Developing an in Vivo Based CRISPR-Induced Genetic Screen to Identify Novel Regulators of T-Cell Dysfunction in the Context of Tumors

Abstract

T-cell dysfunction is a central characteristic of the immune system's response to cancer, and one of the primary mechanisms that allows tumors to escape the immune response and thrive. Within the last decade, there has been rapid progress in immunotherapy treatments that are capable of manipulating the immune system to target and attack cancer cells. Two immunotherapy treatments in particular, antibody checkpoint blockade and chimeric antigen receptor (CAR) modified T-cells, have been especially effective in preventing or reversing T-cell dysfunction in the context of tumors. However, these immunotherapies are not without their limitations, as only certain subsets of patients have robust responses to these treatments and many show no response at all. It is therefore imperative that we discover novel therapeutic targets of T-cell dysfunction in the context of tumors, and this can best be achieved through large-scale loss-of-function genetic screens. The efficiency and affordability of the recently developed CRISPR-Cas9 genomic DNA editing technology makes it an ideal method for performing large scale genetic screens. The primary goal of this thesis was to develop the tools necessary to create an in vivo based CRISPR-mediated system that could be used to generate a genetic screen to discover novel negative regulators of T-cell dysfunction in the tumor microenvironment. Targets discovered in such a screen will be useful to identify novel therapeutic targets for use in both checkpoint blockade and CAR T-cell therapies.