MOLECULAR STUDY OF MPS I--GENE THERAPY

我们建议用分子生物学的方法来研究 这种遗传性疾病称为粘多糖I型(MPS I)。 这些综合症包括赫勒综合征、赫勒/谢伊综合征和谢伊综合征。 以及一种类似于赫勒/谢伊的犬病。所有形式的 MPS I是由溶酶体酶缺乏活性引起的 α-L-艾杜糖醛酸酶。我们的目标有两个：(1)刻画 准确地说，导致人类和犬MPS的突变是 (2)尝试用基因疗法治疗犬病 骨髓细胞的修饰。该计划的基本原理 治疗性试验是犬MPS I的先验证据 对异基因骨髓的生化和临床反应 移植。 需要克隆编码α-L艾杜糖醛酸酶的c DNA 两个目的都是。我们会将这种酶提纯到均一， 用抗血清筛选人和犬的lambda GT11文库 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的放射性标记，我们将 确定MPS I患者的细胞是否能够合成和 处理交叉反应蛋白；如果不是，我们将通过 南方杂交和北方杂交与核跑动 转录：α-L-艾杜糖苷酶基因是否完整 抄写的。我们计划精确定位到基因组 对那些被认为信息量最大的突变进行排序。 我们将在MPS I中表达α-L艾杜糖醛酸酶的全长基因 细胞，使用逆转录病毒递送系统 穆利根和他的同事。一次表达(即，出现 溶酶体内a-L-艾杜糖酸酶活性)已在 成纤维细胞和造血细胞，我们将继续表达 在活体内。骨髓将从MPS I犬身上采集，浓缩 在干细胞中，感染了含有α-L的逆转录病毒- 艾杜糖醛酸酶基因，并返回给供体。集成和 将在循环中监测cDNAs的表达 白细胞。在试点可行性试验后，该协议将 以最近的骨髓移植研究为蓝本。 4至6只MPS I级犬，通过照射或 化疗药物，将自己用来治疗 转基因骨髓。他们将被比作 未经治疗的患者和正常对照 生化、超微结构和临床参数，以便 确定这种基因疗法的长期后果。

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.