Ground-up construction of a bioproduction locus for protein expression in mammalian cells

Protein-based therapeutics, ranging from antibodies to growth hormones, represent an important class of pharmaceuticals. Recombinant DNA technologies massively expedited the process of large-scale protein manufacturing and allowed to add molecules that do not occur in nature - such as fusion proteins - to the arsenal of medicine. Large proteins and proteins that require complex post-translational modifications are normally produced in mammalian cell lines. Chinese hamster ovary (CHO) cells have become the mammalian system of choice for recombinant protein expression. The workflow currently adopted for the development of a production cell line relies on the random integration of a transgene expressing the protein-of-interest (POI) into the host cell line’s genome, normally coupled to a selection-based transgene amplification system that results in increased transgene dosage and protein expression. While high productivities are normally achieved, the establishment of a production cell line with this method requires a large screening effort that can take months to complete in order to identify high-producing, stable clones among a vast number of candidates that revert to low expression levels when the administration of selection drugs is discontinued. Random transgene integration and the use of cytotoxic selection agents have been reported to exacerbate chromosomal instability and clone-to-clone variability. With the advent of novel genome-editing tools, there is tremendous potential for revolutionising the cell line development workflow, making it more predictable and ultimately increasing the speed-to-clinic of novel biopharmaceuticals. The work presented in this thesis proceeds along this direction. The ground-up design of multi-gene expression constructs and their targeted integration via recombinase-mediated cassette exchange (RMCE) into pre-validated loci in the CHO genome is described. Up to ten gene cassettes built from unique genetic elements and expressing the same POI were concatenated in a single assembly step by homologous recombination in yeast. The use of unique regulatory and coding sequences enabled rapid multi-gene assemblies and is expected to be beneficial for chromosomal stability upon genomic integration. Gene dosage increase achieved with this ‘controlled amplification’ strategy resulted in a concurrent increase in expression levels of the mCitrine fluorescent reporter and Interferon gamma (IFNg) in transient transfection experiments. Multi-gene constructs expressing mCitrine were integrated in the CHO genome and two RMCE sites could be targeted simultaneously without selection, showing the feasibility of engineering complex bioproduction loci from the bottom up