MOLECULAR STUDY OF MPSI-GENE THERAPY IN CANINE MODEL

粘多糖样沉积症I型（MPS I）包括一组常染色体 由溶酶体酶活性缺乏引起的传播性疾病， α-L-艾杜糖醛酸酶。 除了人MPS I（Scheie，Hurler/Scheie 和Hurler综合征，按照临床严重程度增加的顺序）， 犬的疾病。 MPS I一直被认为是潜在的 外源性酶可治疗，同种异体骨的中度成功 骨髓移植在减缓疾病进程方面使MPS I 通过造血干细胞进行基因替代治疗的候选者。 的 分子工具（克隆的人和犬α-L-艾杜糖醛酸酶cDNA和 基因），现在可以开发这两种形式的治疗，并测试这些 在犬类模型中。 目的1（基因置换）-克隆的cDNA编码 将α-L-艾杜糖醛酸酶插入逆转录病毒载体用于转移 从MPS I犬培养的骨髓中;以允许cDNA整合 干细胞在分化为最早的祖细胞时， 在各种细胞因子存在下培养骨髓， 干细胞因子 在优化载体、培养条件和 干细胞富集，犬MPS I的体细胞基因治疗将是 在体内进行。 自体移植的效果 转基因骨髓对疾病的过程中将是 与先前研究的骨髓移植效果相比。 目的2（酶替代）-将编码α-L-艾杜糖醛酸酶的cDNA 置于强启动子的控制下， 引入哺乳动物细胞并随后扩增。 的 由这种过表达细胞系分泌的重组α-L-艾杜糖醛酸酶 将纯化并给予受MPS I影响的犬。 结果将 与基因置换和骨髓移植相比。 目标3（MPS I的分子研究）-MPS I基础突变将 为了了解疾病的分子基础， 特别是为了解释不同的 人MPS I的形式。 预期分子异质性， 将使用能够定位两个等位基因上的突变的方法。 建立基因型/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.