866. Modification of the Expression Vector for Systemic Gene Therapy of MLD

Abstract

Metachromatic leukodystrophy (MLD) is a lysosomal storage disorder caused by a deficiency of arylsulfatase A (ASA). Progressive demyelination associated with accumulation of sulfatide results in a variety of neurologic symptoms. No effective therapy is available at this moment. Gene therapy may be an important option for treatment of MLD. Since sulfatide deposits in both the central and peripheral nervous system, local injection of ASA expression vectors may have limited merit. In order to establish a systemic gene therapy protocol for MLD, we modified the ASA expression vector. First, we tested the utility of SUMF1 for overexpression of functional ASA. SUMF1 is a post-translational modifying enzyme essential for sulfatase activity. Coexpression of SUMF1 with ASA in 293 cells resulted in a several folds increase of ASA activity. When the ASA and SUMF1 expression plasmids were injected into liver by the hydrodynamics-based technique, a striking synergistic increase of ASA activity was observed in both liver and serum. Second, we synthesized the ASA-Tat fusion protein to improve the biodistribution of the enzyme. It was reported that the Tat motif (YGRKKRRQRRR) allows the fusion protein to cross the cell membrane and the blood brain barrier. The ASA-Tat fusion protein had full ASA activity and was secreted from cells transfected with the expression vector. When the conditioned media were incubated with MLD patient's fibroblasts, the ASA-Tat was efficiently taken up to the cells, but its uptake was only partially inhibited by the addition of mannose 6 phosphate (M6P). The M6P independent uptake of ASA-Tat was also confirmed by immunohistochemical staining. These results suggest that combination of SUMF1-coexpression and Tat-modification may be important for systemic delivery of ASA for MLD gene therapy.