The overall objective of the proposal research is to investigate the biochemical and molecular genetics of the human acid sphingomyelinase (hASM), the lysosomal hydrolase deficient in the neuronopathic and non- neuronopathic forms of Niemann-Pick disease (Types A and B NPD, respectively), and to use the murine model of NPD to develop and evaluate enzyme replacement and somatic gene transfer strategies for the treatment of this lysosomal storage disease. To accomplish these studies we have: 1) isolated, sequenced and expressed the full-length cDNA and genomic sequences encoding hASM, 2) stably produced hASM in Chinese hamster ovary (CHO) cells using the p91023(B) eukaryotic expression system and developed a pilot purification scheme, 3) collected cell lines from over 80 unrelated NPD families and identified the first molecular lesions which result in Types A and B NPD, and 4) achieved in vitro correction of the metabolic defect in murine NPD fibroblasts using retroviral-mediated gene transfer. For the proposed studies, the p91023(B) expression system will be used to produce large amounts of purified recombinant hASM for physical and kinetic characterization, and the production of monospecific anti-hASM antibodies. In addition, site-directed mutagenesis of the full-length hASM cDNA will be performed to identify important functional domains in the hASM polypeptide and to engineer a more active and/or stable hASM for subsequent enzyme and gene replacement trials in the NPD mouse. We will also continue to identify the molecular lesions which result in Types A and B NPD and evaluate their effects on enzyme activity, stability and/or lysosomal targeting. These studies should permit improved diagnosis for this lysosomal storage disease and, together with the site-directed mutagenesis analyses, provide insights into hASM structure and function. Concurrent with these studies, the NPD mouse will be used as a model system to develop and evaluate enzyme replacement and somatic gene transfer for the treatment of non-neurologic lysosomal storage diseases, such as Type B NPD. Efforts will be directed to identify the precise molecular lesion in the NPD mice by PCR amplification and sequencing of murine ASM cDNA and/or genomic sequences, and to correct the metabolic defect in murine NPD by germline gene transfer. In addition, the in vivo effectiveness of bone marrow- mediated somatic gene transfer will be evaluated and a combination of protein engineering (e.g., site-directed mutagenesis of nu-glycosylation sites), gene transfer (e.g., amplifiable retroviral vectors) and alternative organ delivery (e.g., skin grafting) systems will be used to develop novel approaches to somatic gene therapy. These studies should provide valuable information on the molecular genetics of ASM and NPD, as well as the effectiveness of enzyme replacement and somatic gene transfer for the treatment of NPD as a prototype for other inborn errors of metabolism.
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