Preclinical evaluation of AAV-mediated DARS1 gene supplementation in novel models of the leukodystrophy HBSL

Leukodystrophies are a large group of genetic disorders that impact the white matter (myelinated nerve tracts) of the central nervous system (CNS), often resulting in severe neurological dysfunction, physical disability, and premature mortality. The leukodystrophy Hypomyelination with Brainstem and Spinal cord involvement and Leg spasticity (HBSL) is caused by biallelic loss-of-function mutations in the cytosolic aspartyl-tRNA synthetase gene DARS1. The underlying disease mechanism is poorly understood, and no curative treatments are available. Mammalian models of HBSL have been challenging to establish and are essential for in vivo testing of candidate therapies. Chapters 2 and 3 of this thesis comprehensively characterise two novel HBSL mouse models, generated through Cre-loxP-mediated Dars1 knockout in neurons (Dars1NeuroKO) and oligodendrocytes (Dars1OligoKO). Both models exhibited acute pathological phenotypes including severe motor dysfunction and reduced lifespan. Histological analysis of CNS tissue revealed that cell loss, white matter abnormalities and neuroinflammation were key features in both models. These results indicate the necessity of DARS1 for neuronal and oligodendroglial survival and function, suggesting that both cell populations must be targeted by an effective therapy. As a monogenic disorder, HBSL is an ideal candidate for gene therapy. Our group has developed a set of PHP.eB serotype adeno-associated viral vectors (AAVs) for CNS-directed delivery of an optimised human DARS1 coding sequence. In Chapters 4-6, the efficacy of AAV-mediated DARS1 gene supplementation was tested using the Dars1NeuroKO and Dars1OligoKO models. Systemic administration of AAV.DARS1 equipped with the constitutive CAG promoter achieved long-term phenotypic rescue in Dars1NeuroKO mice, providing proof-of-concept for this treatment strategy. Treatment of Dars1OligoKO mice with DARS1 driven by the ubiquitous CAG or oligodendrocyte-specific MBP and MAG promoters did not achieve significant therapeutic benefits, likely due to insufficient oligodendrocyte transduction. Collectively, our findings support the potential of AAV-mediated DARS1 gene augmentation as a therapy for HBSL, while highlighting the critical need to enhance oligodendroglial targeting for effective treatment of this cell population. Furthermore, the targeted gene delivery and disease modelling platforms established here will aid development of AAV-based gene replacement therapies for other leukodystrophies and similar monogenic CNS diseases.