Electroporation Mediated Gene Transfer of Human Interleukin-10 to Skeletal Muscle Reduces Acute Rejection in Rat Cardiac Allografts

CARDIAC GENE AND CELL THERAPY inexpensiveness, ease in handling, but the major disadvantage is that the transfection efficiency has been relatively poor. However, in the context of a secreted protein, this technical hurdle might be less prominent than when concerning a structural protein. We assessed the in vivo expression of naked plasmid DNA aiming at preservation of the function in a chronically failing heart. Plasmid IGF-I was delivered to the cardiac muscle by direct intracardiac injection. Multiple injections are needed in order to obtain sufficient in vivo modulation of the cardiac function. No severe side effects directly related to multiple injections were observed. Furthermore, high doses of plasmid DNA had to be injected locally, but no adverse reaction due to the amount of DNA was observed at the sites of injection. Protein expression was assessed by immunohistochemistry. IGF-1 treatment appeared to induce cardiac preservation in the cardiomyopathic hamster model, as cardiac muscle mass remained unchanged; we observed a lesser dilatation of the cardiac cavities and greater wall thickness. Moreover, vehicle-treated hamsters exhibited significant decrease in left ventricular functional parameters. At steady state, in plasmid-treated animals, cardiac output raised, mainly due to increased stroke volume, when heart rate remained unchanged. The left ventricular (LV) dP/dt(max) was enhanced by IGF-1, which reflected increased contractility, as the LV filling pressure decreased. The increasing degree of fibrosis correlated inversely with LV function but was unaffected by IGF-1. Enhancing cellular function in cardiomyopathic hamster hearts appears to have beneficial effects on global cardiac function. These studies show that plasmid IGF-I delivery may improve cardiac function and such a strategy might be used for otherwise untreatable patients. The aim is to preserve the ventricular contractile mass by stimulating the hypertrophic pathway and hereby to rescue cardiac function.

925. Electroporation Mediated Gene Transfer of Human Interleukin-10 to Skeletal Muscle Reduces Acute Rejection in Rat Cardiac Allografts Reza Tavakoli,2 Amiq Gazdhar,1 Jaroslaw Pierog,1 Anna Bagdannova,3 Mathias Gugger,4 Steven Hyde,5 Michele Genoni,2 Ralph Schmid.1 1 Division of General Thoracic Surgery, University Hospital, Bern, BE, Switzerland; 2Division of Cardiac Surgery, Trimeli Hospital, Zurich, ZH, Switzerland; 3Institute of Veterinary Physiology, University of Zurich, Zurich, ZH, Switzerland; 4Institute of Pathology, University of Bern, Bern, BE, Switzerland; 5Nuffield Department of Clinical Laboratory Science, John Radcliff Hospital, University of Oxford, Oxford, OX, United Kingdom. Background Electroporation-mediated transfer of plasmid DNA has been demonstrated to enhance gene transduction dramatically. The aim of this study was to investigate the impact of overexpression of human IL-10 (hIL-10) on acute rejection of cardiac allografts in the rat. Methods Heterotopic heart allotransplantation was performed in 12 animals (Brown Norway to Fischer F344) divided in two groups. Plasmid DNA (PCIKhIL10) was injected IM into the tibialis anterior muscle of the recipient followed by electroporation (4x10ms pulses at 200V/cm), 24h prior to the transplantation. In group A (n=6) 2.5 µg of pCIK hIL10 and in group B (n=6) distilled water was injected. Graft function was assessed by daily palpation. At day 7 animals were sacrificed and pripheral blood was drawn for ELISA measurement of hIL-10. Tissue samples were preserved for myeloperoxidase measurement and histology. Results In all animals in group A heart allografts were still beating at day 7 whereas they stopped beating at day 5±1 in group B (p<0.05). Serum hIL-10 levels were 60±31 pg/ml in group A vs 0 in group B. Myeloperoxidase activity was significantly lower in group S354

A than in group B (18±7 vs 32±14 mU/mg protein, p=0.05). Histological examination revealed severe rejection in all animals in group B in contrast to low moderate rejection in group A.Conclusions Electroporation mediated intramuscular gene transfer of hIL-10 reduces acute rejection and improves survival of heterotopic heart allografts in the rat. This was accompanied by a high serum expression of hIL-10 in treated animals and significant reduction of neutrophil infiltration and rejection grade of the graft. The application of electroporation mediated gene transfer to various fields of gene therapy to avoid the side effects related to the use of viral vectors warrants further investigation

926. Bcl-xL Gene Transfer Improves Graft Survival and Prolongs the Cold-Preservation Time in the Heart Transplantation Jinghua Huang,1 Yoshinori Ito,1,2 Takeshi Uzuka,1,3 Masayoshi Kobune,4 Yukiko Honma,2,3 Kiminori Nakamura,1 Hiroaki Uchida,5 Hironari Dehari,1,6 Hirofumi Hamada.1 1 Molecular Medicine, Sapporo Medical University, Sapporo, Japan; 2Gene Therapy, Sapporo Medical University, Sapporo, Japan; 3Thoracic and Cardiovascular Surgery, Sapporo Medical University, Sapporo, Japan; 44th Dept. of Internal Medicine, Sapporo Medical University, Sapporo, Japan; 5Surgery and Bioengineering, Institute of Medical Science, University of Tokyo, Tokyo, Japan; 6Oral Surgery, Sapporo Medical University, Sapporo, Japan. Background: Ischemia and reperfusion (I/R) injury is one of the important causes of early graft dysfunction and graft loss. Among factors that may be involved in the etiology of I/R injury, apoptosis is an early event after I/R injury of heart. Thus, inhibition of myocardial apoptosis might be a rational therapeutic strategy to reduce the preservation injury in the heart transplantants. We previously reported that Bcl-xL gene transfer can confer resistance against warm I/R injury and result in the improvement of cardiac function and the reduction of infarct size in rat heart. These evidences indicate that Bcl-xL would be also useful as a cardiac protective alternative in heart cold preservation. In this study, we employed adenoviral Bcl-xL gene transfer to the heart, and evaluated the cardiac protection of Bcl-xL in the heart transplantation after long cold preservation. Materials and Methods:Myocardial gene transfer was achieved by pre-administration of adenoviral vector (1010 particle units, pu). Adenoviral vector encoding human Bcl-xL (AxCAhBclxL) in 100 µl of saline was infused into the Lewis rat heart by coronary root injection. The aorta and pulmonary artery are clamped for 30 seconds after adenovirus injection and then released. Saline or adenoviral vector encoding LacZ (AxCAZ3) was used as control. Four days after gene-transduction, the heart was excised and perfused with cold UWS, and then stored in the UWS at 4ºC for 24 h. The heterotopic heart transplantation was performed for the evaluation of graft survival. Western blot analysis of Bcl-xL after 24 h of cold preservation and after 15 min of heart transplantation was performed. The histological analysis of heart after 24 h of transplantation was also evaluated. Results: The heart transduced with Bcl-xL gene revealed prolonged survival and 90% of the heart in the Bcl-xL group can survive for more than 2 weeks, while the majority of the control heart stopped beating immediately after heart transplantation (within 15 min). In the Bcl-xL transduced heart, robust Bcl-xL expression was observed before cold-preservation and this remarkable Bcl-xL expression was maintained after the warm reperfusion (after heart transplantation). On the other hand, Bcl-xL expression in the control heart was clearly reduced 15 min after heart transplantation. The histological analysis indicated obvious inflammatory cells infiltration Molecular Therapy Volume 9, Supplement 1, May 2004

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