Date of Award:

12-2022

Document Type:

Thesis

Degree Name:

Master of Science (MS)

Department:

Biological Engineering

Committee Chair(s)

Charles D. Miller

Committee

Charles D. Miller

Committee

Elizabeth Vargis

Committee

Anhong Zhou

Abstract

Between 2014 and 2018, the global market for therapeutic monoclonal antibodies (mAbs) rose from $60 billion to $115.2 billion with a projected value of $300 billion by 2025. These molecules are used to effectively treat some of the most challenging illnesses from auto-immune diseases to cancer. While mAbs are highly valuable with potent applications, their production at scale remains an outstanding challenge. These molecules are largely produced in Chinese Hamster Ovary (CHO) cells that require highly specific conditions to produce a useful product.

Genetic engineering presents one solution to overcome productivity limits. With the advent of CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated proteins) systems, engineering the CHO genome has never been easier. CRISPR/Cas9 allows for site-specific editing and gene integration. Within the CHO genome, a variety of sites have been identified that warrant further investigation for editing. Among these sites is the gene C12orf35. The deletion of C12orf35 has been shown to lead to increased productivity in CHO cells. Additionally, C12orf35 has been identified as a site with a high transcription rate, implying that genes at this site are likely to be expressed more frequently.

The gene coding for mammalian target of rapamycin (mTOR) has been demonstrated to alter CHO cell phenotype characteristics such as cell size, viable cell density, and antibody productivity when expressed transiently. This study aims to evaluate the potential synergism of deleting the gene C12orf35 by editing the gene coding for mTOR between a cut site made in C12orf35. Splicing the gene coding for mTOR at this site has the potential combined benefit of disrupting C12orf35 while simultaneously stably expressing the mTOR gene at a highly transcribed site in the CHO genome.

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