Deficiency of fumarylacetoacetate hydrolase (FAH) causes tyrosinemia type I (hepatorenal tyrosinemia, HT1) in humans and is characterized by severe liver dysfunction, renal tubular damage and liver cancer. In the murine model of HT1 hepatocytes stably expressing Fah have a strong selective growth advantage. Fah mutant mice are therefore uniquely suited for the study of the biology of integrating gene therapy vectors in the liver. Recently, we have found that in vivo selection of Fah+ cells also occurs in the renal proximal tubular epithelium. Therefore, HT1 mice are well suited to study hepatic and renal gene therapy with integrating vectors. In the past funding period, we were able to demonstrate gene repair in hepatocytes and renal proximal using AAV2/2 and AAV2/8 vectors harboring 4 kb of genomic homology to an Fah point mutation mouse. In addition, we developed a novel AAV vector designed to integrate cDNA expression cassettes into the ribosomal DNA rDNA) locus by homologous recombination. We found site-specific integration in vivo in multiple tissues including liver, kidney, heart, lung and skeletal muscle. We also succeeded in the synthesis of a potent small molecule inhibitor of Fah (CEHPOBA), which works in vivo. Because of the powerful selection found in genetic Fah deficiency, pharmacological inhibition of Fah may be a general strategy for the in vivo selection of genetically modified hepatocytes. We have demonstrated that hepatocytes deficient for homogentisic acid dioxygenase (Hgd) could be positively selected in vivo after transplantation into CEHPOBA treated mice. The overall theme of this application is the utilization of the tyrosine catabolic pathway for in vivo metabolic selection of genetically modified epithelial cells in the liver and kidney. Specifically , we will use the model to further develop AAV-based vectors for in vivo gene repair and targeted site-specific integration. We will also explore the use of CEHPOBA for in vivo selection of transgene modified hepatocytes. Public Health Relevance: This research investigates novel strategies of general significance for gene therapy of many genetic hepatic and renal disorders. Improved AAV vectors for gene repair as well as site-specific chromosomal integration will be generated. In addition, a broadly applicable platform for pharmacological growth selection of genetically modified hepatocytes is being developed.

Public Health Relevance

This research investigates novel strategies of general significance for gene therapy of many genetic hepatic and renal disorders. Improved AAV vectors for gene repair as well as site-specific chromosomal integration will be generated. In addition, a broadly applicable platform for pharmacological growth selection of genetically modified hepatocytes is being developed.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK048252-19
Application #
8207994
Study Section
Gene and Drug Delivery Systems Study Section (GDD)
Program Officer
Mckeon, Catherine T
Project Start
1993-12-01
Project End
2013-12-31
Budget Start
2012-01-01
Budget End
2013-12-31
Support Year
19
Fiscal Year
2012
Total Cost
$327,348
Indirect Cost
$106,114
Name
Oregon Health and Science University
Department
None
Type
Organized Research Units
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
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Dorrell, Craig; Tarlow, Branden; Wang, Yuhan et al. (2014) The organoid-initiating cells in mouse pancreas and liver are phenotypically and functionally similar. Stem Cell Res 13:275-83
Yin, Hao; Xue, Wen; Chen, Sidi et al. (2014) Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol 32:551-3
Hickey, Raymond D; Mao, Shennen A; Glorioso, Jaime et al. (2014) Fumarylacetoacetate hydrolase deficient pigs are a novel large animal model of metabolic liver disease. Stem Cell Res 13:144-53
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Hickey, Raymond D; Lillegard, Joseph B; Fisher, James E et al. (2011) Efficient production of Fah-null heterozygote pigs by chimeric adeno-associated virus-mediated gene knockout and somatic cell nuclear transfer. Hepatology 54:1351-9
Paulk, Nicole K; Wursthorn, Karsten; Wang, Zhongya et al. (2010) Adeno-associated virus gene repair corrects a mouse model of hereditary tyrosinemia in vivo. Hepatology 51:1200-8
Willenbring, Holger; Sharma, Amar Deep; Vogel, Arndt et al. (2008) Loss of p21 permits carcinogenesis from chronically damaged liver and kidney epithelial cells despite unchecked apoptosis. Cancer Cell 14:59-67
Azuma, Hisaya; Paulk, Nicole; Ranade, Aarati et al. (2007) Robust expansion of human hepatocytes in Fah-/-/Rag2-/-/Il2rg-/- mice. Nat Biotechnol 25:903-10
Held, Patrice K; Olivares, Eric C; Aguilar, Christina P et al. (2005) In vivo correction of murine hereditary tyrosinemia type I by phiC31 integrase-mediated gene delivery. Mol Ther 11:399-408

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