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Topic

Advancing T cell-based immunotherapies through targeted engineering with CRISPR-Cas9

Department of Biochemistry and Microbiology

Date & location

• Wednesday, November 13, 2024
• 10:00 A.M.
• Engineering & Computer Science Building, Room 130

Examining Committee

Supervisory Committee

• Dr. Julian Lum, Department of Biochemistry and Microbiology, University of Victoria (Supervisor)
• Dr. Perry Howard, Department of Biochemistry and Microbiology, UVic (Member)
• Dr. Francis Nano, Department of Biochemistry and Microbiology, UVic (Member)
• Dr. Stephanie Willerth, Department of Mechanical Engineering, UVic (Outside Member)

External Examiner

• Dr. Jonathan Bramson, Department of Pathology and Molecular Medicine, ºìÐÓÊÓƵ

Chair of Oral Examination

• Dr. Kathy Gaul, School of Exercise Science, Physical and Health Education, UVic

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.