Woodruff, Rosie; (2023) Non-viral delivery methods for the manufacture of reprogrammed chimeric antigen receptor (CAR) T-cells. Doctoral thesis (Ph.D), UCL (University College London).

Preview

Text 20230411_R_Woodruff_Thesis_Final.pdf - Accepted Version Download (28MB) | Preview

Abstract

CAR T-cell therapy involves the genetic manipulation of T-cells, to redirect specificity to tumour-associated antigens, and subsequent expansion in an ex vivo process. Most CAR T-cell therapies rely on the use of viral transduction or transposition to insert heterologous DNA sequences into the genome; however, there are inherent risks with these approaches, including insertional mutagenesis and lack of copy number control. This thesis explores the use of non-viral delivery methods to manufacture CAR T-cells and approaches to prevent the premature differentiation of T-cells to terminal effector cells, which is known to reduce the efficacy of CAR T-cell therapy. Proprietary transfection technologies, including nucleofection and soluporation, were evaluated for gene delivery to T-cells. Nucleofection (electroporation) is a highly efficient process to facilitate the introduction of DNA (~50%), RNA (~50%) or ribonucleic protein complexes (RNPs) (~90%). The transfection of DNA significantly impacted cell viability (~75%) which was vastly improved by the transfection of RNA (~90%). Soluporation was investigated as a method to improve cell viability, however, the viability was comparable to electroporation (~90%), transfection efficiencies of RNA/RNPs were typically lower (~25% and ~50%, respectively), and the introduction of dsDNA was extremely inefficient (<10%). Novel viral approaches to deliver the CAR or accessory genes were investigated as a comparison to the explored non-viral systems. The soluporation of lentivirus did not enhance the efficiency of integration (~30%), and transduction by non-integrating vectors was inefficient (~20%) and resulted in low transgene expression in primary T-cells. The nucleofection of RNA was found to be well tolerated by T-cells and can be exploited for their reprogramming. We explored the use of circular RNA, which has greater stability than linear mRNA, in two different applications. The first involved the reprogramming of T-cells to a less differentiated state by the expression of transcription factors which were known to be involved in controlling lineage commitment. We demonstrate a maintenance of the naïve and stem cell-like subsets, thereby slowing T-cell commitment to the effector cell lineage during ex vivo modification and expansion. The second approach delivered the cytosine base editor, BE4max, to disrupt splicing sites or to introduce premature stop codons into the genes of inhibitory receptors, including PD-1 and TIM3, to disrupt expression and prevent T-cell exhaustion. Building on the established RNA delivery protocols, we combined base editing with the introduction of an anti-CD30 CAR, for the treatment of Hodgkin lymphoma. Our optimised protocol aims to address challenges associated with the limited efficacy of CAR T-cells, including T-cell inhibition and exhaustion, to improve product persistence and therapeutic outcomes.

Downloads since deposit

Loading...

45Downloads

Download activity - last month

Export as XMLJSONCSV

Loading...

Download activity - last 12 months

Export as JSONCSVXML

Loading...

Downloads by country - last 12 months

Export as XMLCSVJSON

Loading...

Archive Staff Only

View Item