FABRY DISEASE: MOLECULAR AND MODEL THERAPEUTIC STUDIES

法布里病：分子和模型治疗研究

• 批准号: 3232420
• 负责人: Robert J Desnick
• 金额: $ 23.14万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1984
• 资助国家: 美国
• 起止时间: 1984-05-01 至 1992-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3232420
• 关键词: Escherichia coli; Fabry's disease; affinity chromatography; alpha galactosidase; bacteriophage lambda; biotechnology; complementary DNA; disease /disorder model; enzyme structure; enzyme therapy; gel electrophoresis; genetic library; genetic manipulation; human tissue; immunoelectrophoresis; lysogeny; messenger RNA; molecular cloning; molecular pathology; nucleic acid chemical synthesis; nucleic acid sequence; orphan disease /drug; synthetic enzyme

The overall objective of the proposed research is to use recombinant DNA strategies to produce human Alpha-galactosidase A (Alpha-Gal A) to evaluate its use for the treatment of Fabry disease, a prototype inherited metabolic disorder. Previous studies have demonstrated the ability of exogenous Alpha-Gal A to correct the metabolic deffect in Fabry fibroblasts, and clinical trials have indicated the biochemical effectiveness of enzyme replacement. Using an animal model system, human Alpha-Gal A expressed in bacteria will be evaluated for enzyme replacement in Fabry disease. Methods that permit the purification of milligram quantities of homogeneous Alpha-Gal A have been devised in our laboratory. The N-terminal amino acid sequence for Alpha-Gal A has been determined and a mixture of oligonucleotides has been synthesized based on an amino acid sequence with minimal codon redundancy. We end-labelled this oligonucleotide mixture and used it as a probe to identify and isolate for in vitro translation a 1.6 to 1.9 kb mRNA fraction from a preparative agarose-urea gel. In addition, the probe was used to isolate specific clones from the cDNA library of Okayama and Berg. The cDNA segments in this library are present in a SV40-derived plasmid vector that permits replication in bacteria and efficient expression in mammalian cell culture. The production of plasmid encoded Alpha-Gal A will be monitored by extremely sensitive enzyme assay and immunoprecipitation techniques. The oligonucleotide mixture has also been used to select clones from an X-chromosome-specific genomic library constructed in phage lambda. Inserts present in these genomic and cDNA clones are currently being screened and the segments hybridizing to the probes will be used for DNA sequence analysis. We anticipate that one or more of the clones already isolated will be shown to contain Alpha-Gal A sequences. Therefore, a major goal of this proposal will be to maximize expression in bacteria using several vectors specifically designed for this purpose. A second major goal will be to examine the stability, hydrolytic capacity and in vivo fate of microbially synthesized Alpha-Gal A in the low Alpha-Gal mouse model system. In addition, the genomic and cDNA sequences of Alpha-Gal A will be used to investigate the nature of the molecular defect(s) in cell lines from unrelated patients and variants with Fabry disease.

拟议研究的总体目标是使用重组 DNA 生产人类 α-半乳糖苷酶 A (Alpha-Gal A) 的策略进行评估 它用于治疗法布里病，这是一种遗传性代谢病的原型 紊乱。 先前的研究已经证明了外源性 Alpha-Gal A 可纠正法布里成纤维细胞的代谢缺陷，以及 临床试验表明酶的生化功效 替代品。 使用动物模型系统，人类 Alpha-Gal A 表达于 将评估细菌在法布里病中的酶替代情况。 允许纯化毫克量均质物质的方法 Alpha-Gal A 是我们实验室设计的。 N端氨基酸 Alpha-Gal A 的序列已确定，并且是 寡核苷酸是根据氨基酸序列合成的 最小的密码子冗余。 我们对这种寡核苷酸混合物进行末端标记并 用它作为探针来识别和分离体外翻译a 1.6 从制备型琼脂糖-尿素凝胶中提取 1.9 kb mRNA 片段。 此外， 该探针用于从 cDNA 文库中分离出特定的克隆。 冈山和伯格。 该文库中的 cDNA 片段存在于 SV40 衍生的质粒载体，允许在细菌和 在哺乳动物细胞培养物中有效表达。 质粒的产生 编码的 Alpha-Gal A 将通过极其灵敏的酶测定进行监测 和免疫沉淀技术。 寡核苷酸混合物还具有 用于从 X 染色体特异性基因组文库中选择克隆 在噬菌体 lambda 中构建。 这些基因组和 cDNA 中存在插入片段 目前正在筛选克隆，并将片段与 探针将用于 DNA 序列分析。 我们预计一个或 更多已分离的克隆将显示含有 Alpha-Gal A 序列。 因此，该提案的一个主要目标是最大化 使用专门为此设计的几种载体在细菌中表达 目的。 第二个主要目标是检查稳定性、水解性 微生物合成的α-Gal A在低浓度下的能力和体内命运 Alpha-Gal 小鼠模型系统。 此外，基因组和 cDNA 序列 Alpha-Gal A 将用于研究分子的性质 来自不相关患者的细胞系缺陷和法布里变异 疾病。