Long-term amelioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes.
Ex vivo gene therapy, in which hepatocytes are harvested from mutants, retrovirally transduced with a normal gene and transplanted back into the donor, has been used for correction of inherited metabolic defects of liver. Major drawbacks of this method include limited availability of autologous hepatocytes, inefficient retroviral transduction of primary hepatocytes, and the limited number of hepatocytes that can be transplanted safely. To obviate these problems, we transduced primary hepatocytes derived from inbred bilirubin-UDP-glucuronosyl-transferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen (tsT). The immortalized cells were then transduced with a second recombinant retrovirus expressing human B-UGT, and a clone expressing high levels of the enzyme was expanded by culturing at permissive temperature (33 degrees C). At 37 degrees C, tsT antigen was degraded and the cells expressed UGT activity toward bilirubin at a level approximately twice that present in normal rat liver homogenates. For seeding the cells into the liver bed, 1 x 10(7) cells were injected into the spleens of syngeneic Gunn rats five times at 10-day intervals. Excretion of bilirubin glucuronides in bile was demonstrated by HPLC analysis and serum bilirubin levels were reduced by 27 to 52% in 40 days after the first transplantation and remained so throughout the duration of the study (120 days). None of the transplanted Gunn rats or SCID mice transplanted with the immortalized cells developed tumors.
Tada
K
K
Department of Medicine, Albert Einstein College of Medicine, Bronx, NY 10462, USA.
Roy-Chowdhury
N
N
Prasad
V
V
Kim
B H
BH
Manchikalapudi
P
P
Fox
I J
IJ
van Duijvendijk
P
P
Bosma
P J
PJ
Roy-Chowdhury
J
J
D000818:Animals; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D002472:Cell Transformation, Viral; D017690:Cell Transplantation; D002478:Cells, Cultured; D018014:Gene Transfer Techniques; D015316:Genetic Therapy; D014453:Glucuronosyltransferase / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics*; D006801:Humans; D008099:Liver / Q000473:pathology* / Q000502:physiology*; D016031:Liver Transplantation; D051379:Mice; D051381:Rats; D011911:Rats, Gunn; D013539:Simian virus 40
D000957:Antigens, Viral, Tumor; C019787:bilirubin glucuronoside glucuronosyltransferase; D014453:Glucuronosyltransferase