Aldahshan, Hala; (2024) Chimeric antigen receptors cells targeting T cell malignancies. Doctoral thesis (Ph.D), UCL (University College London).

Text Hala Aldahshan thesis 1May2024.pdf - Other Access restricted to UCL open access staff until 1 June 2025. Download (7MB)

Abstract

T cell leukaemias are characterised by the abnormal growth and dysfunction of T cells at various stages of development. Novel treatments for T cell malignancies, including adoptive cell transfer and chimeric antigen receptor (CAR) therapies, are being investigated. CAR-T cell therapy introduces a CAR transgene into T cells using viral or non-viral methods and redirects them against malignant cells. CAR-T cells have shown promise against B cell malignancies but face challenges in treating T cell malignancies due to fratricide during production and the risk of T cell aplasia post-administration. Genome editing allows for mitigating these challenges. Trials are underway testing base edited CAR7-T cells, targeting T cell receptor beta (β) chain (TRBC), CD7 and CD52, for elimination of graft-versus-host disease (GVHD), fratricide resistance and protection from serotherapy. This project investigated alternative base editor (BE) platforms incorporating rat or human cytidine deaminases that allow targeted C•G>T•A conversions in the genome independent of DNA breaks. These were tested for the multiplexed disruption of the TCRαβ and CD7 loci in combination with lentiviral transduction strategies for expression of CAR7. All editors achieved on-target conversion with undetectable chromosomal translocations and no ectopic RNA aberrations. Optimal delivery timing for each BE mRNA was next investigated with respect to lentiviral CAR7 transduction to ensure orderly removal of shared antigens. Finally, site-specific ‘knock-in’ of a CAR transgene using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was investigated as it may favour more physiological levels of CAR expression and disruption of endogenous TCRαβ. A virus-free manufacturing timeline for anti-CD7 CAR-T cells was established using Streptococcus pyogenes (Sp) Cas9 ribonucleoproteins (RNPs) with single guide RNA (sgRNA) guides in combination with double-stranded (ds) DNA CAR templates flanked by regions of homology to T cell receptor alpha (α) chain (TRAC), CD3ζ or CD7. Phenotypic profiling revealed CAR7+ expression and molecular analysis confirmed site-specific integration of CAR7 into CD3ζ, CD7, but not TRAC. CD3ζ-CAR7 effector cells exhibited potent cytotoxicity against CD7+ targets, comparable to lentivector expressed CAR7, warranting further investigation.

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