Growth Factor and Antiapoptotic Gene Delivery to Human Islets

描述（由申请人提供）：人胰岛移植作为治疗胰岛素依赖型糖尿病的有效手段具有巨大潜力。原发性无功能是胰岛移植失败的主要原因，它导致需要多供体移植。肝细胞生长因子（HGF）可促进胰岛细胞增殖，促进胰岛血管再生，而白细胞介素1受体拮抗剂（hIL-1 Ra）可抑制胰岛细胞凋亡。Caspase-3是不同凋亡途径的汇聚点，因此其沉默保护胰岛免于凋亡。我们将检验这样的假设，即用编码hHGF和hIL-1 Ra cDNA或hHGF cDNA和caspase-3-shRNA的复制缺陷型腺病毒（Adv）载体离体转导人胰岛改善胰岛移植治疗1型糖尿病的结果。在初步研究中，我们通过将hVEGF或hHGF和hIL-1 Ra编码序列和polyA信号克隆到Adenoquick质粒中的单独CMV启动子下，构建了Adv-hHGF-hIL-1 Ra。Adv-hHGF-hIL-1 Ra转染人胰岛后，这些基因的表达呈剂量和时间依赖性。转导的胰岛是活的，如葡萄糖激发后胰岛素释放所证明的。共表达hHGF和hIL-1 Ra的胰岛细胞表现出caspase-3活性降低和细胞凋亡诱导的炎性细胞因子鸡尾酒。与未处理的胰岛相比，在移植到NOD-SCID小鼠的肾包膜下之前用Adv-hHGF-hIL-1 Ra转导胰岛降低血糖并增加葡萄糖激发后的血清胰岛素水平和c肽水平。转染Adv-caspase-3-shRNA的胰岛细胞也能抑制细胞因子诱导的胰岛细胞凋亡，提高胰岛移植的成功率。这些结果表明，二分腺病毒载体有效地表达生长因子和抗凋亡基因，减少细胞凋亡，改善胰岛移植的结果。我们的具体目的是确定i）用Adv-hHGF-hIL-1 Ra进行的离体转导是否能预防移植后原发性胰岛无功能和早期移植物排斥，ii）？i）Adv-hHGF-hIL-1 Ra将改善门静脉内移植后的胰岛功能; ii）在移植后保护胰岛移植物方面，用Adv-hHGF-胱天蛋白酶-3-shRNA的离体转导上级于Adv-hHGF-hIL-1 Ra;和iii）用Adv-hHGF-hIL-1 Ra和Adv-hHGF-胱天蛋白酶-3-shRNA的离体转导在免疫缺陷小鼠和免疫活性小鼠中保护胰岛免受非特异性炎症和适应性免疫应答。这项研究的广泛意义是通过人类胰岛移植实现1型糖尿病治疗效率的显着改善的潜力。 公共卫生相关性：所提出的用编码生长因子和抗凋亡基因的复制缺陷型腺病毒载体离体转导人胰岛将通过促进血管再生和抑制门静脉内移植后快速凋亡胰岛细胞死亡来增加人胰岛的存活和功能。这将减少I型糖尿病患者达到正常血糖所需的胰岛数量。

DESCRIPTION (provided by applicant): Transplantation of human islets has great potential as an effective means of treating insulin dependent diabetes mellitus. Primary non-function is the main cause of islet graft failure and it results in the need for multi-donor transplants. Hepatocyte growth factor (HGF) increases 2-cell proliferation and promotes revascularization of islets, while interleukin-1 receptor antagonist (hIL-1Ra) inhibits islet cell apoptosis. Caspase-3 is the converging point of different apoptotic pathways and thus its silencing protects islets from apoptosis. We will test the hypothesis that ex vivo transduction of human islets with a replication deficient adenoviral (Adv) vector encoding hHGF and hIL-1Ra cDNAs or hHGF cDNA and caspase-3-shRNA improves the outcome of islet transplantation for treating type 1 diabetes. In preliminary studies, we constructed Adv-hHGF-hIL-1Ra by cloning hVEGF or hHGF and hIL-1Ra coding sequences and polyA signal under separate CMV promoters in Adenoquick plasmid. There was a dose and time dependent expression of these genes after transduction of Adv-hHGF-hIL-1Ra into human islets. Transduced islets were viable as evidenced by insulin release upon glucose challenge. Co-expression of hHGF and hIL-1Ra by islets showed decrease in caspase-3 activity and apoptosis induced by the inflammatory cytokine cocktail. Compared to non-treated islets, transduction of islets with Adv-hHGF-hIL-1Ra prior to transplantation under the kidney capsules of NOD-SCID mice reduced blood glucose and increased the level of serum insulin and c-peptide levels upon glucose challenge. Transduction of islets with Adv-caspase-3-shRNA also prevented islets from cytokine induced apoptosis and improve islet transplantation. 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. Our specific aims are to determine whether ex vivo transduction with i) ex vivo transduction with Adv- hHGF-hIL-1Ra prevent primary islet nonfunction and early graft rejection after transplantation, ii)? i) Adv- hHGF-hIL-1Ra will improve islet function after intraportal transplantation; ii) ex vivo transduction with Adv- hHGF-caspase-3-shRNA be superior to Adv-hHGF-hIL-1Ra in protecting islet grafts after transplantation; and iii) ex vivo transduction with Adv-hHGF-hIL-1Ra and Adv-hHGF-caspase-3-shRNA protect islets from non- specific inflammatory and adaptive immune responses in both immunodeficient and immunocompetent mice. The broad significance of this research is the potential to achieve dramatic improvement in the treatment efficiency of type 1 diabetes by human islet transplantation. PUBLIC HEALTH RELEVANCE: The proposed ex vivo transduction of human islets with replication deficient adenoviral vectors encoding growth factor and antiapoptotic genes will increase the survival and function of human islets by promoting revascularization and inhibiting the rapid apoptotic islet cell death after Intraportal transplantation. This will decrease the number of islets required to achieve normoglycemia in type I diabetic patients.