MOLECULAR STUDY OF MPSI GENE THERAPY

粘多糖病I(MPS I)由一组常染色体组成 由于溶酶体酶缺乏活性而引起的传播性疾病， α-L-艾杜糖醛酸酶。除了人类MPS I(The Scheie，Hurler/Scheie 和赫勒综合征，按临床严重程度递增的顺序)， 犬型的疾病。国会议员I长期以来一直被认为是潜在的 可用外源性酶治疗，异体骨移植适度成功 骨髓移植延缓了病程，使MPS I 通过造血干细胞进行基因替代治疗的候选者。这个 分子工具(克隆人和犬的α-L艾杜糖醛酸酶cDNA和 基因)现在可用于开发这两种形式的治疗方法并对其进行测试 在犬类模型中。AIM 1(基因置换)-克隆的编码基因 将α-L-艾杜糖酸酶插入逆转录病毒载体进行转移 从MPS I犬培养的骨髓中；允许整合c DNA 作为最早的祖细胞，干细胞将被刺激通过 在包括犬在内的各种细胞因子存在的情况下培养骨髓 干细胞因子。对载体进行优化后，培养条件和 干细胞浓缩、体细胞基因治疗犬MPS I将被 在活体内进行。自体肾移植的疗效观察 转基因骨髓在病程中将被 与以往研究的骨髓移植效果进行了比较。 目的2(酶置换)-编码α-L艾杜糖醛酸酶的c DNA将被 被置于载体中的强启动子的控制下， 被引入哺乳动物细胞，随后被扩增。这个 这种高表达细胞分泌的重组α-L艾杜糖醛酸酶 将被提纯并注射给受MPS I影响的狗。结果将会是 可与基因置换和骨髓移植相比较。 目标3(MPS I的分子研究)-MPS I的潜在突变将是 以了解该病的分子基础，以及 特别是要考虑到不同的临床差异 人类MPS的不同类型I.分子异质性的预测和筛选 可以在两个等位基因上定位突变的程序将被采用。 建立基因/表型相关性将有助于提供 预后和适当的治疗。

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.