Targeted gene integration for improved production of therapeutic proteins in CHO cells
Abstract
Recombinant therapeutic proteins are crucial medicines for the treatment of human diseases including cancer, infections, inflammatory and autoimmune diseases. The production of these proteins relies on mammalian cell factories - Chinese hamster ovary (CHO) cells. For each protein, a stable CHO cell line with high productivity and protein quality must be developed. Traditionally, CHO cell line development has been long and unpredictable and required massive resources to find a single cell line with the best performance. The recent development of precise genome editing tools for mammalian cells enabled a potential paradigm shift in CHO cell line engineering, offering more rapid and predictable ways of biopharmaceuticals production. This thesis aimed to develop and optimize new CHO cell line development methods based on targeted gene integration and encourage the change to nextgeneration cell line development platforms. To advance CHO cell line development, we created a targeted gene integration platform using CRISPR/Cas9 and recombinases and showed its use for the production of valuable therapeutic proteins, including a vaccine against SARS-CoV-2. We demonstrated that this platform minimizes clonal variation, which in turn reduces the need for screening of cell lines and thus shortens the timeline of CHO cell line development. This platform enables robust comparative studies of CHO cells, which was illustrated by the analysis of CHO transcriptomes. Furthermore, we increased the productivity of cell lines using multi-copy targeted integration, reaching industriallyrelevant titers of therapeutic proteins. We investigated the response to increased protein production in multi-copy cell lines using RNA-seq and revealed a transcriptional limitation in protein expression that appears at high copy numbers. Overall, the thesis proves that targeted integration is an advantageous method for CHO cell line generation that minimizes genetic heterogeneity thus making cell line generation faster, more robust and predictable. It can clearly improve CHO cell line development, reducing time, resources and cost of delivering new biopharmaceuticals to the patients.
Info
Thesis PhD, 2020
UN SDG Classification
DK Main Research Area
Science/Technology
