Multi-Landing Pad DNA Integration Platform for Mammalian Cell Engineering

Landing Pad (LP) technology is a novel strategy for site-specific chromosomal DNA integration in mammalian cells based on the construction of stable isogenic chassis LP cell lines that form a framework for targeted integration of large DNA payloads into a well-defined chromosomal context. We show the discovery and construction of novel LP cell lines in Chinese Hamster Ovary (CHO) cells, the preferred mammalian host for therapeutic proteins production, and their application for efficient and stable production of monoclonal antibodies. Using random lentiviral genome targeting we identified 21 novel stable integration sites in CHO cell genome and subsequently generated single-, double- and triple-LP cell lines by CRISPR/Cas9 targeted integration. By using a highly efficient BxB1 recombinase along with different selection markers and fluorescent reporters at each site, we were able to direct recombinase-mediated insertion of heterologous DNA to different sites, or target all three with a single transfection. We used this method to precisely integrate up to 9 copies of a monoclonal antibody, representing about 100 kb of heterologous DNA encoding 21 transcriptional units. Because the integration was targeted to pre-validated loci, recombinant protein expression remained stable for weeks and additional copies of the antibody cassette in the integrated payload vector resulted in a linear increase in antibody expression. Overall, this foundational technology allows fast and efficient development of recombinant mammalian cell lines that stably express large payloads and multi-component genetic networks in preselected chromosomal sites, and has broad applications for mammalian synthetic biology, gene therapy, recombinant protein production, and bio-manufacturing.