MOLECULAR STUDY OF MPS I--GENE THERAPY

我们建议应用分子生物学的方法来研究 被称为粘多糖样变性I（MPS I）的遗传性疾病。 这些包括Hurler、Hurler/Scheie和Scheie综合征， 以及类似于Hurler/Scheie的犬科疾病。 一切形式 MPS I是由溶酶体酶缺乏活性引起的 α-L-艾杜糖醛酸酶。 我们的目标是双重的：（1）描述 正是这种突变导致了人类和犬类的MPS I （2）尝试通过基因治疗犬疾病 骨髓细胞的修饰。 的理由 治疗试验是先前证明犬MPS I 对同种异体骨髓的生化和临床反应 移植 需要编码α-L-艾杜糖醛酸酶的克隆cDNA， 两个目的。 我们要把酶提纯到均匀， 用抗血清筛选λ GT 11文库（人和犬） 和/或寡核苷酸探针，表征和测序 cDNA。 在细胞培养中使用生物合成放射性标记，我们将 确定来自MPS I患者的细胞是否能够合成， 处理交叉反应蛋白;如果没有，我们将通过 Southern和北方杂交及核结合 转录是否α-L-艾杜糖醛酸酶基因是完整的， 转录。 我们计划精确定位到基因组 测序那些被认为最有价值的突变。 我们将在MPS I中表达全长α-L-艾杜糖醛酸酶cDNA， 细胞，使用逆转录病毒递送系统，所述逆转录病毒递送系统的类型由 Mulligan及其同事 一旦表达（即，外观 溶酶体内α-L-艾杜糖醛酸酶活性）已在 成纤维细胞和造血细胞，我们将继续表达 in vivo. 将从MPS I犬中采集骨髓，进行富集 在干细胞中，感染了含有α-L- 艾杜糖醛酸酶cDNA并返回给供体。 一体化和 将在循环中监测cDNA的表达， 白细胞 经过可行性试验，该议定书将 以最近的骨髓移植研究为模型。 4 - 6只MPS I犬，通过辐照或 化疗药物，将与他们自己的治疗 转基因骨髓 他们将与 未治疗的受累和正常对照组 生化、超微结构和临床参数，以便 确定这种基因治疗的长期后果。

We propose to apply methods of molecular biology to the study of the genetic disorders known as Mucopolysaccharidosis I (MPS I). These include the Hurler, Hurler/Scheie and Scheie syndromes, as well as a canine disease analogous to Hurler/Scheie. All forms of MPS I are caused by lack of activity of the lysosomal enzyme alpha-L-iduronidase. Our goal is two-fold: (1) to characterize precisely the mutation that give rise to human and canine MPS I and (2) to attempt therapy of the canine disease by genetic modification of bone marrow cells. The rationale for the therapeutic trial is the prior demonstration that canine MPS I responds biochemically and clinically to allogeneic bone marrow transplantation. Cloned cDNA encoding the alpha-L-iduronidase is required for both purposes. We shall purify the enzyme to homogeneity, screen lambda GT11 libraries (human and canine) with antisera and/or oligonucleotide probes, characterize and sequence the cDNA. Using biosynthetic radiolabeling in cell culture, we shall determine if cells from MPS I patients are able to synthesize and process cross-reactive protein; if not, we shall determine by Southern and Northern hybridization and nuclear run-on transcription whether the alpha-L-iduronidase gene is intact and transcribed. We plan to localize precisely to the genomic sequence those mutations deemed most informative. We will express full-length alpha-L-iduronidase cDNA in MPS I cells, using a retroviral delivery system of the type described by Mulligan and colleagues. Once expression (i.e., appearance of intralysosomal a-L-iduronidase activity) has been demonstrated in fibroblasts and hematopoietic cells, we will proceed to expression in vivo. Bone marrow will be collected from MPS I dogs, enriched in stem cells, infected with the retrovirus containing the alpha-L- iduronidase cDNA and returned to the donors. Integration and expression of the cDNA will be monitored in circulating leukocytes. After a pilot feasibility experiment, the protocol will be modeled on the recent bone marrow transplantation studies. Four to six MPS I dogs, prepared by irradiation or chemotherapeutic drugs, will be treated with their own genetically modified bone marrow. They will be compared with untreated affected and normal controls with respect to biochemical, ultrastructural and clinical parameters, in order to determine the long term consequences of such genetic therapy.