Prolongation of Cardiac Allograft Survival With Intracoronary Viral Interleukin-10 Gene Transfer C.K. Wang, X.J. Zuo, D. Carpenter, S. Jordan, E. Nicolaidou, M. Toyoda, L.S.C. Czer, H. Wang, and A. Trento E X VIVO gene transfer has the potential to deliver biologically active molecules to either the graft itself or defined sites in the recipient, leading to the expression of molecules capable of inhibiting immune response, resulting in local modulation of immunity after grafting. 1 The repli- cation-deficient adenovirus (Adv) has increasingly been used as a vehicle for gene delivery due to its high level of transfectability to target cells, regardless of the cell cycle, and also due to its high viral titers. Recent studies have shown that IL-10 inhibits monocyte production of IL-12, an important cytokine for Th1 cell differentiation and cell- mediated immunity. 3,4 Viral IL-10 (vIL-10), a product encoded by Epstein–Barr virus, is structurally homologous to murine and human IL-10, and shares many of their biological properties. 6 In this study, we evaluated the use of adenoviral vector encoding vIL-10 in a major histoincom- patible rat heterotopic cardiac transplant model to examine the effect of transfected vIL-10 in prolongation of allograft survival. MATERIALS AND METHODS Animals Inbred male ACI (RT-1 a ) rats (200 to 250 g) and Lewis rats (RT-1 1 ) (200 to 250 g) were used as recipient and donor, respectively. Viral Vectors Replication-deficient recombinant adenovirus-encoded -galacto- sidase (Adv--gal) and viral IL-10 (Adv-vIL-10) were produced, respectively. Viral titers were determined by plaque assays on monolayer 293 cells and expressed as plaque-forming units per milliliter (pfu/mL). The ability of the vector to express the encoded transgene was confirmed by measurement of cultured supernatant 24 hours after infection for the encoded protein. Intracoronary Gene Transfer The donor aorta was cannulated ex vivo. Viral vector (0.3 mL) contained 2  10 9 pfu/mL titer that was administered by slow infusion via cannula, and the heart remained preserved in cold Euro-Collins solution for 1 hour prior transplantation. Heterotopic Cardiac Transplantation Heterotopic cardiac transplantation was performed according to the method described previously. 5 The experimental groups were designed as follows: group A: control; group B: Ad--gal; group C: Adv-vIL-10; group D: CsA; group E: Adv-vIL-10 combined with CsA. Allograft survival was determined daily palpation. X-Gal Staining and Histologic Analysis The graft was then removed from the recipient animal and the blood was flushed out cold with heparinised saline and the solution was infused into the graft fixed in 0.2% glutaraldehyde with 2% formaldehyde in PBS for 30 minutes. For histologic staining, the grafts were quick frozen embedded in OCT, then sectioned at 10 m, fixed at room temperature in 0.2% glutaraldehyde in PBS solution for 20 to 30 minutes, rinsed three times with PBS, and incubated at 37°C overnight in X-gal solution (provided by Boehr- inger Mannheim). Detection of vIL-10 Gene Expression Grafts were removed 7 days after transplantation, RNA was isolated, and mRNA expression using RT-PCR was assessed for detection of expression of vIL-10 gene in the graft. RESULTS Gene Expression in Cardiac Grafts Syngeneic and allgeneic grafts transfected with Adv--gal showed expression of -galactosidase activity when assayed at 7 days posttransplantation in allografts and at 7 and 21 days posttransplantation in syngeneic grafts. Positive stain- ing of -galactosidase was seen in transplanted cardiac myocytes. vIL-10 gene expression detected in two grafts showed clear mRNA expression, which reconfirmed that mRNA expressed was from transfected DNA by PCR (data not shown). Adv-vIL-10 Gene Transfer Prolongs Cardiac Allograft Survival Allografts transfected with Adv-vIL-10 at the time of trans- plantation demonstrated a significant prolongation of graft survival at 19.6  0.65 days compared with 10.5  0.50 days From the Cardiothoracic Surgical Research Lab, Division of Cardiothoracic Surgery, Cedars Sinai Medical Center, Los An- geles, California, USA. Address reprint requests to Dr Charles Wang, Davis Research 6086, 8700 Beverly Boulevard, Los Angeles, CA 90048. © 1999 by Elsevier Science Inc. 0041-1345/99/$–see front matter 655 Avenue of the Americas, New York, NY 10010 PII S0041-1345(98)01851-X Transplantation Proceedings, 31, 951–952 (1999) 951