Growth Factor and Antiapoptotic Gene Delivery to Human Islets

描述（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很大的潜力。原发性无功能是胰岛移植失败的主要原因，因此需要多供体移植。肝细胞生长因子（HGF）增加2细胞增殖，促进胰岛血运重建，而白细胞介素-1受体拮抗剂（hIL-1Ra）抑制胰岛细胞凋亡。Caspase-3是不同凋亡途径的交汇点，因此它的沉默可以保护胰岛细胞免于凋亡。我们将验证一个假设，即用编码hHGF和hIL-1Ra cDNA或hHGF cDNA和caspase-3-shRNA的复制缺陷腺病毒（Adv）载体在体外转导人胰岛可改善胰岛移植治疗1型糖尿病的结果。在前期研究中，我们通过克隆腺快速质粒中不同CMV启动子下的hVEGF或hHGF和hIL-1Ra编码序列及polyA信号，构建Adv-hHGF-hIL-1Ra。Adv-hHGF-hIL-1Ra转导到人胰岛后，这些基因的表达有剂量和时间依赖性。经转导的胰岛在葡萄糖刺激下可通过胰岛素释放而存活。hHGF和hIL-1Ra在胰岛的共表达表明，炎症细胞因子混合物诱导的caspase-3活性和细胞凋亡降低。与未处理的胰岛相比，移植前在NOD-SCID小鼠肾胶囊下用Adv-hHGF-hIL-1Ra转导胰岛可降低血糖，并在葡萄糖刺激下提高血清胰岛素和c肽水平。Adv-caspase-3-shRNA转导胰岛也能阻止细胞因子诱导的胰岛凋亡，改善胰岛移植。上述结果表明，二部Adv载体能有效表达生长因子和抗凋亡基因，减少细胞凋亡，改善胰岛移植的预后。我们的具体目的是确定i) Adv- hHGF-hIL-1Ra体外转导是否能预防移植后原发性胰岛功能丧失和早期移植排斥；ii)？i) Adv- hHGF-hIL-1Ra可改善门静脉内移植后胰岛功能；ii)体外转导Adv- hHGF-caspase-3-shRNA对移植后胰岛移植物的保护作用优于Adv- hhgf - hil - 1ra；iii) Adv-hHGF-hIL-1Ra和Adv-hHGF-caspase-3-shRNA的体外转导保护免疫缺陷和免疫正常小鼠的胰岛免受非特异性炎症和适应性免疫反应。这项研究的广泛意义在于，有可能通过人类胰岛移植显著提高1型糖尿病的治疗效率。

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.