Article

Hepatic repopulation with stably transduced conditionally immortalized hepatocytes in the Gunn rat.

Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA.
Journal of Hepatology (impact factor: 9.26). 08/2008; 49(1):99-106. DOI:10.1016/j.jhep.2008.02.020 pp.99-106
Source: PubMed

ABSTRACT Conditionally immortalized hepatocytes offer a renewable source of hepatocytes, but although preparative maneuvers have been developed for hepatic repopulation with primary hepatocytes, extensive proliferation of transplanted immortalized hepatocytes has not been accomplished heretofore. Our aim was to achieve ex vivo gene therapy of uridinediphosphoglucuronate glucuronosyltransferase-1A1 (UGT1A1)-deficient jaundiced Gunn rats (model of Crigler-Najjar syndrome type-1) by hepatic repopulation with genetically modified and conditionally immortalized hepatocytes.
Gunn rat hepatocytes were conditionally immortalized by stable transduction with a thermolabile mutant simian virus 40 T-antigen ((ts)Tag(A58)) and further transduced with UGT1A1. These hepatocytes proliferate at 33 degrees C, but at 37 degrees C the (ts)Tag(A58) is degraded and the cells become quiescent. The cells were transplanted into Gunn rat livers after preparative hepatic irradiation (50 Gy) and 66% hepatectomy.
The engrafted UGT1A1-positive immortalized hepatocytes replaced approximately 80% of the host hepatocytes in 20 weeks, leading to normalization of hyperbilirubinemia. Liver histology, and serum albumin and alanine aminotransferase levels remained normal.
We achieved complete cure of hyperbilirubinemia in Gunn rats by ex vivo gene therapy via genetically modified and conditionally immortalized hepatocytes.

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    Article: Long-term reduction of jaundice in Gunn rats by nonviral liver-targeted delivery of Sleeping Beauty transposon.
    Xia Wang, Debi P Sarkar, Prashant Mani, Clifford J Steer, Yong Chen, Chandan Guha, Voshavar Chandrasekhar, Arabinda Chaudhuri, Namita Roy-Chowdhury, Betsy T Kren, Jayanta Roy-Chowdhury
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    ABSTRACT: Asialoglycoprotein receptor (ASGPR)-mediated endocytosis has been used to target genes to hepatocytes in vivo. However, the level and duration of transgene expression have been low because of lysosomal translocation and degradation of the DNA and lack of its integration into the host genome. In this study we packaged the DNA of interest in proteoliposomes containing the fusogenic galactose-terminated F-glycoprotein of the Sendai virus (FPL) for targeted delivery to hepatocytes. After the FPL binds to ASGPR on the hepatocyte surface, fusogenic activity of the F-protein delivers the DNA into the cytosol, bypassing the endosomal pathway. For transgene integration we designed plasmids containing one transcription unit expressing the Sleeping Beauty transposase (SB) and another expressing human uridinediphosphoglucuronate glucuronosyltransferase-1A1 (pSB-hUGT1A1). The latter was flanked by inverted/direct repeats that are substrates of SB. In cell culture, FPL-mediated delivery of the E. coli beta-galactosidase gene (LacZ) resulted in transduction of ASGPR-positive cells (rat hepatocytes or Hepa1 cell line), but not of ASGPR-negative 293 cells. Intravenous injection of the FPL-entrapped pSB-hUGT1A1 (4-8 microg/day, 1-4 doses) into UGT1A1-deficient hyperbilirubinemic Gunn rats (model of Crigler-Najjar syndrome type 1) resulted in hUGT1A1 expression in 5%-10% of hepatocytes, but not in other cell types. Serum bilirubin levels declined by 30% +/- 4% in 2 weeks and remained at that level throughout the 7-month study duration. With histidine containing FPL, serum bilirubin was reduced by 40% +/- 5%, and bilirubin glucuronides were excreted into bile. No antibodies were detectable in the recipient rats against the F-protein or human UGT1A1. Conclusion: FPL is an efficient hepatocyte-targeted gene delivery platform in vivo that warrants further exploration toward clinical application.
    Hepatology 06/2009; 50(3):815-24. · 11.66 Impact Factor
Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.

Keywords

alanine aminotransferase levels
 
conditionally immortalized hepatocytes
 
Crigler-Najjar syndrome type-1
 
engrafted UGT1A1-positive immortalized hepatocytes
 
ex vivo gene therapy
 
extensive proliferation
 
Gunn rat hepatocytes
 
Gunn rat livers
 
Gunn rats
 
host hepatocytes
 
hyperbilirubinemia
 
immortalized hepatocytes
 
normalization
 
preparative hepatic irradiation
 
preparative maneuvers
 
primary hepatocytes
 
renewable source
 
serum albumin
 
UGT1A1)-deficient jaundiced Gunn rats
 
uridinediphosphoglucuronate glucuronosyltransferase-1A1