Long-term amerlioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes

Kouji Tada, Namita Roy-Chowdhury, Vinayaka R. Prasad, Byung Ho Kim, P. Manchikalapudi, Ira J. Fox, Peter Van Duijvendijk, Piter J. Bosma, Jayanta Roy-Chowdhury

Research output: Contribution to journalArticle

49 Citations (Scopus)

Abstract

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-glucuronosyltransferase (BUGT)-deficient Gunn rats by infection with a recombinant retrovirus expressing temperature-sensitive mutant SV40 large T antigen ((ts)T). 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 (33Γ). At 37°C, (ts)T 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 107 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.

Original languageEnglish (US)
Pages (from-to)607-616
Number of pages10
JournalCell Transplantation
Volume7
Issue number6
DOIs
StatePublished - Nov 1998

Fingerprint

Gunn Rats
Bilirubin
Rats
Hepatocytes
Defects
Liver
bilirubin glucuronoside glucuronosyltransferase
Viral Tumor Antigens
Retroviridae
Antigens
Polyomavirus Transforming Antigens
Gene therapy
Temperature
SCID Mice
Bile
Genetic Therapy
Tumors
Spleen
Enzymes
Clone Cells

Keywords

  • Bilirubin
  • Bilirubin-UDP-glucuronosyltransferase
  • Ex vivo gene therapy
  • Gunn rats
  • Immortalized hepatocytes
  • Retrovirus

ASJC Scopus subject areas

  • Cell Biology
  • Transplantation

Cite this

Long-term amerlioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes. / Tada, Kouji; Roy-Chowdhury, Namita; Prasad, Vinayaka R.; Kim, Byung Ho; Manchikalapudi, P.; Fox, Ira J.; Van Duijvendijk, Peter; Bosma, Piter J.; Roy-Chowdhury, Jayanta.

In: Cell Transplantation, Vol. 7, No. 6, 11.1998, p. 607-616.

Research output: Contribution to journalArticle

Tada, Kouji ; Roy-Chowdhury, Namita ; Prasad, Vinayaka R. ; Kim, Byung Ho ; Manchikalapudi, P. ; Fox, Ira J. ; Van Duijvendijk, Peter ; Bosma, Piter J. ; Roy-Chowdhury, Jayanta. / Long-term amerlioration of bilirubin glucuronidation defect in Gunn rats by transplanting genetically modified immortalized autologous hepatocytes. In: Cell Transplantation. 1998 ; Vol. 7, No. 6. pp. 607-616.
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