Annual Meeting of the European Society of Pediatric Gastroenterology and Nutrition
Most of the congenital errors of liver metabolism affect one single pathway and spare the general function and structure of the liver. Liver transplantation is a possible treatment, but the number of donor organs is limited and this therapy has a considerable rate of mortality. Gene therapy might be an alternative for the treatment of many inborn errors of liver metabolism. We study gene therapy for Crigler-Najjar disease type I. This disease is due to mutations in the UDP dependent glucuronyl transferase (B-UDPGT) in the liver. Plasmid based vectors were made and tested using a rat model (Gunn-rat) of the disease.
For the gene transfer into primary rat hepatocytes the cationic liposome DOTAP™ (Boehringer Mannheim) and the synthetic peptide P2lip/HA (M Wilke et al submitted) were tested in transient transfection experiments. Efficient transfection of primary Gunn rat hepatocytes (20-30% LacZ +, > 106 U/mg luciferase) could be achieved under optimal conditions. The transfection efficiencies depended on the presence of mitogens in the culture medium with both transfection systems. This shows that synthetic peptides can be used for the development of gene transfer protocols into hepatocytes. For the complementation experiments in the Gunn-rat model we cloned three different expression vectors. We tested constructs with and without intron sequences as well as a liver-specific vector. After the transfection of Gunn rat hepatocytes complementation of the bilirubin conjugation defect could be shown by HPLC analysis. In these experiments the vector containing the intron showed the highest level of expression. The success of the in vitro experiments enables us to investigate the possibilities of in vivo gene transfer.
In vivo gene transfer to the liver with plasmid vector depends on an efficient route of delivery, the targeting of the DNA/transfection complexes to parenchymal cells. Pilot experiments with fluorescent microspheres in the rat showed, that both retrograde perfusion of the bile duct and isolated perfusion through the portal vein are suitable routes of delivery. The advantage of peptide derived transfection compounds is, that they can be modified, e.g. by adding receptor ligands such as galactose. This ensures liver-specific gene expression in in vivo experiments.