Thenge, Prateek Pradeep2025-01-282025-01-282024-06https://hdl.handle.net/11299/269509University of Minnesota M.S. thesis. June 2024. Major: Pharmacology. Advisor: Beau Webber. 1 computer file (PDF); iii, 77 pages.Next-generation cancer therapies using genome-edited immune cells are a rapidly evolving yet promising avenue for personalized medicine. Human induced pluripotent stem cells (iPSCs) are a genetically tractable platform for scalable in vitro production of engineered immune cells; however, multiplex editing using Cas9 nucleases to enhance effector function and overcome the immunosuppressive tumor microenvironment (TME) poses significant risks for genotoxicity and chromosomal translocations. To mitigate risks associated with double-stranded breaks (DSBs) induced by CRISPR/Cas9 nucleases, we utilized base editing (BE) to install precise nucleotide edits without DSB induction, achieving highly efficient, single-step multiplexed genome editing in human iPSCs with minimal genotoxic side effects. Comparing Cas9 nuclease and adenosine base editor (ABE8e) delivered as synthetic mRNA, we simultaneously edited 9 genes previously shown to enhance function in patient-derived immune cells. Cas9 and ABE8e achieved >90% editing efficiencies in single plex at a single target (B2M). Conversely, in the multiplex setting, ABE8e achieved high editing rates (70% +/- 30%) compared to only 9% (+/- 8%) for Cas9. Cell toxicity was nearly undetectable with ABE8e but was notably higher with Cas9, as evidenced by lower cell recovery and a (5-35x fold) induction in levels of p21 transcription, a protein involved in DNA damage response and cell cycle arrest. This result confirmed substantial impacts on genomic stability, stress, and DNA damage with Cas9 that are absent with ABE8e. Furthermore, cytogenetic analysis of Cas9-edited populations revealed chromosomal abnormalities, inversions, and random loss events linked to targeted loci that were notably absent in cells edited with ABE8e. These findings confirm that base editing allows single-step, highly multiplexed editing of human iPSCs without the genotoxicity associated with nuclease-induced DSBs. Our platform supports the rapid production of highly multiplex-engineered iPSCs using ABE8e, which is ideal for the in vitro characterization of diverse engineered immune cells with enhanced function for cancer immunotherapy and beyond. After the differentiation of these cells, we observed better Haemopoietic (CD34+CD43+) and improved NK cell fate (CD56+CD7+CD3-), along with significantly higher cytotoxicity.enAdoptive cell transferBase editingGenome engineeringimmunotherapyiPS engineeringThesis or Dissertation