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


METHODS
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.


RESULTS
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.


CONCLUSIONS
We achieved complete cure of hyperbilirubinemia in Gunn rats by ex vivo gene therapy via genetically modified and conditionally immortalized hepatocytes.
Kawashita
Yujo
Y
Department of Medicine, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, New York, NY 10461, USA.
Guha
Chandan
C
Moitra
Rituparna
R
Wang
Xia
X
Fox
Ira J
IJ
Roy-Chowdhury
Jayanta
J
Roy-Chowdhury
Namita
N
D000818:Animals; D001663:Bilirubin / Q000097:blood; D015153:Blotting, Western; D002455:Cell Division; D002461:Cell Line, Transformed; D003414:Crigler-Najjar Syndrome / Q000150:complications* / Q000235:genetics; D005260:Female; D015870:Gene Expression; D015316:Genetic Therapy / Q000379:methods*; D020719:Glucuronides / Q000378:metabolism; D014453:Glucuronosyltransferase / Q000235:genetics / Q000378:metabolism; D006085:Graft Survival; D006498:Hepatectomy; D022781:Hepatocytes / Q000166:cytology / Q000637:transplantation*; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology / Q000378:metabolism; D008107:Liver Diseases / Q000209:etiology* / Q000473:pathology / Q000628:therapy*; D008111:Liver Function Tests; D008297:Male; D051381:Rats; D011911:Rats, Gunn; D013696:Temperature; D014161:Transduction, Genetic; D019737:Transplants; D000099148:UGT1A1 Enzyme
D001663:Bilirubin; D020719:Glucuronides; D014453:Glucuronosyltransferase; C499946:Ugt1a1 protein, rat; D000099148:UGT1A1 Enzyme