Giardina, Enzo Marco; (2025) Design of a retinoid-responsive, toxin gene therapy approach for neuroblastoma treatment. Doctoral thesis (Ph.D), UCL (University College London).

Text Enzo Giardina - PhD Thesis.pdf - Submitted Version Access restricted to UCL open access staff until 1 July 2026. Download (15MB)

Abstract

Retinoic acid (RA) has been shown to improve clinical outcomes for neuroblastoma patients by inducing differentiation in tumour cells. However, its use has been limited by toxicity and tumour-acquired resistance after prolonged treatment, following which patients relapse and this is often fatal. To increase RA’s efficacy, this project aimed to design a toxin gene therapeutic that is transcriptionally activated by RA, converting its activity from cytostatic to indirectly cytotoxic. Using recombinant DNA technology, truncated forms of the highly potent diphtheria toxin were linked to a RAresponsive promoter in plasmids and their cytotoxicity was tested at a range of RA concentrations in culture. MicroRNA-response-elements (MREs) were cloned into plasmids to minimise off-target expression in liver cells. Immunotoxin constructs, targeting the neuroblastoma-specific antigen GD2, were generated and tested on their GD2-selectivity and bystander activity in non-transfected tumour cells. Liposomes coated with neuroblastomatargeting peptides were assessed as delivery vectors to cells in culture and in xenograft mice. The RA-driven diphtheria toxin A plasmid, after transfection into neuroblastoma cells, was highly sensitive to RA, requiring minimal exogenous RA (<100 nM) for maximal activity. Compared to RA alone, it showed greater toxicity at all RA concentrations, shown by the complete loss of positively transfected neuroblastoma cells following RA treatment. Liver-specific MREs knocked down transcription by 98% in the hepatoma-derived Huh7 cell line but had minimal effect on cytotoxicity. Immunotoxin constructs were successfully generated and secreted by neuroblastoma cells, but toxicity was seen to act independently of cell GD2- status and a bystander effect was not demonstrated. Lipid nanoparticle delivery was not neuroblastoma-specific in culture, but repeated in vivo tests are needed to truly determine their potential. This project demonstrated the potential of a RA-responsive toxin gene system for neuroblastoma treatment. With further development, this system could decrease relapse frequencies and improve long-term survival of high-risk patients.

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