Gene Transfer and NMR Studies in Alpha-Mannosidosis Brain. We will investigate the ability of systemic delivery of a novel adeno-associated virus (AAV) gene transfer vector to treat the central nervous system (CNS) disease in a cat model of the human lysosomal storage disease (LSD) alpha-mannosidosis (AMD). In most LSDs genetic correction of a relatively small number of mutant cells results in secretion of the normal enzyme and receptor-mediated uptake by surrounding cells, resulting in metabolic correction of the non- transduced cells. The major problem for treating the brain is that pathology is present throughout the CNS because the metabolic defect is present in all cells. Thus, treating the whole brain requires global distribution of the therapeutic normal alpha-mannosidase (MANB) enzyme. This problem is exacerbated by the enormous size difference between mouse and human brains (~3,000 fold). The domestic cat brain is an excellent intermediate in size as it is ~100 times larger than a mouse brain, while the human brain between birth and 1 year (when treatment is expected to be the most effective) is only 10-30 times larger than the cat brain. The cat brain also has a gyrencephalic cerebral cortex that is structurally much more similar to the human brain than the rodent's. Thus, the underlying premise of this translational project is that strategies developed to globally correct the AMD cat brain are more likely to translate effectively into clinical trials. We have shown in this project that AAV gene transfer into the AMD cat brain either by multiple intraparenchymal injections or by infusion into the CSF can improve clinical and histological parameters, but the correction is incomplete. Certain AAVs can enter the CNS after systemic intravascular delivery in mice and mediate widespread transduction, but in large animal brains vector distribution is much more limited. We have developed a novel AAV vector for systemic delivery that transduces neurons throughout the cerebral cortex and other regions of both cat and monkey brains. The new vector also has the novel property of efficiently delivering the gene into large brains with a single-stranded AAV vector genome, whereas previous large animal intravascular AAV delivery experiments have used self-complementary (sc)-AAVs, which are too small to accommodate the MANB cDNA (~3kb). The novel vector will be compared to other AAVs to determine if complimentary patterns of transduction occur in the CNS and other organs to improve the therapeutic effect. We also have developed MR-based imaging assays to non-invasively measure brain pathology and preliminary experiments show they can quantitatively monitor the response to AAV treatment.
The specific aims are directed towards optimizing delivery, minimizing dose, evaluating clinical and lifespan improvements, and assessing the accuracy of non- invasive imaging parameters by correlation with histopathology. Although significant progress has been made on this project to date, the treatment is still incomplete and further improvements are needed to ensure the most effective translation into a clinical protocol for human patients.
The lysosomal storage diseases are a large group of inherited disorders that have severe disease in the brain. We are studying gene therapy methods to correct the brain disease. The proposed studies are directed towards improving gene and protein delivery sufficiently to make them feasible for human clinical trials.
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