ANALYSIS OF ADENOVIRAL MEDIATED GENE TRANSFER TO PANCREATIC ISLETS BY VASCULAR PERFUSION OF THE INTACT PANCREAS PRIOR TO ISLET ISOLATION

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161 Genetic manipulation of islet tissue may correct β-cell dysfunction and improve islet allograft survival. However, gene transfer to 3-dimensional cellular complexes using co-culture results in transfection of cells at the periphery only. Since β cells reside at the core of the islet and islets have a rich microvascular network, we hypothesized that vascular perfusion of adenoviral vectors to intact pancreata prior to islet isolation might allow efficient gene transfer to all cells of the islet complex. In this study, we compared gene transfection rates using co-culture and vascular perfusion methodology, and examined islet function and transgene expression of transfected islets. Methods: Gene transfer was performed by either 1) co-culture (CC) where isolated rat islets were co-cultured with adLacZ virus at MOI of 1:1000 for 16hrs, or 2) vascular perfusion (VP) where intact Lewis rat pancreata were slowly perfused via the artery with cold 0.9% saline containing ad-LacZ (1010 pfu) and stored at 4°C for one hour prior to islet isolation. The transfection rate was assessed by staining islet tissue sections and trypsin-dispersed single islet cells with X-gal solution. Islet function post-transfection was evaluated in vitro by insulin release during static incubation and in vivo by transplantation of islets under the kidney capsule of STZ-diabetic nude mice or Lewis rats with or without immunosuppression (1.5ml anti-lymphocyte serum, ALS). Islet grafts were analyzed by immunohistological analysis of infiltrating cells and X-gal staining to determine LacZ gene expression. Results: Efficient gene transfer was demonstrated in 95-100% (n=6) of cells of the islet complex in VP-transfected islets versus 25-36% (n=7) in CC-transfected islets. Fully VP-transfected islets maintained normal insulin response in static incubation, similar to untreated control or CC-transfected islets. Transplantation of CC- or VP-transfected islets (300 islets) normalized blood glucose in STZ-diabetic nude mice (N=6/each). X-gal staining of transfected islet grafts in nude mice showed stable β-galactosidase expression for more than 100 days in both groups. When non-treated control or CC-transfected islets (1000 islets) were transplanted to diabetic syngeneic Lewis rats, hyperglycemia was reversed in all cases (n=6/each). However, transplantation of VP-transfected islets (1000 islets) did not reverse hyperglycemia (n=6/each). Immunohistologic examination of islet isografts showed a profound leukocyte infiltration in both CC- and VP-transfected islets, characterized by ED-1 positive macrophages at an early stage, followed later by CD4 or CD8 positive lymphocytes. At day 5 to 10 post-transplantation, VP-transfected islets were completely destroyed, while CC-transfected islet tissue survived with gradual loss of transgene expression. Transient immunosuppression (one dose of ALS) in Lewis rat recipients of VP-transfected islets prevented islet loss and normoglycemia was restored. Stable transgene expression for 100 days was achieved with VP-transfected islets in immunocompetent recipients who received ALS at the time of transplantation. Conclusions: Gene transfer to all cells of the pancreatic islet complex can be achieved by intra-arterial delivery of adenoviral vectors. Fully transfected islets maintain normal islet function in vitro and in vivo in immunodeficient animals, with stable long-term transgene expression. Virally infected islets evoke a profound inflammatory response which results in complete destruction of fully transfected islets in immunocompetent animals, but this detrimental effect can be overcome by transient immunosuppressive therapy.