429. AAV5-Mediated Delivery of Human Aryl Sulfatase A (hARSA) Prevents Sufatide Storage and Neuropathological Phenotype in Metachromatic Leukodystrophy (MLD) Mice

Abstract

Metachromatic leukodystrophy (MLD) is an inborn lysosomal storage disease caused by arylsulfatase A (ARSA) enzyme deficiency. Lack of ARSA results in the storage of galactosyl-3-sulfate ceramide (sulfatide) in neurons and glial cells that leads to progressive demyelination and neuronal damage in the CNS. The most severe form of MLD is the late infantile form that results in rapid loss of motor and cognitive functions and death within 2-5 years. No efficient therapy is available in infantile form of MLD and allogeneic hematopoietic cell transplantation has limited effects in the juvenile forms of MLD. MLD mice exhibit phenotypic abnormalities reminiscent of the human disease that include sulfatide storage in neurons and glial cells and neuromotor impairment. AAV5 vector encoding human ARSA was injected in the internal capsules and cerebellum of 3 month-old MLD mice (3 x109 vp /injection; n= 14) at the time they are beginning to develop CNS disease. Treated mice and mock-treated mice (AAV5-LacZ) were sacrificed at 6 and 18 months of age. Functional recombinant hARSA was detected in almost all areas of the forebrain, cerebellum and brainstem of AAV5-hARSA treated mice, using an ELISA assay that recognizes specifically the hARSA, immunohistochemistry and measurement of hARSA activity with the artificial substrate p-nitrocatechol sulfate. As expected, hARSA was mostly expressed in neurons, but also in a significant percentage of astrocytes (up to 14% arround the injection site) and in microglial cells. The widespread diffusion of hARSA resulted from both vector and enzyme diffusion within the CNS. Quantitative PCR in AAV5-ARSA vector showed that vector genomes were concentrated at the injection sites but spreads over a distance from injection sites. ARSA expression using ELISA test was proportional to the number of vector genomes but extended far beyond PCR-positive brain regions. The increase of ARSA positive neurons and astrocytes from 6 to 18 months of age confirmed that a significant amount of AAV5-encoded ARSA was secreted by transduced cells and captured by neurons and astrocytes. ARSA delivery prevented sulfatide deposits and reversed the galactosyl ceramide/sulfatide abnormal ratio that reflects sulfatide storage and loss of oligodendrocytes. Eighteen-month old treated mice showed a nearly absence of neuropathological abnormalities (Purkinje cell loss, astrogliosis, microglial activation) and normal motor behavior (at the rotarod test). These results indicate that ARSA delivery from intracerebral sources has the capacity to alleviate most disease manifestation in the MLD model.