Transplantation of Gunn Rats with Autologous Fibroblasts Expressing Bilirubin UDP-Glucuronosyltransferase: Correction of Genetic Deficiency and Tumor Formation

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Abstract
The end product of the breakdown of the heme group of hemoglobin and other heme-containing proteins is bilirubin. Bilirubin is hydrophobic and cannot be excreted as such. Therefore, mammals have a liver enzyme bilirubin UDP-glucuronosyltransferase (B-UGT), which conjugates bilirubin with glucuronic acid, thereby making the molecule much more water soluble. Bilirubin glucuronides are secreted into bile. Patients with Crigler-Najjar (CN) disease have a deficiency in bilirubin UDP-glucuronosyltransferase and accumulate high serum levels of bilirubin. An animal model for CN disease is the Gunn rat. The obvious target for gene therapy for CN disease is the liver, but because liver cells do only divide infrequently, they are difficult to transduce. To investigate whether cells that are easily transduced can be used to develop gene therapy for CN disease, we have transduced Gunn rat fibroblasts with B-UGT, using a recombinant retrovirus. Gunn rat fibroblasts expressing B-UGT were able to glucuronidate bilirubin present in cell culture media. In this study, we describe the intraperitoneal transplantation of Gunn rats with Gunn rat fibroblasts expressing B-UGT. Transplantation of the fibroblasts corrected the genetic deficiency of the Gunn rats, serum bilirubin concentrations of the transplanted Gunn rats were reduced to normal, and bilirubin glucuronides appeared in bile. However, due to the prolonged period of cell culture, the transplanted fibroblasts were transformed, and the experimental animals developed tumors after transplantation. Gunn rats and patients with Crigler-Najjar (CN) disease both have a defect in the hepatic enzyme, bilirubin UDP-glucuronosyltransferase (B-UGT). The result of this defect is high serum concentrations of the toxic endogenous compound unconjugated bilirubin. We have expressed B-UGT in Gunn rat fibroblasts; these fibroblasts took up and glucuronidated bilirubin, and excreted the conjugated bilirubin back into the culture media. When Gunn rat fibroblasts expressing B-UGT were transplanted into Gunn rats, serum bilirubin levels dropped to normal values. Transplantation of untransduced control fibroblasts did not change serum bilirubin concentrations. Bilirubin conjugates were detected in the bile of Gunn rats that were transplanted with transduced Gunn rat fibroblasts expressing B-UGT. Gunn rats transplanted with control and transduced fibroblasts developed tumors; therefore, a high degree of caution must be exercised when considering gene therapy with ex vivo-transduced autologous fibroblasts.
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