Lysosomal storage diseases (LSD) result from deficiencies of enzymes involved in the degradation of substances within lysosomes. Many of these disorders have central nervous system (CNS) and peripheral manifestations. Recombinant adeno-associated virus (rAAV) vectors have been shown to be capable of efficiently transferring genes to the CNS and skeletal muscle. To investigate the potential of using these vectors in the treatment of LSD, we are studying β-glucuronidase deficiency (mucopolysaccharidosis type VII; MPS VII) because of the availability of a relevant animal model (MPS VII mice) to test gene transfer. Methods: A rAAV vector was engineered to contain an expression cassette consisting of the cytomegalovirus-immediate-early promoter and the murine β-glucuronidase gene. HeLa cells were transduced with this vector (rAAV-gus.1.1) and enzyme activity was measured in cellular extracts, culture media, and cells in situ. To test for secretion and uptake of the enzyme, humanβ-glucuronidase-deficient fibroblasts were co-cultured with transduced HeLa cells and enzymatic activity within the fibroblasts determined. Electron microscopy was performed to evaluate whether this vector was capable of reversing the storage abnormality within the β-glucuronidase-deficient fibroblasts. To test the vector for in vivo activity, stereotaxic injections of the rAAV-gus.1.1 vector into the cortex and caudate/putamen region of the brain of MPS VII mice were performed. Control animals received a similar rAAV vector expressing β-galactosidase. Results: Following transduction with rAAV-gus.1.1, active β-glucuronidase was expressed in cultured cells and secreted into the culture medium. The secreted enzyme was appropriately taken up by β-glucuronidase-deficient fibroblasts. rAAV-mediated expression of β-glucuronidase within the deficient fibroblasts resulted in a decrease in the size of the abnormal lysosomes as compared to non-infected control cells. MPS VII mice were sacrificed one month following vector administration. β-glucuronidase activity was detected within the region of injection in animals receiving the rAAV-gus.1.1 vector and was absent in the contralateral hemisphere and control animals. Conclusions: These data demonstrate that a rAAV vector carrying the β-glucuronidase gene produces a functional protein that is appropriately processed and capable of correcting the lysosomal defect present in deficient cells. In addition, the in vivo administration of this vector can complement the enzymatic defect present in an MPS VII mouse disease model.