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Efficient Ex Vivo Transduction of Pancreatic Islet Cells With Recombinant Adeno-Associated Virus Vectors

¹ Genetics Institute
² Powell Gene Therapy Center, and the
³ Departments of Medicine
⁴ Pediatrics
⁵ Pathology, and
⁶ Neuroscience, University of Florida, Gainesville
⁷ Diabetes Research Institute, University of Miami, Miami, Florida
⁸ Molecular Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, Maryland

The ability to transfer immunoregulatory, cytoprotective, or antiapoptotic genes into pancreatic islet cells may allow enhanced posttransplantation survival of islet 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 previous gene delivery studies using viral transfer vectors. We demonstrate here that recombinant adeno-associated 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 cytokine 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 infection of 100. Although adenovirus (Ad) coinfection was required for efficient ex vivo expression at early time points, islets transduced without Ad expressed efficiently 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 ß- and non–ß-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.

Abbreviations: AAV, adeno-associated virus; Ad, adenovirus; CB, CMV enhancer/chicken ß actin hybrid; CMV, cytomegalovirus; GFP, green fluorescent protein; IL, interleukin; ITR, inverted terminal repeat; i.u., infectious units; MOI, multiplicity of infection; nlacZ, nuclear-targeted ß-galactosidase; PBS, phosphate-buffered saline; rAAV, recombinant AAV; rAAV2, rAAV serotype 2; rAAV5, rAAV serotype 5; RSV, Rous sarcoma virus; SV40, simian virus 40

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