The overall objective of the proposed research is to use recombinant DNA strategies to produce human Alpha-galactosidase A (Alpha-Gal A) to evaluate its use for the treatment of Fabry disease, a prototype inherited metabolic disorder. Previous studies have demonstrated the ability of exogenous Alpha-Gal A to correct the metabolic deffect in Fabry fibroblasts, and clinical trials have indicated the biochemical effectiveness of enzyme replacement. Using an animal model system, human Alpha-Gal A expressed in bacteria will be evaluated for enzyme replacement in Fabry disease. Methods that permit the purification of milligram quantities of homogeneous Alpha-Gal A have been devised in our laboratory. The N-terminal amino acid sequence for Alpha-Gal A has been determined and a mixture of oligonucleotides has been synthesized based on an amino acid sequence with minimal codon redundancy. We end-labelled this oligonucleotide mixture and used it as a probe to identify and isolate for in vitro translation a 1.6 to 1.9 kb mRNA fraction from a preparative agarose-urea gel. In addition, the probe was used to isolate specific clones from the cDNA library of Okayama and Berg. The cDNA segments in this library are present in a SV40-derived plasmid vector that permits replication in bacteria and efficient expression in mammalian cell culture. The production of plasmid encoded Alpha-Gal A will be monitored by extremely sensitive enzyme assay and immunoprecipitation techniques. The oligonucleotide mixture has also been used to select clones from an X-chromosome-specific genomic library constructed in phage lambda. Inserts present in these genomic and cDNA clones are currently being screened and the segments hybridizing to the probes will be used for DNA sequence analysis. We anticipate that one or more of the clones already isolated will be shown to contain Alpha-Gal A sequences. Therefore, a major goal of this proposal will be to maximize expression in bacteria using several vectors specifically designed for this purpose. A second major goal will be to examine the stability, hydrolytic capacity and in vivo fate of microbially synthesized Alpha-Gal A in the low Alpha-Gal mouse model system. In addition, the genomic and cDNA sequences of Alpha-Gal A will be used to investigate the nature of the molecular defect(s) in cell lines from unrelated patients and variants with Fabry disease.

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National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
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Mount Sinai School of Medicine
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