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。原发无功能是胰岛移植失败的主要原因，需要多个供体的移植。肝细胞生长因子(HGF)促进胰岛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)则抑制胰岛细胞凋亡。Caspase-3是不同凋亡途径的汇合点，因此其沉默可以保护胰岛免受细胞凋亡的影响。我们将验证这样一种假设，即用编码hHGF和hIL-1ra cDNAs的复制缺陷型腺病毒(ADV)载体或hHGF cDNAs和caspase-3-shRNA体外转导人胰岛可改善胰岛移植治疗1型糖尿病的结果。在初步研究中，我们通过克隆hVEGF或hHGF和hIL-1ra编码序列以及在不同CMV启动子作用下的PolyA信号，构建了AdV-hHGF-hIL-1ra。将AdV-hHGF-hIL-1ra导入人胰岛后，这些基因的表达呈剂量和时间依赖性。葡萄糖刺激后胰岛素释放证明转导后的胰岛是存活的。联合表达hHGF和hIL-1ra的胰岛caspase-3活性降低，炎性细胞因子诱导的细胞凋亡减少。与未经处理的胰岛相比，在NOD-SCID小鼠的肾被膜下移植前用AdV-hHGF-hIL-1ra转导胰岛可降低血糖，并在葡萄糖攻击时升高血清胰岛素和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细胞凋亡，促进胰岛移植。这些结果表明，两段ADV载体能有效表达生长因子和抗凋亡基因，减少细胞凋亡，改善胰岛移植的效果。我们的具体目的是确定体外转导联合i)体外转导联合Adv-hHGF-hIL-1ra能否预防移植后的原发胰岛无功能和早期排斥反应，ii)？1)AdV-hHGF-hIL-1Ra可改善门脉内移植后的胰岛功能；ii)体外转导AdV-hHGF-caspase-3-shRNA对移植后胰岛的保护作用优于AdV-hHGF-hIL-1Ra；iii)体外转导AdV-hHGF-hIL-1ra和AdV-hHGF-caspase-3-shRNA对免疫缺陷和免疫活性小鼠胰岛的非特异性炎症和获得性免疫反应均有保护作用。这项研究的广泛意义在于，通过人类胰岛移植可以显著提高1型糖尿病的治疗效率。 公共卫生意义：建议将携带生长因子和抗凋亡基因的复制缺陷型腺病毒载体体外转导人胰岛，通过促进门脉内移植后的血管重建和抑制胰岛细胞的快速死亡来增加人胰岛的存活率和功能。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.