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.

拟议研究的总体目的是使用重组DNA 生产人α-半乳糖苷酶A（alpha-gal a）的策略来评估 它用于治疗Fabry病，一种原型的遗传代谢 紊乱。 先前的研究表明了外源的能力 α-gal A校正Fabry成纤维细胞中的代谢脱离，并 临床试验表明酶的生化有效性 替代品。 使用动物模型系统，以人α-gal a表示 将评估细菌在法布里疾病中替代酶。 允许净化毫克量的均质的方法 Alpha-Gal A已在我们的实验室中设计。 N末端氨基酸 已经确定了α-gal A的序列，并混合了 寡核苷酸已根据氨基酸序列合成 最小密码子冗余。 我们结束了这种寡核苷酸的混合物和 将其用作识别和隔离体外翻译A 1.6的探针 从制备琼脂糖 - 尿素凝胶中至1.9 kb mRNA。 此外， 该探针用于将特定克隆与cDNA库分离 冈山和伯格。 该库中的cDNA片段存在于 SV40衍生的质粒载体，允许在细菌和 哺乳动物细胞培养中的有效表达。 质粒的产生 编码的α-gal A将通过极敏感的酶测定法监测 和免疫沉淀技术。 寡核苷酸混合物也具有 用于从X染色体特异性基因组库中选择克隆 用噬菌体兰伯达建造。 这些基因组和cDNA中存在的插入物 克隆目前正在筛选，并将片段与 探针将用于DNA序列分析。 我们预计这是一个或 已经显示出更多的克隆将显示包含alpha-gal a 序列。 因此，该提案的主要目标是最大化 使用专门为此设计的几个载体在细菌中的表达 目的。 第二个主要目标是检查稳定性，水解 在低的微生物合成α-gal a的容量和体内命运 alpha-gal鼠标模型系统。 另外，基因组和cDNA序列 Alpha-gal A的a将用于研究分子的性质 来自无关患者的细胞系中的缺陷和Fabry的变体 疾病。