Yoon, Jongwon; (2025) Immunocompetent in vivo CRISPR screening to identify key transcription factors which enhance persistence and efficacy of CAR-T cells in cancer. Doctoral thesis (Ph.D), UCL (University College London.

Text Thesis_sig_removed.pdf - Accepted Version Access restricted to UCL open access staff until 1 December 2027. Download (4MB)

Abstract

Chimeric antigen receptor (CAR) T cell therapy has emerged as a revolutionizing advancement in immunotherapy over the past decade, particularly for the treatment of haematological malignancies such as B-cell acute lymphoblastic leukaemia (B-ALL). Nevertheless, the limited persistence of CAR T cells in patients remains a significant obstacle to achieving durable remission. While recent genome-wide screens have identified several transcription factors potentially crucial for enhancing CAR T cell persistence, these findings are predominantly derived from in vitro screens or in vivo studies using immunodeficient animal models. Moreover, research has largely focused on identifying negative regulators through knockout approaches, while positive regulatory factors remain comparatively underexplored. To address these limitations, I hypothesized that novel targets could be obtained through CRISPR-based gain-of-function (GOF) approaches. Therefore, I performed in vivo CRISPR activation screens in an immunocompetent animal model of B-ALL using CD19-directed CAR T cells, aiming to identify putative transcription factors influencing CAR T cell persistence and therapeutic durability. Specifically, I designed and generated a gRNA library targeting 1610 mouse transcription factors with co-expression of anti-CD19 CAR. The library was introduced into primary T cells derived from a dCas9-SAM transgenic animal strain, and the engineered cells were transferred into tumour-bearing mice. By tracking the relative enrichment of gRNAs in persisting CAR T cells compared with the baseline population, critical transcription factors influencing persistence could be identified. The screen revealed several promising candidates, including novel regulators such as ONECUT2, HNF1A, BATF2, and MYRF, alongside established factors such as BATF, TCF7, and T bet, which validates the approach. In conclusion, this study provides a comprehensive in vivo screen of transcriptional regulators that regulate CAR T cell persistence in an immunocompetent setting and offer valuable insight into potential targets for enhancing the persistence and efficacy of CAR T cell therapy.

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