Privolizzi, Riccardo; (2022) In vivo evaluation of novel synthetic promoters for CNS gene therapy. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Dopamine transporter deficiency syndrome (DTDS) is a pharmacoresistant, life-limiting childhood neurotransmitter disease caused by loss-of-function mutations in the solute carrier family 6 member 3 (SLC6A3) gene encoding the dopamine transporter (DAT). There is an unmet clinical need for paediatric patients presenting parkinsonism to adults with early-onset Parkinson’s disease. Seminal works using transgenic mice overexpressing Slc6a3 in non-dopaminergic neurons, and mice overexpressing Slc6a3 in midbrain dopaminergic (mDA) neurons resulted in mDA neuronal toxicity and lethality. Previous research in our laboratory using the DAT knockout (DAT-KO) mouse model of DTDS receiving neonatal intracerebroventricular injections with rAAV9 vectors driving SLC6A3 under the transcriptional control of the pan-neuronal human synapsin 1 (hSyn) promoter resulted in phenotype correction, but ectopic DAT expression caused neurotoxicity, motor dysfunction, weight loss, requiring euthanisation. Non-specific transgene expression represents a major safety risk as most gene therapy clinical trials use ubiquitous promoters. We therefore proposed that development of highly active and cell type-selective promoters will improve efficacy of gene therapies. I partnered with AskBio Europe to assess novel synthetic CNS promoters generated using their bioinformatics-based platform PromPT®. My hypothesis is that gene expression from a rAAV9-based gene therapy vector can be restricted to target the CNS and CNS cell types under the transcriptional control of novel promoter candidates designed by PromPT®. This thesis presents the detailed characterisation of 10 novel CNS candidate promoters and the therapeutic validation of one candidate with superior mDA activity than hSyn in DAT-KO mice. Gene therapy showed significant differences in behavioural anxiety and motor coordination tests, but not in locomotor activity or survival. Immunofluorescence showed low expression of human DAT in mDA neurons as well as non-dopaminergic expression, that may account for incomplete phenotype rescue. This work demonstrates PromPT®’s ability to generate functional synthetic CNS promoters and encourages its further development to achieve refined specificity and selectivity for future gene therapies.

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