Caspase-3 Gene Silencing for Inhibiting Apoptosis in Insulinoma Cells and Human Islets

Abstract

Abstract:Although islet transplantation has great potential to treat type I diabetes, most islet grafts do not function due to the host immune rejection, nonspecific inflammatory response and poor revascularization. Since caspase-3 plays a crucial role in apoptosis of transplanted islet cells, we used chemically synthesized small interfering RNAs (siRNAs) to silence caspase-3 in insulinoma (INS-1E) cells and human islets, and then determined whether caspase-3 gene silencing can prevent these cells from cytokine-induced apoptosis. Transfection of INS-1E cells and islets with siRNAs reduced caspase-3 transcripts by 50-67% and 50%, respectively. Additionally, apoptosis in transfected insulinoma cells was markedly inhibited. Since gene silencing did not last beyond two days, we converted potent siRNA into shRNA and constructed replication deficient adenoviral (Adv) vectors encoding these shRNAs driven by a U6 or H1 promoter. Compared to chemically synthesized siRNA, Adv-caspase-3-shRNA efficiently transduced islets, showed relatively higher and prolonged levels of gene silencing beyond five days, with higher gene silencing with a U6 promoter, and protected islets from cytokine-induced apoptosis. Finally, return to normoglycemia was achieved at 1 day post-transplantation of Advcaspase-3-shRNA transduced islets under the kidney capsules of streptozotocin induced nonobese diabetic-severe combined immunodeficiency (NOD-SCID) mice and maintained beyond two weeks. Blood glucose levels returned to g325 mg/dL upon removal of the islet graft-bearing kidney at 32 days after transplantation, confirming that transplanted islets were functional.

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

“…Two of the most important limitations to the widespread of human islet transplantation are the limited supply of islets for transplantation and the inadequate means for preventing islet loss after transplantation [2]. Moreover, because of the extensive posttransplantation challenges, a patient needs at least 10,000 islet equivalents (IEs) per kilogram of body weight (extracted from two or more donor pancreases) for an optimal transplantation outcome, making the current shortage in islet supply even worse [3,6]. The major reasons for islet graft failure and useful interventions are listed in Table 1-1.…”

“…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].…”

“…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]. Therefore, despite its effectiveness, the clinical application of gene therapy is still hindered by the high risk of immunogenicity of viral vectors.…”

“…Although cytomegalovirus (CMV) promoter drives considerable transgene expression in human islets, the discoveries of new isletspecific promotes and enhancers boost the expression of therapeutic proteins significantly. For example, we previously reported that U6 promoter is transcriptionally more effective than H1 promoter in human islets [6]. Sharma et al demonstrated that transgene expression can be drastically increased by incorporating an islets-specific enhance into the construct [18].…”