ADENOVIRUS-MEDIATED GENE-TRANSFER IN THE TRANSPLANT SETTING .2. SUCCESSFUL EXPRESSION OF TRANSFERRED CDNA IN SYNGENEIC LIVER GRAFTS

These experiments establish a model for gene transfer to transplanted liver grafts ex vivo using adenoviral vectors. Rat liver grafts (n=8) were harvested, and preserved in UW or lactated Ringer's. The grafts w ere infected ex-vivo via portal vein perfusion with replication-defect ive Ad vectors encoding the beta-galactosidase (beta-gal) gene diluted in UW solution. The infected grafts were stored at 4 degrees C for 1 hr, then transplanted into syngeneic hosts. Liver biopsies were taken at 1, 7, and 15 days after transplantation. Infection rate was assesse d by histochemical staining for beta-gal. Liver DNA and RNA were assay ed for the beta-gal sequences, and recombinant protein production meas ured at 24 hr and 7 days after transplantation. Under conditions mimic king Liver graft cold storage, efficient gene transfer was achieved wi th an infection rate of 10-15%, as assessed by X-gal staining. Viral D NA and RNA presence in the graft was confirmed; gene expression with p rotein production were verified by western blots and a functional prot ein assay. Ah studies were negative in control Livers. Gene expression persisted for at least 2 weeks after transplantation. We conclude tha t efficient adenovirus-mediated gene insertion and expression of gene products can be accomplished in whole-liver grafts under hypothermic p reservation conditions currently used in clinical transplantation.