Keegan, Anna; (2025) Gene Therapy for Pyruvate Dehydrogenase Complex Deficiency. Doctoral thesis (Ph.D), UCL (University College London).

Text Anna Keegan Gene therapy for Pyruvate Dehydrogenase Complex Deficiency.pdf - Accepted Version Access restricted to UCL open access staff until 1 August 2026. Download (44MB)

Abstract

Pyruvate dehydrogenase complex deficiency (PDHD) is a rare mitochondrial disorder of carbohydrate metabolism leading to a decrease in adenosine triphosphate (ATP) production. The pyruvate dehydrogenase (PDH) complex is responsible for converting pyruvate into acetyl-CoA, which enters the tricarboxylic acid (TCA) cycle to ultimately lead to ATP production via the electron transport chain and oxidative phosphorylation system. Most patients harbour a mutation in the X-linked gene encoding the PDH complex E1α-subunit (PDHA1). Patients experience developmental delay, neurological abnormalities, lactic acidosis, and early childhood death. Treatments are limited. A brain-specific hemizygous knockout (KO) mouse model of PDHD closely recapitulates the human disease in males. In this study, I aimed develop an adeno-associated viral vector (AAV) gene therapy for PDH and conduct pre-clinical studies to deliver the AAV gene therapy to the brain of the newborn PDHD mice. I then developed a novel gene therapy, in which I have used AAV9 and AAV-F to deliver a brain-specific transcript of the human PDHA1 gene driven by a cytomegalovirus enhancer fused to the chicken beta-actin promoter (CAG) promoter, to restore the PDH complex enzyme activity to prevent onset of the disease symptoms. Firstly, safety and biodistribution of the two novel AAV vectors were investigated by ICV biodistribution studies to neonatal wild-type mice at a range of doses. The mice were monitored for 35 days while undergoing open field testing at P18, and P35. Transgenic PDHA1 gene expression in the brain and peripheral organs as well as vector causing neuropathology. The AAV doses which demonstrated an appropriate safety profile were used to treat the KO Pdha1 mice via neonatal ICV. On post-natal day 100 these animals were sacrificed with brain and visceral organs were collected for gene expression analysis, gas chromatography-mass spectrometry, PDH enzyme activity assay and immunohistochemistry. Biodistribution studies showed that the neonatal ICV gene transfer of AAV9 and AAV-F encoding hPDHA1 driven by the CAG promoter effectively targets the brain. I also showed that AAV-F had a greater hPDHA1 expression profile in the discrete brain regions compared to titre-matched AAV9. The gene therapy study showed that administration of a high dose of 5x1010vg of AAV9 and a medium dose of 5x109 vg AAV-F (log lower than high dose AAV9) to newborn Pdha1 KO mice resulted in the most significant increase in survival, improvements in weights and behavioural assessment. Furthermore, AAV9 and AAV-F restored PDHA1 gene expression, PDH enzyme activity, improved metabolite concentrations and neuropathology in the brain. Although there were improvements in phenotype there was a lack of cerebellum targeting which resulted in pathology. This proof-of-concept study has demonstrated promising data for the use of AAV9 and AAVF vectors as gene therapy for PDHD. I have shown that AAV9 high dose and AAV-F medium dose can prevent further onset of disease in the PDHD mouse model.

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