This grant application proposes to develop new approaches to somatic cell gene therapy for the central nervous system (CNS) pathology of lysosomal storage diseases. In these diseases, deficiencies in activities of specific lysosomal enzymes result in the accumulation of un-degraded substrates in lysosomes of many cell types, often including neurons. In general, treatments that have had beneficial effects on some organ systems, e.g. bone marrow transplantation to provide a source of the missing enzyme to patients, have not been effective for the CNS component of these diseases. Treatment of the CNS is important because its involvement in many lysosomal storage diseases results in mental retardation in human patients. As a model lysosomal storage disease for these studies, we will investigate beta-glucuronidase (beta-gus) deficiency, which results in mucopolysaccharidosis type VII (MPS VII, SLY disease) in mice, dogs, and humans. Two strategies for treating lysosomal storage in the CNS in vivo will be investigated: 1) Direct gene transfer to the CNS using a novel neurotropic viral vector system. The vector is based on Herpes simplex virus (HSV)-1 and uses the promoter form the HSV gene encoding the latency associated transcript (LAT). We have demonstrated that the LAT gene is expressed at high levels in latently infected neurons in vivo, but is not necessary for maintenance of latency. Therefore, the LAT promoter is an excellent candidate to drive transcription of foreign genes in the CNS in vivo. The HSV vector system will also be tested for its ability to repair the mutation of MPS VII cells by homologous recombination, which may occur between vector and genomic beta-gus sequences because the HSV genome exists as unintegrated episomal DNA in the host cell nucleus. 2) Indirect transfer of the gene product into the CNS by engraftment of the brain with genetically corrected cells. We have developed retroviral vectors that express beta-gus at normal levels in MPS VII cells and degrade the specific accumulated substrates. The vector-corrected cells can export beta-gus to mutant cells (cross-correction). The completely negative background of beta-gus activity in the MPS VII mice will enable us to determine the fate of the transplanted cells, and the uptake of beta-gus by host cells, as well as evaluate the expression of beta-gus from the HSV vectors. We will develop these gene therapy methods using the MPS VII mouse, then use effective vectors and transfer protocols to treat the MPS VII dog as a model for applying these methods to human patients. The availability of both an inbred mouse model and an outbred large animal model that are true homologues of a human genetic disease, plus the cloned gene and vectors, make this a powerful and unique model for gene therapy studies.
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