MOLECULAR STUDY OF MPSI GENE THERAPY

粘多糖病I型（MPS I）由一组常

Mucopolysaccharidosis I (MPS I) comprises a group of autosomally transmitted disorders caused by lack of activity of the lysosomal enzyme, alpha-L-iduronidase. In addition to human MPS I (the Scheie, Hurler/Scheie and Hurler syndromes, in order of increasing clinical severity), there is a canine form of the disease. MPS I has long been considered potentially treatable by exogenous enzyme, and the moderate success of alloogeneic bone marrow transplantation in slowing the course of the disease has made MPS I a candidate for gene replacement therapy via hematopoietic stem cells. The molecular tools (cloned human and canine alpha-L-iduronidase cDNAs and genes) are now available to develop both forms of therapy and to test these in the canine model. Aim 1 (gene replacement) - Cloned cDNA encoding alpha-L-iduronidase will be inserted into retroviral vectors for transfer into bone marrow cultured from MPS I dogs; to allow integration of the cDNA into the earliest progenitors, stem cells will be stimulated to divide by culture of the marrow in the presence of various cytokines including canine stem cell factor. After optimization of vectors, culture conditions and stem cell enrichment, somatic gene therapy of canine MPS I will be undertaken in vivo. The effects of autologous transplantation of genetically modified bone marrow on the course of the disease will be compared to the previously studied effects of bone marrow transplantation. Aim 2 (enzyme replacement) - cDNA encoding alpha-L-iduronidase will be placed under the control of strong promoters in vectors that will be introduced into mammalian cells and subsequently amplified. The recombinant alpha-L-iduronidase secreted by such overexpressing cell lines will be purified and administered to MPS I affected dogs. The results will be compared to those of gene replacement and bone marrow transplantation. Aim 3 (molecular studies of MPS I) - Mutations underlying MPS I will be identified in order to understand the molecular basis of the disease, and particularly to account for the clinical differences between the different forms of human MPS I. Molecular heterogeneity is anticipated and screening procedures that can localize the mutation on both alleles will be employed. Establishing genotype/phenotype correlations will assist in providing prognosis and appropriate treatment.