Gaucher Disease: In addition to the accumulation of glucocerebroside in the brain of patients with neuronopathic Gaucher disease, significantly increased quantities of glucosylsphingosine (GlcSph), the deacylated congener of glucocerebroside, are known to be present in the brain of these patients. In order to determine whether GlcSph contributes to the pathologenesis of this disorder, we examined the effect of GlcSph on cultured LA-N-2 cholinergic neuronal cells. We found that GlcSph is highly toxic to these cells in concentrations that are present in the brain of patients with both the acute and chronic neuronopathic forms of Gaucher disease. We deduce that GlcSph plays an important role in the neuronal dysfunction and destruction in these patients. We then carried out an investigation on the biosynthesis of GlcSph and found that there is an enzyme in the brain of neonatal animals that catalyzes the formation of this neurotoxin. In addition, we have shown that certain compounds that inhibit the formation of glucocerebroside also block the synthesis of GlcSph. Because the inhibitors of GlcSph formation are low molecular weight substances, we believe that these materials will cross the blood-brain barrier and improve the clinical course of patients with neuronopathic Gaucher disease. Accordingly, we have initiated a clinical trial with one of these inhibitors, N-butyldeoxynojirimycin, in patients with chronic neuronopathic (Type 3) Gaucher disease. Fabry disease: We extended our pre-clinical investigations on gene therapy for this hereditary metabolic disorder using the alpha-galactosidase A (alpha-Gal A) knock-out animal model of Fabry disease created by this Branch. We constructed an adeno-associated virus (AAV) vector containing the chicken alpha-actin promoter and the human alpha-Gal A gene. A single intravenous injection of this vector into the Fabry mice caused long-term enzymatic and functional correction in multiple organs of these animals. Alpha-gal A activity in the liver, heart and spleen of the recipients became higher than that in wild-type mice of the same strain. Significant increases of alpha-Gal A activity also occurred in the kidney, lung and small intestine of the injected mice. Following injection of the vector, there was complete clearance of accumulating globotriaosylceramide (Gb3) in the liver, spleen and heart of the Fabry mice that lasted more than 6 months. Gb3 decreased in the lung and small intestine, but the levels were not completely restored to that of normal mice. Gb3 in the kidney was reduced to nearly normal by 8 weeks after injection of the vector. However, by 24 weeks after administering the vector, Gb3 had begun to reaccumulate in this organ. There were no signs of toxicity to AAV. These findings provide a realistic basis and an extraordinarily strong incentive to explore gene therapy in patients with Fabry disease. They further indicate that this technique merits examination for the treatment of many other hereditary disorders of metabolism. Delivery of Genes to the Central Nervous System: Many hereditary disorders are characterized by brain pathology and developmental abnormalities. We created a novel lentiviral vector construct to deliver therapeutic genes to the central nervous system. This vector has the ability to deliver genes to non-dividing cells such as neurons. We incororated the human gene for glucocerebrosidase (GC) into the lentiviral vector and found that it functions very well in vitro. High expression of GC was detected in culture cells derived from a patient with Gaucher disease. In a collaborative effort with colleagues in Korea, it was shown that intraportal administration of this recombinant lentiviral vector resulted in efficient transduction of liver cells and expression of GC for more than 6 weeks. We have engineered the vector to include the herpes simplex virus type 1 tegument protein VP-22. The addition of VP-22 facilitates intercellular delivery of protein products of genes from transduced cells to non-infected cells. This technique greatly enhances the effectiveness of gene therapy in the CNS. Many investigators have requested this vector from DMNB. We have also developed a lentivirus based gene trap vector. This vector markedly facilitates the identification of previously uncharacterized genes and elucidation of their biological functions. It also has significant advantages for studies on differentiation and cell lineage. In addition, it may prove useful for examining the localization of specific gene products in various types of cells and their function in vivo. This construct has also been frequently requested by investigators at NIH and extramural scientists. Mucolipidosis IV: We previously reported cloning the gene that is mutated in patients with this disorder. This gene produces a protein called mucolipin. Mucolipin is a member of the TRP family of proteins. This discovery implies that the dominant pathophysiological alteration in mucolipidosis IV (MLIV) is a channelopathy. We are developing testable in vitro and in vivo models of this human neurogenetic disorder. The first of these investigations consists of electrophysiological studies in artificial lipid bilayers into which normal and mutated forms of mucolipin are inserted. This study revealed that ion channel dysfunction is produced by disease-related mutations in the MLIV gene. The alteration is characterized by an abnormal sensitivity of ion channels containing mutated mucolin to reduction in pH. The second approach employed telomerase-immortalized skin fibroblasts derived from normal humans and from MLIV patients that contain specific point mutations in the MLIV gene. Studies with these cells provided additional insight into the ion channel properties of mucolipin. The third investigation involves selective silencing of mucolipin RNA to block expression of the MLIV gene in a continuous human parietal cell line. These investigations are underway at this time. Experiments with this cellular model are expected to be extraordinarily significant since the major documented biochemical defect in MLIV is complete absence of acid secretion by parietal cells. This investigation should lead to an understanding of the role of mucolipin in acid production. The fourth line of investigation centers on the creation of a murine model of MLIV. We have prepared an appropriately targeted gene construct, and we have begun blastocyst injections with it in order to create an MLIV knock-out mouse.