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

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–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°C). At 37°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 × 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.

Introduction

Patients with Crigler-Najjar syndrome type 1 (CN-I) and homozygous Gunn rats lack hepatic bilirubin conjugating activity because of mutations in the UGT1A1 gene, which encodes the UDP–glucuronosyl–transferase isoform that catalyzes the glucuronidation of bilirubin (BUGT) (2). Because glucuronidation is essential for efficient biliary excretion of bilirubin, CN-I patients and Gunn rats do not excrete bilirubin glucuronides in bile and accumulate unconjugated bilirubin in plasma, resulting in neurological damage (20). Currently, liver transplantation is the only definitive treatment available for Crigler-Najjar syndrome type 1. In this study, we used Gunn rats as an animal model for developing an efficient ex vivo gene therapy method for inherited metabolic liver diseases.

In the current ex vivo gene therapy methods for metabolic liver diseases developed in our laboratory 18, 24 and elsewhere (8), a segment of the liver is surgically removed from the mutant, primary hepatocytes are isolated and cultured, the cells are transduced with a retrovirus capable of transferring a corrective gene, and finally, the cells are autologously transplanted into the donor. However, the efficiency of the currently used technique is limited by the number of hepatocytes that can be harvested, inefficient transduction by retroviral vectors because of the low mitosis rate of primary hepatocytes, and difficulty of transplanting a large number of cells in a single session.

To address these major problems associated with ex vivo liver-directed gene therapy, we conditionally immortalized the hepatocytes before transduction with the human BUGT gene. The conditional immortalization was performed by infecting primary hepatocytes isolated from a resected liver lobe of a Gunn rat with a recombinant retrovirus capable of transferring the gene for a thermolabile mutant SV-40 large T antigen (^tsT) (16). At a permissive temperature (33°C), the transduced cells express the ^tsT antigen, which results in their proliferation. At 37–39°C, thermolabile T antigen is degraded, and the cells stop growing and exhibit differentiated function. In a second step, a human BUGT gene was introduced using another recombinant retrovirus, while the cells are rapidly dividing at 33°C, allowing highly efficient transduction. The transduced cells were expanded in culture and repeatedly transplanted into Gunn rats by intrasplenic injections. Long-term engraftment and function of the transplanted cells was shown by the excretion of bilirubin glucuronides in bile and persistent reduction of serum bilirubin levels. Thus, we describe a new strategy for improving the efficiency of ex vivo liver-directed gene therapy for metabolic diseases.