Assesment of Structure, Function, and Microevolutionary Dynamics of Extrachromosomal Circular DNA in Chinese Hamster Ovary Cells

Author

Dylan ChitwoodFollow

Date of Award

5-2023

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Bioengineering

Committee Chair/Advisor

Dr. Sarah Harcum

Committee Member

Dr. Christopher Saski

Committee Member

Dr. Angela Alexander-Bryant

Committee Member

Dr. Mark Blenner

Abstract

Chinese hamster ovary (CHO) cell lines are among the most popular expression hosts used in biopharmaceutical manufacturing due to relative ease of culture, capacity to perform human-like post-translational modifications, and non-susceptibility to viruses. However, the intrinsic plasticity of the CHO genome can lead to undesired genetic rearrangements, phenotypic shifts, reduced product quality, and early culture termination that prevents continuous biomanufacturing. A characteristic of plastic and unstable genomes that is poorly understood in CHO cells is extrachromosomal circular DNA (eccDNA). EccDNAs are focal amplifications of the genome that reside in the extranuclear space. These plasmid-like entities are structurally complex and are capable of contributing to a wide variety of biological functions including gene overexpression, regulation of nuclear-encoded genes, immunostimulation, and adaptive stress responses.

The objective of this work is to establish the foundational knowledge of eccDNA structure, function, and microevolutionary dynamics in CHO cells under various conditions. This work characterizes eccDNA content in CHO cells grown in bioreactors for two weeks under control and lactate-stressed conditions, two CHO K-1-derived cell lines of different ages, and CHO cells gradually adapted to high extracellular lactate levels. More than 2,000 genes were observed to be encoded on eccDNAs and summaries of gene function are presented using Gene Ontology and KEGG pathway analyses. RNA-seq data is utilized to identify potential changes in gene expression mediated by eccDNAs. Furthermore, the study presents a broad characterization of eccDNA sequence structures and biogenesis sites that may be used as targets in future work.

Recommended Citation

Chitwood, Dylan, "Assesment of Structure, Function, and Microevolutionary Dynamics of Extrachromosomal Circular DNA in Chinese Hamster Ovary Cells" (2023). All Dissertations. 3296.
https://open.clemson.edu/all_dissertations/3296