Fabry Disease: We have investigated the protein expression profiles in cells derived from patients with Fabry disease using microarray technology. Fabry disease is secondary to deficiency of the lysosomal enzyme alpha-galactosidase A, leading to altered glycosphingolipid metabolism and accumulation that is often associated with endothelial dysfunction. Current evidence suggests that there is impairment of the vascular nitric oxide pathway, with abnormalities evident in the cerebral circulation and in the dermal vasculature of patients with Fabry disease. Some of these findings have been confirmed in a mouse model of Fabry disease and are under investigation in the Fabry patient population. Future developments in array technology for proteins and DNA single nucleotide polymorphism analysis, together with gene expression microarray analysis, will open a new chapter in our understanding of the biology of lysosomal storage disorders. Additional studies are planned to investigate the role of rare lipids in the etiology of this disease.? ? Gaucher Disease: We extended our basic investigations on the pathogenesis of the neuronopathic forms of Gaucher disease. Both glucocerebroside and glucosylsphingosine (GlcSph) are elevated in the brain of these patients. GlcSph is neurotoxic and contributes significantly to the dysfunction and destruction of brain cells. Our investigations have identified six compounds that inhibit the enzymatic synthesis of GlcSph and ranked them in the order of their effectiveness. Using compounds that reach the brain in an effective and non-toxic concentration, we propose to conduct clinical trials with these agents to improve the debilitating clinical course in patients with neuronopathic Gaucher disease. ? ? Our genomic investigations into the changes that occur in Gaucher disease have identified statistically significant increases in expression of 10-fold or more over normal in 203 genes and decreases in 78 genes. This information is critical to understanding the genesis of the hepatosplenomegaly and other pathophysiological changes that occur in patients with Gaucher disease and may lead to improved treatment strategies.? ? Protein Transduction Domains: We are extending our strategies to extend the organ and tissue distribution of exogenous enzymes for the treatment of both Gaucher disease and Fabry disease to augment the effectiveness of enzyme replacement therapy. We have previously expressed a TAT-GC fusion protein in which recombinant glucocerebrosidase (GC) is fused to 11-amino acid peptide from the HIV-1 transactivator protein (TAT) which functions to facilitate the transport of the enzyme across the plasma membrane of a variety of cell-types in a receptor-independent manner. We have extended these studies to include additional GC constructs containing flexible spacers to increase enzyme activity in addition to a variety of TAT analogs in order to test the effect of sequence variation on the uptake of the enzyme. Further studies have also been undertaken to construct similar fusions with the enzyme alpha-galactosidase A (AGA) as a possible strategy for wider distribution of this enzyme for the treatment of Fabry disease. Efforts are underway to introduce these fusion proteins into animals to test their distribution and compare these findings with that of the native constructs. Recombinant GC-TAT and AGA-TAT were expressed in eukaryotic cells from which catalytically active, normally glycosylated enzyme fusion proteins were obtained and tested for receptor independent uptake into cultured cells. It is expected that GC-TAT will be more efficiently delivered than unmodified GC to cells in the bone marrow and lung, and perhaps additional cells that lack the mannose lectin and thereby enhance the clinical responses of patients with Gaucher to enzyme replacement therapy We anticipate that the AGA-TAT enzyme may be capable of entering heart and kidney more efficiently than the native enzyme.? ? Enzyme Replacement Therapy: In collaboration with members of the Surgical Neurology Branch (SNB), we determined the safety of intracerebral injection of GC in non-human primates using the convection-enhanced delivery (CED) technique developed by SNB. This was followed by clinical administration of this enzyme to a patient with Type 2 Gaucher disease. Alterations in the enzyme aimed at improving the distribution and stability of the administered enzyme are currently under investigation. ? ? Gene Therapy: We are developing methods to improve gene therapy for patients with Gaucher disease and Fabry disease using adeno-associated viral vectors in conjunction with the abovementioned protein transduction domains.. The first of these constructs increased the level of glucocerebrosidase activity significantly in multiple organs and tissues when injected into experimental animals. Moreover, it very effectively transduces bone-marrow stem and progenitor cells ex vivo. We demonstrated long-term expression of GC in experimental animals following transplantation of bone-marrow-derived cells transformed by a GC lentivirus vector. It is anticipated that autologous cells transduced with this, or a related lentiviral vector, may be appropriate for gene therapy trials in patients with Gaucher disease since successful bone marrow transplantation can cure patients with type 1 (non-neuronopathic) Gaucher disease.? ? Chaperone Therapy: We have identified a number of patients with Fabry disease in whom the reduced catalytic activity of alpha-galactosidase A is increased in the presence of the molecular chaperone 1-deoxy-galactonojirimycin (DGJ). This technique is based on the ability of certain compounds to interact with the catalytic site of mutated enzyme and escort it from the endoplasmic reticulum where it is produced through the Golgi apparatus to lysosomes. The low pH and the reduced concentration in lysosomes causes the chaperone to dissociate from the enzyme and allows it to carry out its catalytic function. We have examined the ability of DGJ to augment residual alpha-galactosidase A activity in cultured skin fibroblasts, T cells and peripheral blood mononuclear cells derived from patients with Fabry disease. A standardized protocol has been developed for the screening of patients for whom chaperone therapy with DGJ may be an effective way of increasing the enzyme levels. Further investigations are planned to evaluate variations in test conditions with DGJ as the chaperone and to test additional agents as potential chaperones for patients whose alpha-galactosidase A activity is not enhanceable using DGJ. A protocol has been initiated for a Phase 1 safety and dose-response trial with DGJ in patients with Fabry disease. When it is completed, we shall examine the clinical effectiveness of active site-specific chaperone therapy in patients with enhanceable alpha-galactosidase A activity (cf. lead investigator's report). ? ? Additional studies have demonstrated the potential for the use of chaperone therapy for the treatment of Gaucher disease. Using a similar test model as designed for chaperone therapy in Fabry disease, candidate active-site-directed molecules are under investigation. All surviving patients with Gaucher disease have residual glucocerebrosidase activity and appear to be excellent candidates for molecular chaperone therapy. Such studies underscore the possibility that small molecules may be used in place of or in conjunction with enzyme replacement therapy as it is currently practiced.
| Shin, Sang H; Kluepfel-Stahl, Stefanie; Cooney, Adele M et al. (2008) Prediction of response of mutated alpha-galactosidase A to a pharmacological chaperone. Pharmacogenet Genomics 18:773-80 |
| Goldin, Ehud; Caruso, Rafael C; Benko, William et al. (2008) Isolated ocular disease is associated with decreased mucolipin-1 channel conductance. Invest Ophthalmol Vis Sci 49:3134-42 |
| Shen, Jin-Song; Meng, Xing-Li; Moore, David F et al. (2008) Globotriaosylceramide induces oxidative stress and up-regulates cell adhesion molecule expression in Fabry disease endothelial cells. Mol Genet Metab 95:163-8 |
| Shen, Jin-Song; Edwards, Nancy J; Hong, Young Bin et al. (2008) Isofagomine increases lysosomal delivery of exogenous glucocerebrosidase. Biochem Biophys Res Commun 369:1071-5 |
| Shin, Sang-Hoon; Murray, Gary J; Kluepfel-Stahl, Stefanie et al. (2007) Screening for pharmacological chaperones in Fabry disease. Biochem Biophys Res Commun 359:168-73 |
| Shen, Jin-Song; Meng, Xing-Li; Schiffmann, Raphael et al. (2007) Establishment and characterization of Fabry disease endothelial cells with an extended lifespan. Mol Genet Metab 92:137-44 |
| Askari, Hasan; Kaneski, Christine R; Semino-Mora, Cristina et al. (2007) Cellular and tissue localization of globotriaosylceramide in Fabry disease. Virchows Arch 451:823-34 |
| Moore, David F; Kaneski, Christine R; Askari, Hasan et al. (2007) The cerebral vasculopathy of Fabry disease. J Neurol Sci 257:258-63 |
| Lonser, R R; Schiffman, R; Robison, R A et al. (2007) Image-guided, direct convective delivery of glucocerebrosidase for neuronopathic Gaucher disease. Neurology 68:254-61 |
| Yoshimitsu, M; Higuchi, K; Ramsubir, S et al. (2007) Efficient correction of Fabry mice and patient cells mediated by lentiviral transduction of hematopoietic stem/progenitor cells. Gene Ther 14:256-65 |
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