Bipartite Vector Encoding hVEGF and hIL-1Ra for ex Vivo Transduction into Human Islets

Abstract

Ex vivo gene transfer can improve the outcome of islet transplantation for treating type I diabetes. Earlier we have shown co-expression of human vascular endothelial growth factor (hVEGF) and human interleukin-1 receptor antagonist (hIL-1Ra) after transfection of plasmid DNA encoding these two genes. Due to poor transfection efficiency of plasmid DNA and the better known islet transduction efficiency of adenoviral (Adv) vectors, in this study, we constructed Adv-hVEGF-hIL-1Ra by cloning hVEGF and hIL-1Ra coding sequences and polyA signal under separate cytomegalovirus (CMV) promoters in Adenoquick plasmid (Ad 13.1). There was dose and time dependent expression of these genes after transduction of Adv-hVEGF-hIL1Ra into human islets. The mRNA expression of hVEGF and hIL-1Ra was more than 100 times higher than that of the non-transduced and bipartite plasmid transfected control islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Co-expression of hVEGF and hIL-1Ra by islets showed decrease in caspase-3 activity and apoptosis induced by a cocktail of inflammatory cytokines such as TNF-α, IL-1β and IFN-γ. Compared to non-treated or Adv-LacZ transduced islets, transduction of islets with Adv-hVEGF-hIL-1Ra prior to transplantation under the kidney capsules of diabetic NOD-SCID mice reduced the blood glucose levels, and increased serum insulin and c-peptide levels. Immunohistochemical staining of the islet bearing kidney sections at day 20 after transplantation was positive for human insulin, hVEGF and von Willebrand factor. These results indicate that the bipartite Adv vector efficiently expresses both growth factor and antiapoptotic genes, decreases apoptosis and improves the outcome of islet transplantation.

Beta Cell Regeneration: A Novel Strategy for Treating Type 1 Diabetes

“…Although these factors were also proven to be helpful in preserving the beta cell mass in T1D animals [11,12], they were absent from the intrinsic pathways governing the development and proliferation of pancreatic beta cells. Therefore, instead of working specifically on pancreatic beta cells, these factors seemed to work predominantly through combination with the native mechanism of beta cell regeneration in a synergistic or additive manner [12,13].…”

Gene Therapy and Stem Cell Therapy to Improve the Outcome of Human Islet Transplantation

“…Immunosuppressive regimens are capable of preventing immune rejection from months to years, but the agents used in these treatments may induce significant side effects, resulting in progressive decline in graft function. Some of the most commonly used immunosuppressive agents such as tacrolimus (also known as FK-506 or Fujimycin), mycophenolic acid (MPA), and sirolimus (also known as rapamycin) are also deleterious to islet function and insulin secretion [4,5]. PNF is characterized as the loss of islet viability and function caused by non-immune reactions, such as the disruption of islet microvasculature during islet isolation and purification process, hypoxia in the core of islet grafts and production of inflammatory cytokines at the transplantation sites.…”

“…For example, Giannoukakis et al first reported that adenoviral gene transfer of interleukin 1 receptor antagonist (IL-1Ra) prevented IL-1β-mediated nitric oxide production from human islets in vitro as well as the suppression of insulinproducing β cell function as determined by glucose-stimulated insulin production [3]. In our lab, Narang et al and Panakanti et al combined the gene expression of vascular endothelia growth factor (VEGF) and IL-1Ra to improve the therapeutic effects of single gene therapy while Li et al and Cheng et al targeted downstream pro-apoptotic factors of IL-1β pathway and used small heparin RNA to inhibit the expression of iNOS and caspase 3, respectively [4][5][6][7]. During the time we worked on the apoptosis of human islets, we identified a potent anti-apoptotic factor X-linked inhibitor of apoptosis (XIAP), which binds to and inhibits caspase 3, 7 and 9 at the same time.…”

“…We and others have demonstrated adenoviral vector-based gene therapy to be effective in promoting the revascularization and engraftment of human islets posttransplantation [12,15]. For example, growth factor gene expression can significantly improve the islet revascularization after transplantation [4,8,12], while anti-apoptotic protein expression can increase the resistance of islet grafts to multiple posttransplantation challenges in human islets [5,6,15]. The recent success in constructing bipartite viral vectors which can simultaneously promote revascularization and increase the resistance of transplanted islets may highlight the promising future of gene therapy [4,8].…”

“…For example, growth factor gene expression can significantly improve the islet revascularization after transplantation [4,8,12], while anti-apoptotic protein expression can increase the resistance of islet grafts to multiple posttransplantation challenges in human islets [5,6,15]. The recent success in constructing bipartite viral vectors which can simultaneously promote revascularization and increase the resistance of transplanted islets may highlight the promising future of gene therapy [4,8]. However, because of the cluster-like property of islet, multiplicity of infection (MOI) of higher than 500 is usually required to achieve optimal transduction efficiency as suggested from many reports [4,6,8,16].…”