Porphyria and Human Heme Biosynthesis

卟啉症和人血红素生物合成

• 批准号: 8072299
• 负责人: Robert J Desnick
• 金额: $ 10万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-06-01
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8072299
• 关键词: Active Sites; Acute; Acute Intermittent Porphyria; Alleles; Aminolevulinate; Aminolevulinic Acid; Anabolism; Antibodies; Biochemistry; Blood Circulation; Bone Marrow; Breeding; Cell Culture Techniques; Complementary DNA; Complex; Confocal Microscopy; Cutaneous; Dermatologic; Disease; Enhancers; Enzymes; Erythroid; Erythropoietic Porphyria; Escherichia coli; Evaluation; Functional disorder; Future; Galactosidase; Gene Conversion; Gene Expression; Gene Targeting; Generations; Genotype; Goals; Hepatic; Hepatic Porphyrias; Hepatocyte; Heterozygote; Housekeeping; Human; Hydro-Lyases; Hydroxymethylbilane Synthase; Inborn Genetic Diseases; Kinetics; Knock-in Mouse; Knock-out; Knockout Mice; Label; Life; Liver; Location; Missense Mutation; Mitochondria; Modeling; Monitor; Multienzyme Complexes; Mus; Mutation; Neurologic; Phenobarbital; Photosensitivity; Plasma; Plasmids; Porphobilinogen; Porphobilinogen Synthase; Porphyrias; Porphyrins; Portal vein structure; Reaction; Recombinants; Relative (related person); Reporter Genes; Research; Residual state; Role; Serotyping; Signal Transduction; Solutions; Structure; Surface; Synthase I; Therapeutic; Tissues; Toxic effect; Transplantation; UV induced; Ultraviolet Rays; Uroporphyrinogen Decarboxylase; Uroporphyrinogen III Synthetase; Uroporphyrinogens; Viral; Viral Genes; adeno-associated viral vector; albino mouse; analog; base; blastocyst; coproporphyrinogen III; embryonic stem cell; enzyme substrate; gene therapy; heme biosynthesis; hydroxymethylbilane; intravenous drip; molecular pathology; mouse model; overexpression; prevent; promoter; skin lesion; three dimensional structure; uptake; urinary; vector

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.

描述（由申请人提供）：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，分子病理学和潜在的治疗方法，AIP是一种常染色体显性肝性卟啉症，由于羟甲基双烷合成酶（HMBS）活性半正常，而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，但不是缺乏，尿卟啉原III合成酶（UROS）的活性。提出了三个具体目标：1)核磁共振研究将表征人类UROS的结构和反应机制及其在UROS/HMBS细胞质复合物中的相互作用。将研究UROS/HMBS复合物与5-氨基酸脱水酶（ALAD）和尿卟啉原脱羧酶（UROD）在多酶复合物或“代谢”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确定。2)对于AIP，努力将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。在对各种肝脏特异性启动子/增强子组合进行评估的基础上，将制备两种含有HMBS cDNA（含/不含强α -半乳糖苷酶A先导序列）的最佳启动子/增强子结构，并评估其在流体动力学递送后的肝脏表达和分泌情况。将具有囊膜血清型（1、5和8）的最佳表达载体(s)注射到“AIP小鼠”的门静脉中，并通过血浆和尿液ALA和卟啉胆色素原（PBG）水平监测其预防苯巴比妥诱导的急性卟啉发作的能力。3)对于CEP，将使用表达0.1-10%野生型活性的4个小鼠UROS错义突变生成可行的UROS敲入小鼠模型。正在筛选转染的胚胎干细胞克隆，阳性克隆将被超选择到纯合子，以评估其生存能力和剩余UROS活性。阳性杂合克隆将用于产生每次突变的创始小鼠，这些小鼠将被培育成纯合子，彼此之间，以及UROS杂合零小鼠，可能产生具有0.05-10%野生型活性的敲入小鼠。这些小鼠将被表征生化，病理和临床，特别是他们的血液学和皮肤病学的表现。可行的CEP小鼠应该允许研究疾病的病理生理和未来的治疗努力。

项目成果

Regional assignment of the human uroporphyrinogen III synthase (UROS) gene to chromosome 10q25.2----q26.3.

人尿卟啉原 III 合酶 (UROS) 基因在染色体 10q25.2----q26.3 上的区域分配。

• DOI: 10.1007/bf01213085
• 发表时间: 1991
• 期刊: Human genetics
• 影响因子: 5.3
• 作者: Astrin,KH;Warner,CA;Yoo,HW;Goodfellow,PJ;Tsai,SF;Desnick,RJ
• 通讯作者: Desnick,RJ

Congenital erythropoietic porphyria. A mild variant with low uroporphyrin I levels due to a missense mutation (A66V) encoding residual uroporphyrinogen III synthase activity.

先天性红细胞生成性卟啉症。

• DOI: 10.1001/archderm.128.9.1243
• 发表时间: 1992
• 期刊: Archives of dermatology
• 作者: Warner,CA;Poh-Fitzpatrick,MB;Zaider,EF;Tsai,SF;Desnick,RJ
• 通讯作者: Desnick,RJ

Nonoverlapping clusters: approximate distribution and application to molecular biology.

非重叠簇：近似分布及其在分子生物学中的应用。

• DOI: 10.1111/j.0006-341x.2001.00420.x
• 发表时间: 2001
• 期刊: Biometrics.
• 作者: Su,X;Wallenstein,S;Bishop,D
• 通讯作者: Bishop,D