Advancing T cell-based immunotherapies through targeted engineering with CRISPR-Cas9
| dc.contributor.author | Carleton, Gillian | |
| dc.contributor.supervisor | Lum, Julian | |
| dc.date.accessioned | 2024-11-29T23:41:43Z | |
| dc.date.available | 2024-11-29T23:41:43Z | |
| dc.date.issued | 2024 | |
| dc.degree.department | Department of Biochemistry and Microbiology | |
| dc.degree.level | Doctor of Philosophy PhD | |
| dc.description.abstract | T cell-based immunotherapies such as chimeric antigen receptor T (CAR-T) cell therapy have undoubtably revolutionized the treatment of cancer. However, the broad effectiveness of CAR-T cell therapy is hindered by several unresolved problems, most notably a lack of therapeutic efficacy in treating solid tumor cancers. A second challenge stems from the widespread use of viral vectors in CAR-T manufacturing, which poses safety risks to patients receiving treatment. Here, we showed that genome editing with CRISPR-Cas9 can be used to overcome both of these issues. As the solid tumor microenvironment (TME) is known to be metabolically suppressive, we devised a single-step editing method to enhance the metabolism and effector function of CAR-T cells. This approach combined CRISPR-mediated homology-directed repair with a gene-trap approach to link CAR integration with simultaneous deletion of a metabolic gene of interest. For proof-of-concept, we targeted the folate receptor alpha (aFR) CAR to the locus of the essential autophagy gene ATG5, and showed that editing at ATG5 could be achieved with a high level of on-target specificity. Functionally, deletion of ATG5 led to alterations in glucose and glutamine metabolism and enhanced CAR-T cell efficacy under nutrient-restricted conditions in vitro and in vivo. To address the safety concerns associated with viral transduction, we developed a process for nonviral manufacturing of clinical-grade CAR-T cells for B-cell malignancies. This approach used electroporation of a Cas9 ribonucleoprotein complexed with a linear double-stranded DNA template to facilitate site-specific insertion of a CD22 CAR at the T cell receptor alpha chain (TRAC) locus. In vitro, nonviral CD22 CAR-T cells exhibited comparable antitumor activity to lentiviral CD22 CAR-T cells. thereby establishing feasibility of our nonviral manufacturing process. Taken together, the results of these studies highlight the broad applicability of CRISPR-Cas9 as a tool for engineering safer, more effective T cell-based immunotherapies for patients with cancer. | |
| dc.description.embargo | 2025-11-13 | |
| dc.description.scholarlevel | Graduate | |
| dc.identifier.bibliographicCitation | McPhedran SJ, Carleton GA, Lum JJ. Metabolic engineering for optimized CAR-T cell therapy. Nature Metabolism. 2024;6(3):396-408. | |
| dc.identifier.uri | https://hdl.handle.net/1828/20812 | |
| dc.language | English | eng |
| dc.language.iso | en | |
| dc.rights | Available to the World Wide Web | |
| dc.subject | Cancer immunotherapy | |
| dc.subject | Genome editing | |
| dc.subject | CRISPR | |
| dc.title | Advancing T cell-based immunotherapies through targeted engineering with CRISPR-Cas9 | |
| dc.type | Thesis |