Porphyria and Human Heme Biosynthesis

描述（申请人提供）：拟议研究的总体目标是调查急性间歇性卟啉症（AIP）的生物化学，分子病理学和潜在治疗，AIP是一种常染色体显性肝卟啉症，由于羟甲基胆烷合酶（HMBS）的半正常活性，先天性红细胞生成性卟啉症（CEP）是一种常染色体隐性疾病，由于明显缺乏，但并非缺失，尿卟啉原III合酶（UROS）的活性。本文提出了三个具体目标：1）NMR研究将表征人UROS的结构和反应机制及其在UROS/HMBS胞质复合物中的相互作用。将研究UROS/HMBS复合物与5-氨基酮戊酸酯酶（ALAD）和尿卟啉原脱羧酶（UROD）在多酶复合物或“代谢子”中的可能相互作用。UROS/HMBS复合物的亚细胞位置将用荧光抗酶抗体测定。2)对于AIP，将努力确定是否可以通过肝脏靶向基因治疗来预防危及生命的急性神经系统发作。基于对各种肝特异性启动子/增强子组合的评价，将制备含有HMBS cDNA（具有/不具有强α-半乳糖苷酶A前导序列）的两种最佳启动子/增强子构建体，并评价流体动力学递送后的肝表达和分泌。将具有包膜血清型（1、5和8）的最佳表达载体注射到“AIP小鼠”的门静脉中，并通过血浆和尿ALA和胆色素原（PBG）水平监测它们预防苯巴比妥诱导的急性卟啉病发作的能力。3)对于CEP，将使用表达0.1-10%野生型活性的四种鼠UROS错义突变产生活的UROS敲入小鼠模型。筛选转染的ES细胞克隆，并将阳性克隆超选择至纯合性，以评估其活力和残留UROS活性。阳性杂合克隆将用于产生每种突变的创始小鼠，其将被培育成纯合性、彼此之间以及UROS杂合无效小鼠，可能产生具有0.05-10%野生型活性的敲入小鼠。将对这些小鼠进行生化、病理学和临床表征，特别是其血液学和皮肤病学表现。有活力的CEP小鼠应允许研究疾病的病理生理学和未来的治疗努力。

DESCRIPTION (provided by applicant): The overall objectives of the proposed research are to investigate the biochemistry, molecular pathology, and potential therapy of acute intermittent porphyria (AIP), an autosomal dominant hepatic porphyria due to the half-normal activity of hydroxymethylbilane synthase (HMBS), and congenital erythropoietic porphyria (CEP), an autosomal recessive disorder due to the markedly deficient, but not absent, activity of uroporphyrinogen III synthase (UROS). Three specific aims are proposed: 1) NMR studies will characterize the structure and reaction mechanism of human UROS and its interaction in the UROS/HMBS cytosolic complex. Possible interaction of the UROS/HMBS complex with 5-aminolevulate dehydratase (ALAD) and uroporphyrinogen decarboxylase (UROD) in a multi-enzyme complex or "metabolon" will be investigated. The subcellular location of the UROS/HMBS complex will be determined with fluorescent anti-enzyme antibodies. 2) For AIP, efforts will determine if the life-threatening, acute neurologic attacks can be prevented by liver-targeted gene therapy. Based on the evaluation of various liver-specific promoter/enhancer combinations, two optimal promoter/enhancer constructs containing the HMBS cDNA (with/without the strong alpha-galactosid nase A leader sequence) will be made and evaluated for hepatic expression and secretion following hydrodynamic delivery. The optimally expressing vector(s) with envelope serotypes (1, 5, and 8) will be injected into the portal vein of the "AIP mice" and their ability to prevent phenobarbital-induced acute porphyric attacks will be monitored by plasma and urinary ALA and porphobilinogen (PBG) levels. 3) For CEP, a viable UROS knock-in mouse model(s) will be generated using four murine UROS missense mutations expressing 0.1-10% of wild-type activity. Transfected ES cells clones are being screened and positive clones will be hyper-selected to homozygosity to assess their viability and residual UROS activity. Positive heterozygous clones will be used to generate founder mice for each muation, which will be bred to homozygosity, to each other, and to UROS heterozygous null mice, potentially generating knock-in mice with 0.05-10% of wild-type activities. These mice will be characterized biochemically, pathologically, and clinically, especially their hematologic and dermatologic manifestations. Viable CEP mice should permit studies of the disease pathophysiology and future therapeutic endeavors.