culture and high expression levels of the biotherapeutics. This strategy also alleviates clonal variation and results in production titers comparable with those of RI (S. Shin, Kim, Lee & Lee, 2021; Tihanyi & Nyitray, 2020).…”

Engineering a material-genetic interface as safety switch for embedded therapeutic cells

“…Finally, so as to apply this design to a wide range of encapsulated cell therapies, a stable insertion of DNA would be required instead of transient transfection. For such purposes, different systems have been described that allow the stable installation of multiple genes in different cell lines [46][47][48] . In addition, since the final product would involve genetically modified cells, it would have to fulfill the biosafety requirements that apply to genetically modified organisms used in the clinic 49 .…”