Correction of Deficient Enzyme Activity in a Lysosomal Storage Disease, Aspartylglucosaminuria, by Enzyme Replacement and Retroviral Gene Transfer

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Abstract
The ability of lysosomal enzymes to be secreted and subsequently captured by adjacent cells provides an excellent basis for investigating different therapy strategies in lysosomal storage disorders. Aspartylglucosaminuria (AGU) is caused by deficiency of aspartylglucosaminidase (AGA) leading to interruption of the ordered breakdown of glycoproteins in lysosomes. As a consequence of the disturbed glycoprotein catabolism, patients with AGU exhibit severe cell dysfunction especially in the central nervous system (CNS). The uniform phenotype observed in these patients will make effective evaluation of treatment trials feasible in future. Here we have used fibroblasts and lymphoblasts from AGU patients and murine neural cell lines as targets to evaluate in vitro the feasibility of enzyme replacement and gene therapy in the treatment of this disorder. Complete correction of the enzyme deficiency was obtained both with recombinant AGA enzyme purified from CHO-K1 cells and with retrovirus-mediated transfer of the AGA gene. Furthermore, we were able to demonstrate enzyme correction by cell-to-cell interaciton of transduced and nontransduced cells. Lysosomal storage diseases display ideal targets for enzyme replacement and gene therapy because they are caused by mutations in a single gene and consequently exhibit deficient activity of a single enzyme with a constant expression level. We have studied aspartylglucosaminuria (AGU), the only known lysosomal amidase deficiency in humans, as a target for both enzyme replacement and retroviral-mediated gene transfer in vitro. Unlike other lysosomal diseases, AGU displays a rather uniform phenotype, which allows reliable evaluation of the success of different treatment strategies. Due to the neuropathological features seen in AGU patients, successful therapy is likely to require in vivo delivery of the gene or the correct enzyme to the central nervous system (CNS). We have successfully performed enzyme replacement and gene therapy both in AGU patients primary fibroblasts and in neural cells of murine origin. Cross-correction between treated and untreated cells could also be achieved, suggesting that only a relatively small number of treated cells would be sufficient to correct the deficient enzyme activity in vivo.