Improving Islet Transplantation Outcome With Akt1

描述(由申请人提供)：胰岛移植正在成为治疗1型糖尿病(T1D)的潜在疗法。然而，移植围术期的有限供应和显著的胰岛丢失被认为是这种治疗策略的主要限制。本项目旨在通过在体外产生胰岛素的2-细胞中引入成分活性Akt1(CA-Akt1)来改善胰岛移植的治疗效果。丝氨酸/苏氨酸蛋白激酶Akt/PKB是PI3Kinase途径的直接下游靶点，具有抗细胞凋亡和诱导细胞增殖的双重功能。在转基因和基因敲除小鼠模型的研究中，以及使用药理学方法，已经证明Akt与2-细胞的存活和增殖有关。然而，为了实现CA-Akt1的治疗潜力，需要一种安全、高效和特异的载体将Akt1导入体外培养的胰岛2细胞。在这方面，基于5型腺病毒(Ad5)的载体引起了极大的兴趣。我们先前的研究表明，修饰后的Ad5载体AdRGDpK7对人胰岛细胞具有显著的基因转移效率。因此，我们建议使用Ad5RGDpK7将Akt1基因导入体外培养的胰岛细胞。为了进一步减少CA-Akt1的潜在不良影响，我们将通过使用2细胞特异性启动子-大鼠胰岛素启动子(RIP)来驱动Akt1的表达来限制外源Akt1在2-细胞中的表达。此外，我们建议将HSV-TK与CA-Akt1共表达，这样它既可以作为一种非侵入性的成像方式来跟踪移植的胰岛，也可以作为自杀基因在恶性发生时使用。因此，我们的具体目标是：1)开发一种2细胞特异性的、感染性增强的Ad5载体，使CA-Akt1和HSV-TK基因能够在体外高效、特异地导入2细胞；2)检测上述Ad5载体在促进胰岛存活和增殖的同时将转化降至最低的能力，从而提高胰岛移植的效率；以及3)评估上述Ad5载体在胰岛移植中的安全性。

DESCRIPTION (provided by applicant): Summary Islet transplantation is becoming a potential cure for type 1 diabetes (T1D). However, the limited supply and significant islet loss in the peritransplant period are cast as the major limitations of this treatment strategy. This project is aimed to improve the therapeutic outcome of islet transplantation by introducing constitutively active Akt1 (CA-Akt1) into the insulin producing 2-cells ex vivo. The serine/threonine protein kinase Akt/PKB is the direct downstream target of PI3 Kinase pathway, and has been found to have dual functions of anti-apoptosis and induction of cell proliferation. Relevance of Akt on 2-cell survival and proliferation has been demonstrated in studies of transgenic and knockout mouse models, as well as using pharmacological methods. Nonetheless, in order to realize the therapeutic potential of CA-Akt1, a safe, efficient and specific vector is needed to deliver Akt1 into islet 2-cells ex vivo. In this regard, adenovirus serotype 5 (Ad5)-based vector is of great interest. Our previous studies have demonstrated the modified Ad5 vector, AdRGDpK7, exhibited significantly higher gene transfer efficiency for the human islet cells. We thus propose to employ Ad5RGDpK7 to deliver Akt1 into islet cells ex vivo. To further diminish the potential adverse effect of CA-Akt1, we will restrict exogenous Akt1 expression in 2-cells by employment of 2-cell specific promoter-rat insulin promoter (RIP) to drive Akt1 expression. In addition, we propose to co-express a dual functional modality, HSV-TK, with CA-Akt1 so that it can be used as both a non-invasive imaging modality to follow the transplanted islets and a suicide gene should malignancy occur. Our specific aims are thus: 1) To develop a 2-cell specific, infectivity-enhanced Ad5 vector that allows efficient and specific CA-Akt1 and HSV-TK gene delivery into 2-cells ex vivo; 2) To examine the capacity of the Ad5 vector developed above to promote islet survival and proliferation while minimizing transformation, thus enhancing the efficacy of islet transplantation; and 3) To evaluate the safety of the Ad5 vector developed above in the context of islet transplantation.