Preece, Roland; (2020) Enhancing Strategies for CRISPR/Cas9 Based T cell Engineering. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Genome editing tools are being rapidly developed, accelerating many areas of cell and gene therapy research, and are now entering clinical phase testing. Each successive genome editing technology promises increased efficacy, improved specificity, reduced manufacturing cost and design complexity; all of which are epitomised by the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9) platform. Implementation of CRISPR/Cas9 in existing methodologies has been instrumental to recent progress in the treatment of cancer, primary immunodeficiency, and infectious diseases. To this end, T cell therapies attempting to redirect antigen recognition have been enhanced through CRISPR/Cas9 genome editing, endowing them with increased potency and persistence, as well as allowing the generation of allogeneic products capable of overcoming barriers to transplant. This project aims to improve CRISPR/Cas9 strategies for T cell engineering through the development of a novel self-inactivating lentiviral vector platform (terminal-CRISPR) that couples expression of CRISPR single guide RNA (sgRNA) and transgene expression, through incorporation of a sgRNA expression cassette within the ΔU3 region of the 3’ long terminal repeat sequence. Initial investigation coupled expression of a chimeric antigen receptor and a T cell receptor alpha constant specific sgRNA, resulting in CAR+ cells devoid of alloreactive T cell receptor (TCR)αβ complexes. Moreover edited TCRαβ-/CAR+ effectors demonstrated enhanced anti-leukemic outcomes in an in vivo tumour model. The terminal-CRISPR configuration further provided a platform for exploring the architecture of the U6 promoter, enabling the discovery of a minimal U6 promoter, which has been applied to the optimisation of a multiplex terminal-CRISPR vector. Moreover, concerns surrounding the generation of double strand DNA breaks prompted the application of emerging cytidine deaminase base editing technologies, showing high levels of targeted base conversions at multiple genomic loci, while also reducing large chromosomal translocation events. The terminal-CRISPR platform was further proved effective at linking delivery of a Hepatitis B virus specific recombinant TCR and sgRNA for disruption of the endogenous TCR. Discriminatory enrichment of antigen specific T cells with removal of the endogenous TCR offers an enhanced, highly targeted T cell therapy. This strategy is currently under evaluation for application in phase 1 clinical testing targeting B cell malignancies.

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