(6hf) Development of Strategies to Combat Genomic Instability in Cell Culture Engineering, Biopharmaceutical Production, Disease and Ageing

Research Interests:

Glyco-engineering of biopharmaceuticals

Glycans are sugar polymers of outstanding pharmaceutical importance. Not only do some of the most widely prescribed drugs represent glycan biopharmaceuticals (e.g. heparin), but glycans also modify and impact the efficacy and safety of protein drugs, such as hormones or antibodies. Unlike proteins, however, glycans are synthesized in highly complex, stochastic reaction networks, which complicates biopharmaceutical manufacturing of these drugs since (i) synthesis does not follow a direct template (as protein synthesis does), and (ii) the genetic factors controlling activity of glycan-synthesizing enzymes remain largely unknown.

During my time as a postdoc I have explored novel approaches to tackle both of these confounding factors of glycan biomanufacturing. To increase the predictability of the glycan reaction network, I developed a computational model that describes the network as a Markov chain, a frequently used class of stochastic processes in engineering applications. This approach enables computational simulation of glycan biosynthesis while requiring few parameters defined by the user. Thus, this facilitates the development of cell engineering strategies to obtain a desired glycan profile during manufacturing. In addition, I utilized screening techniques, such as computational binding site predictions as well as genome-wide CRISPR/Cas9 knock-out libraries, and identified previously unrecognized regulator genes of glycan biosynthesis. In particular, we found that of one these genes (a zinc finger repressor-protein) controls biosynthesis of heparin, a blood anticoagulant of significant pharmaceutical relevance. This finding may pave the way for the engineering of cell lines capable to produce this valuable drug which can currently only be extracted from porcine intestinal mucosa. Thus, clean cell-culture based production will greatly enhance cost-efficiency and biosafety.

Cell line stabilization for biopharmaceutical manufacturing

In the biopharmaceutical industry, cell lines, such as Chinese Hamster Ovary (CHO) cells, are grown at large scales to produce various protein drugs, e.g. therapeutic antibodies, enzymes, and hormones which represent powerful new medicines for metabolic disorders, congenital ailments and high-incidence diseases like cancer or auto-immune disorders. The high prices of these drugs, however, pose a significant burden on patients and health care providers, and hamper the availability of these treatments in developing countries. A key contributing factor to high prices of these drugs is the high production cost, caused by instability of the cell lines producing them, wherein cells fail to maintain an economically viable protein titer over extended periods of time. One major underlying cause is the inherent instability of the CHO genome, such as the propensity of chromosomes to suffer frequent double-strand breaks. This can lead to the loss of genomic material, particularly to the loss of transgene copies which, consequently, results in dramatic loss of expression of the desired protein. So far, effective strategies to mitigate genomic instability in CHO cells have had only very limited success. Thus, genomic instability remains a major drain on resources and time in cell line development and production.

I have deployed a novel approach to restore the cells' ability to repair naturally occurring DNA damage and thus prevent the rapid occurrence of chromosomal instability. We analyzed whole-genome sequencing data of a multitude of commonly used CHO cell lines and identified several potentially deleterious mutations in key DNA repair genes. In particular, we found genes related to double-strand-break repair pathways to be severely affected which is consistent with the observed chromosomal instability. Using genome editing technology, I was able to revert several mutations in these genes, and the resulting cell lines gained improved double-strand repair capability and more stable product titers. We are currently conducting screens using a GFP-reporter system that allows us to more rapidly identify all DNA repair genes that, if restored, will improve DNA repair capability. This will enable us to engineer DNA-repair optimized CHO cell lines with a more stable genome and thus higher and more cost-efficient production capacities. My work on this project forms the backbone of a proposal currently under review in the Cellular and Biochemical Engineering program at NSF.

Future directions

In the future I would like to continue my work on CHO cell line stabilization but also expand the scope of this project into other areas. Genomic instability is also a growing concern in cell therapies, including induced pluripotent stem cells and CAR-T cells, potentially jeopardizing their clinical applicability. Also, genomic instability is a major driver of cancer and cell senescence, the latter being the root cause of ageing. My long-term goal as a future faculty member is to develop approaches to diagnose, mitigate, and prevent genomic instability in these contexts and realize applications in industry and medicine. I strive to maintain a dual methodology, combining both computational and experimental approaches, and would like to continue exploring biotechnological applications as an inspiration and a driver in the search for basic biological discoveries. Such aims will be highly fundable at several NIH institutions (including NCI, NIGMS, and NIAID), NSF, and many other foundations.

Teaching Interests:

As a scientist, learning is my passion. And as one of my high school teachers liked to say, the best way to learn is to teach to others. During my time as a PhD student, participation in teaching was part of my job responsibilities, and one that I greatly enjoyed. I have held classes in front of large student audiences, in both undergraduate and graduate programs. I developed new laboratory classes from scratch, including lectures, class material and exams, covering cell & developmental biology, as well as scientific programming. As a postdoc I helped design and teach at workshops on CHO cell manufacturing and CRISPR/Cas9 screens, aimed at academic and industrial audiences. Both as a PhD student and as a postdoc, I was involved in hiring and mentoring students and volunteers in the laboratory.

I can cover areas like cell & molecular biology, developmental biology, genetic engineering, cell culture engineering, and statistics.