Improving AAV-based gene therapy for Krabbe Disease

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Abstract Krabbe’s Disease (KD) is a rare inherited leukodystrophy caused by mutations in the GALC gene encoding the lysosomal enzyme, galactosylceramidase (GALC). Lack of functional GALC leads to global demyelination and neurodegeneration. Untreated KD presents in infants as developmental delay and regression, failure to thrive, and ultimately death by 2-3 years of age. KD is treated with hematopoietic stem cell transplantation (HSCT) which slows the progression of disease and prolongs life expectancy into the teenage years, but it is not a cure. Adeno-associated viruses (AAV) have been utilized successfully as gene therapy treatment vectors in numerous pre-clinical and clinical trials. The initial success of AAV gene therapy in diseases primarily affecting neuronal cells led to investigations of their efficacy in monogenic diseases affecting multiple cells types such as KD. The Bongarzone lab previously developed a treatment protocol using AAV9- GALC to correct the deficiency in the Twitcher murine model of KD. This treatment completely prevented disease development for the first 6-8 months of life, such that AAV-treated mice were nearly indistinguishable from wild-type mice. Despite these promising results, our study also revealed a slow decline in treatment efficacy over time, including development of disease signs and focal demyelinating plaques in mice of advanced age; similar findings have been noted by other investigators as well. No alternative modifications to improve gene therapy have been proposed thus far. The overarching goal of this application is to establish whether gene therapy efficacy is declining due to exhaustion of AAV DNA in the adult brain and how to optimize gene therapy treatment efficacy and duration in Twitcher mice. As AAV-GALC DNA exists as a non-replicating extra-chromosomal episome after entering a cell, we hypothesize the decline in treatment efficacy is caused by loss of therapeutic vector in replicating cells, particularly oligodendrocyte precursors, in the Twitcher brain, which leads to a decrease in the average AAV- GALC episomes per cell over time. This hypothesis will be investigated by treating mice shortly after birth with AAV9-GALC at decreasing dosages and then observing how long the treatment is efficacious based on clinical score, survival, as well as biochemical and histological analyses. We will then examine if redosing utilizing oligodendrocyte targeted AAV (AAV-001) later in life, but prior to disease sign onset, delays or prevents development of disease signs. Finally, we will determine if utilizing AAV-001 singly will increase the efficacy and duration of treatment in comparison to AAV9. This study is important because we need to better understand what happens to episomal AAV DNA long term before attempting to use AAV based therapies to treat KD patients. Furthermore, our findings will have the intrinsic impact of better characterizing how episomal AAV DNA behaves in similar monogenic disorders.