Efficient Ex Vivo Transduction of Pancreatic Islet Cells With Recombinant Adeno-Associated Virus Vectors Terence Flotte, 1,2,4 Anupam Agarwal, 3 Jianming Wang, 4 Sihong Song, 4 Elizabeth S. Fenjves, 7 Luca Inverardi, 7 Kye Chesnut, 1,2 Sandra Afione, 8 Scott Loiler, 4 Clive Wasserfall, 5 Matthias Kapturczak, 3 Tamir Ellis, 5 Harry Nick, 6 and Mark Atkinson 5 The ability to transfer immunoregulatory, cytoprotec- tive, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of is- let allografts and inhibition of recurrent autoimmune destruction of these cells in type 1 diabetes. However, transient transgene expression and the tendency to induce host inflammatory responses have limited previ- ous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associat- ed virus (rAAV) serotype 2, a vector that can overcome these limitations, effectively transduces both human and murine pancreatic islet cells with reporter genes as well as potentially important immunoregulatory cyto- kine genes (interleukin-4, interleukin-10), although a very high multiplicity of infection (10,000 infectious units/islet equivalent) was required. This requirement was alleviated by switching to rAAV serotype 5, which efficiently transduced islets at a multiplicity of infec- tion of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed effi- ciently when they were transplanted under the renal capsule and allowed to survive in vivo. The rAAV- delivered transgenes did not interfere with islet cell insulin production and were expressed in both - and non–-cells. We believe rAAV will provide a useful tool to deliver therapeutic genes for modulating immune responses against islet cells and markedly enhance long- term graft survival. Diabetes 50:515–520, 2001 A ttempts to use islet cell transplantation for reversing type 1 diabetes have been docu- mented for more than two decades; however, the procedure has been largely unsuccessful (1,2). Concurrent mechanisms believed to underlie this lack of success include rejection, recurrence of anti–islet cell autoimmunity, and nonspecific islet loss because of perturbation of the graft microenvironment (e.g., inflam- mation, ischemia/reperfusion). A number of candidate gene products may prevent immune-mediated destruction and extend graft survival (e.g., interleukin [IL]-4, manganese superoxide dismutase, Bcl-2) (3). Furthermore, these genes may prove safer and more effective than systemic phar- macological immunosuppression because some agents are themselves potentially prodiabetogenic (e.g., cyclosporine, FK506, steroids) through imposition of increased meta- bolic demand. However, such studies have been limited by the lack of gene transfer vectors that are safe, efficient, and long lasting (4). Recombinant adeno-associated virus (rAAV) vectors have recently demonstrated some superiority to other viral and nonviral systems with regard to their in vivo safety, efficiency, and duration of action both in ani- mal models and in early persistent infections in humans without known pathology and with only modest immune responses (5–10). rAAV retains these beneficial properties and therefore has the potential to be an ideal vector for in vivo gene transfer. However, previous studies have failed to demonstrate rAAV transduction of islet cells (3). RESEARCH DESIGN AND METHODS Islet isolation. Pancreatic islet cells were isolated as previously described (11). Briefly, after intraductal injection of a solution containing Liberase (Boehringer-Mannheim Biochemicals, Indianapolis, IN), a whole human pan- creas was subjected to mechanical shaking, and aliquots of eluate were withdrawn at various points during a 2-h period. Purification of the final islet preparation was obtained by centrifugation on discontinuous Eurocollins- Ficoll gradients followed by hand picking. Mouse islets (C57Bl/6; Jackson Research Laboratories, Bar Harbor, ME) were obtained through intraductal injection of collagenase type XI solution (Sigma, St. Louis, MO), followed by purification through repeated washings and hand picking. Islets were main- tained in standard culture conditions (human-CMRL-1,066 with 5% normal human serum; mouse RPMI-1640 with 10% fetal bovine serum; 5% CO 2 , 24°C) until used (within 48 h). Islet purity was assessed by diphenylthiocarbazone staining, and viability was determined by staining with propidium iodide and fluorescein diacetate. Plasmid construction, viral packaging and production, and cellular transduction. The rAAV serotype 2 (rAAV2) vector plasmids used for these experiments are depicted diagrammatically (Fig. 3). Briefly, murine cDNAs for the cytokines IL-4 and IL-10 were cloned into the p43.2 (AAV2-ITR-containing- vector) plasmid between the XbaI site downstream from the cytomegalovirus (CMV) promoter and the XbaII site upstream from the simian virus 40 (SV40) polyadenylation signal. rAAV2 production was performed as previously described (12). The method involves cotransfection with two plasmids by calcium phosphate coprecipitation of a permissive human cell line (HEK293). HEK293 cells were grown as monolayers (initially seeded with 6  10 8 cells) in Dulbecco’s phosphate-buffered saline (PBS) containing 5% fetal bovine serum (37°C, 5% CO 2 ). After 18 h, the cells were transfected with different pairs of plasmids. The first nonrescuable helper plasmid (pDG) contained the rAAV2 comple- menting functions, rep and cap, as well as the Ad helper genes (E2a, VA RNA, From the 1 Genetics Institute, 2 the Powell Gene Therapy Center, and the Departments of 3 Medicine, 4 Pediatrics, 5 Pathology, and 6 Neuroscience, Uni- versity of Florida, Gainesville; the 7 Diabetes Research Institute, University of Miami, Miami, Florida; and the 8 Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland. Address correspondence and reprint requests to Dr. Mark Atkinson, Uni- versity of Florida, Department of Pathology, Box 100275 JHMHC, 1600 SW Archer Road, Gainesville, FL 32610. E-mail: atkinson@ufl.edu. Received for publication 23 June 2000 and accepted in revised form 1 November 2000. AAV, adeno-associated virus; Ad, adenovirus; CB, CMV enhancer/chicken  actin hybrid; CMV, cytomegalovirus; GFP, green fluorescent protein; IL, interleukin; ITR, inverted terminal repeat; i.u., infectious units; MOI, multi- plicity of infection; nlacZ, nuclear-targeted -galactosidase; PBS, phosphate- buffered saline; rAAV, recombinant AAV; rAAV2, rAAV serotype 2; rAAV5, rAAV serotype 5; RSV, Rous sarcoma virus; SV40, simian virus 40. DIABETES, VOL. 50, MARCH 2001 515 Downloaded from http://diabetesjournals.org/diabetes/article-pdf/50/3/515/366729/515.pdf by guest on 04 November 2022