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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026824-19
Application #
6757291
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Badman, David G
Project Start
1980-04-01
Project End
2008-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
19
Fiscal Year
2004
Total Cost
$363,366
Indirect Cost
Name
Mount Sinai School of Medicine
Department
Genetics
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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Clavero, Sonia; Ahuja, Yuri; Bishop, David F et al. (2013) Diagnosis of feline acute intermittent porphyria presenting with erythrodontia requires molecular analyses. Vet J 198:720-2
Balwani, Manisha; Desnick, Robert J (2012) The porphyrias: advances in diagnosis and treatment. Hematology Am Soc Hematol Educ Program 2012:19-27
Balwani, Manisha; Desnick, Robert J (2012) The porphyrias: advances in diagnosis and treatment. Blood 120:4496-504
Hasanoglu, Alev; Balwani, Manisha; Kasapkara, Ci?dem S et al. (2011) Harderoporphyria due to homozygosity for coproporphyrinogen oxidase missense mutation H327R. J Inherit Metab Dis 34:225-31
Machaczka, Maciej; Klimkowska, Monika; Regenthal, Sofie et al. (2011) Gaucher disease with foamy transformed macrophages and erythrophagocytic activity. J Inherit Metab Dis 34:233-5
Clavero, Sonia; Bishop, David F; Giger, Urs et al. (2010) Feline congenital erythropoietic porphyria: two homozygous UROS missense mutations cause the enzyme deficiency and porphyrin accumulation. Mol Med 16:381-8
Cantatore-Francis, Julie L; Cohen-Pfeffer, Jessica; Balwani, Manisha et al. (2010) Hepatoerythropoietic porphyria misdiagnosed as child abuse: cutaneous, arthritic, and hematologic manifestations in siblings with a novel UROD mutation. Arch Dermatol 146:529-33
Yasuda, Makiko; Bishop, David F; Fowkes, Mary et al. (2010) AAV8-mediated gene therapy prevents induced biochemical attacks of acute intermittent porphyria and improves neuromotor function. Mol Ther 18:17-22
Bishop, David F; Schneider-Yin, Xiaoye; Clavero, Sonia et al. (2010) Congenital erythropoietic porphyria: a novel uroporphyrinogen III synthase branchpoint mutation reveals underlying wild-type alternatively spliced transcripts. Blood 115:1062-9

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