Porphyria and Human Heme Biosynthesis

描述(申请人提供)：拟议研究的总体目标是研究急性间歇性卟啉症(AIP)和先天性红细胞生成性卟啉症(CEP)的生化、分子病理学和潜在的治疗方法。AIP是一种常染色体显性遗传性肝卟啉病，其原因是羟甲基胆烷合成酶(HMBS)活性半正常，先天性红细胞生成性卟啉症(CEP)是一种常染色体隐性遗传性疾病，其原因是尿卟啉原III合成酶(UROS)活性明显不足。提出了三个具体的目标：1)核磁共振研究将表征人Uros的结构和反应机理及其在Uros/HMBS胞浆复合体中的相互作用。Uros/HMBS复合体与5-氨基酮戊酸脱水酶(ALAD)和尿卟啉原脱羧酶(UROD)在多酶复合体或“代谢”中的可能相互作用将被研究。Uros/HMBS复合体的亚细胞位置将用荧光抗酶抗体确定。2)对于AIP，努力将决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发作。在评价各种肝脏特异性启动子/增强子组合的基础上，我们将构建两个含有HMBS基因的最佳启动子/增强子载体(带有/不带有较强的α-半乳糖苷酶A前导序列)，并对流体输送后肝脏的表达和分泌情况进行评估。包膜血清型(1、5和8型)的优化表达载体(S)将被注射到“AIP小鼠”的门静脉中，其预防苯巴比妥诱导的急性卟啉发作的能力将通过血浆和尿液ALA和卟啉胆红素(PBG)水平来监测。3)对于CEP，将使用4个表达0.1-10%野生型活性的小鼠UROS错义突变来建立一个可行的UROS敲入小鼠模型(S)。转基因ES细胞克隆正在筛选中，阳性克隆将被超选择为纯合子，以评估它们的活性和剩余的UROS活性。杂合子阳性克隆将被用来为每一次交配产生创始人小鼠，这些小鼠将被培育成纯合子，彼此之间，以及Uros杂合子缺失小鼠，潜在地产生具有0.05-10%野生型活动的敲入小鼠。这些小鼠将具有生化、病理和临床特征，特别是它们的血液学和皮肤病表现。存活的CEP小鼠应该可以研究这种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.