Adeno-Associated Virus Gene Therapy For Maple Syrup Urine Disease

Abstract

Advancements in genetic therapies, including recombinant adeno-associated virus (AAV) technologies, have now reached a point where potential curative treatments are within reach for incurable genetic diseases such as Maple Syrup Urine Disease (MSUD). MSUD is caused by biallelic mutations in one of five autosomal genes required for the enzyme BCKDH. Current treatment options are non-curative. This thesis explored the potential of recombinant AAV technology as a novel therapy for MSUD using a DBT-deficient neonatal lethal murine model. This thesis first explored a liver directed approach with AAV8 pseudoserotyped vectors carrying a human DBT transgene under the transcriptional regulation of the APOe/hAAT enhancer/promoter. These experiments used conventional and hybrid AAV/piggyBac transposase vectors and found that both integrating and non-integrating AAV vectors significantly improved the physical and biochemical phenotype of DBT-deficient mice, though the rescue was only transient with non-integrating vectors. A muscle restricted vector was also developed and included the CK8e promoter/enhancer, a human DBT transgene and was packaged in the muscle trophic AAVMYO capsid. MicroRNA targeting site sequences were also included to de-target transgene expression in the liver and brain. Neonatal injection of the muscle optimised vector successfully rescued DBT-deficient mice to adulthood with biophysical parameters statistically equivalent to those observed in wildtype mice during periods of metabolic stability and transgene expression was highly muscle restricted. Whilst further, rigorous pre-clinical testing is required before an equivalent muscle optimised vector would be safe for human clinical application, this thesis has demonstrated the viability of AAV-mediated gene transfer to skeletal muscle, a post mitotic tissue, in a neonatal lethal MSUD murine model and is an encouraging step in the search for a cure for MSUD patients and their families