Laboratory of Mammalian Cell Design and Engineering
We are a collaborative and energetic team at Ajou University in Korea. Our research goal is to develop state-of-the-art genome editing technologies and mammalian synthetic biology tools to improve mammalian cell engineering efforts and facilitate implementation of next-generation cell line development technology.
Therapeutic proteins are effective drugs for treating many diseases including diabetes, rheumatoid arthritis, clotting disorders, and cancers because of their highly specific functions, reduced side effects, and lack of immune response. For the production of therapeutic proteins, mammalian cells, particularly Chinese hamster ovary (CHO) cells, have been the most popular hosts in the biopharmaceutical industry. As of 2018, 79% of therapeutic proteins approved in the US and/or EU are produced in mammalian cells. This trend in the prominence of mammalian over microbial manufacturing platforms will continue to increase with the proportion of complex molecules in the pipeline at both the qualitative and quantitative levels. Our research group focus on the development of cost-effective and efficient high-yielding mammalian cell-based production processes through genome-scale based methodologies including genome editing technologies and systems biology approaches. For this purpose, we currently have three specific research topics as following:
The bacterial clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system enables rapid, easy and efficient engineering of mammalian genomes. It has a wide range of applications from modification of individual genes to genome-wide screening or regulation of genes. Facile genome editing using CRISPR/Cas9 empowers researchers in the industrial mammalian cell community to elucidate the mechanistic basis behind high level production of proteins and product quality attributes of interest. Previously, we have generated methods for generating gene knockouts, targeted transgene integration and multiplexed genome editing which has accelerated industrial mammalian cell engineering significantly and opened up for new engineering strategies. Now, we will expand our current genome engineering tools to generate multiple gene corrections, targeted integrations, and gene activation for tight regulation of gene expression.
I. Development of efficient and precise CRISPR-Cas9 based genome engineering tools
II. Genome mining for transgene integration site/element screening
The targeted integration of a gene of interest leads to more stable and homogeneous transgene expression in recombinant mammalian cell lines, in contrast to high levels of variation seen in random integration clones. Additionally, homologous recombination-dependent knock-in methods not only insert clean plasmid backbone-free gene expression cassettes in a precise manner, but also allow regulation of knock-in direction and genome-transgene junctions. Removal of plasmid bacterial backbone DNA is beneficial for homogeneity of transgene expression. Efficient targeted knock-in methods are only useful when locations of the transcriptionally active genomic regions, hot spots, are known. In this regard, we are working on knowledge-based prediction of hot spots. Such genome mining efforts may provide much safer and more reasonable candidate integration sites that support high and stable transgene expression while minimizing the risk of perturbing the natural genomic landscape of host cells. Because transgene expression levels are affected by both target locus and regulatory elements, we are also trying to study the crosstalk observed between integration sites and transgene regulatory elements, which can provide a more systematic guideline for the selection of the site of integration and its compatible vector element.
III. Platform development for production of difficult-to-express proteins
The emergence of novel proteins such as bispecific antibodies and fusion proteins have shown superior clinical benefits for treating complex diseases. However, many of these proteins are poorly expressed in mammalian cells. To provide next-generation therapeutics to patients, novel strategies are required to develop cost-effective and efficient high-yielding mammalian cell-based production processes.
Expression can be limited by the folding, assembly, and quality control processes of the cell, which occurs in the endoplasmic reticulum (ER) as part of the secretory pathway. Expression of these difficult-to-express (DTE) proteins can interfere with ER function and exceed the inherent secretory capacity of industrial mammalian cell lines, such as CHO cells, leading to inefficient secretion and low productivity. However, the complexity of this pathway, makes it difficult to engineer mammalian cells by manipulating a limited number of target genes. Therefore, our group is working on the identification of limiting bottlenecks, as well as specific engineering strategies to improve the production of DTE proteins in rCHO cells. Our aim is to enable the CHO cell secretory machinery to cope with DTE protein production, thus support high-level production of DTE proteins and developing customized therapeutic proteins in a rational manner.