Discovery of Novel Cancer Immunotherapy Targets with Next Generation Genome Engineering Methods
Date of Award
Doctor of Philosophy (PhD)
Recent advances in immuno-oncology have led to remarkable clinical benefits in a variety of different cancers; however, a large fraction of patients encounter resistance. Identifying novel targets that can improve T cell functionality can therefore broaden the potential of cancer immunotherapy. We developed methods for discovering such targets using CRISPR gene editing technology. First, we created a hybrid genetic screening system where Sleeping Beauty (SB) transposons and single guide RNA cassettes are nested in an adeno-associated virus (AAV) to enable in vivo AAV-SB-CRISPR screens for membrane protein targets in CD8+ T cells in mouse models of glioblastoma. Second, we developed a CRISPR-based dual perturbation screening platform in antigen-specific co-culture models to identify gene pairs that mediate melanoma cancer cell resistance to T cell killing. Third, we developed a system for efficient generation of modularly engineered chimeric antigen receptor T cells (CAR-T cells) by leveraging tracrRNA-independent Cas12a/Cpf1 and AAVs. Finally, we created a platform for rapid generation of customized scales of CAR-T variants by Cpf1 mRNA electroporation with pooled AAV transduction for selection of variants with favorable phenotypes such as prolonged persistence and enhanced in vivo therapeutic efficacy. In these studies we demonstrate the potential of CRISPR-based screening platforms for target discovery in the context of immuno-oncology. By using state-of-the art genome engineering methods, we are able to gain new insights into anti-tumor immunity and generate innovative cellular immunotherapies.
Park, Jonathan Joon-Young, "Discovery of Novel Cancer Immunotherapy Targets with Next Generation Genome Engineering Methods" (2022). Yale Graduate School of Arts and Sciences Dissertations. 508.