Inhibition of NF-KB to Facilitate Islet Transplantation

描述(申请人提供)：胰岛素依赖型糖尿病(IDDM)的特征是产生胰岛素的胰岛β细胞遭到破坏。同种异体胰岛移植有可能有效治疗糖尿病，甚至有可能成功治愈糖尿病。然而，影响胰岛移植成功的几个因素包括移植所需的大量胰岛，移植胰岛的存活，以及对移植细胞的同种和自身免疫反应的调节。一种促进胰岛移植的方法是通过对胰岛进行基因修饰来表达抗凋亡或免疫调节剂。我们一直在研究不同的病毒载体，包括腺病毒、慢病毒、不同血清型的AAV、HSV和非病毒载体，通过基因转移来修饰小鼠和人类胰岛的能力，以确定合适的载体 临床应用。我们还通过IL-1抑制剂和抗凋亡基因的基因转移，检测了在培养中抑制IL-1β介导的胰岛功能障碍和Fas介导的细胞凋亡的能力。然而，我们的初步结果表明，对培养中的胰岛最有效的保护方法是通过腺病毒基因转移NF-kB的抑制剂IKB。抑制培养的小鼠和人胰岛的核因子-kB活性可完全阻断IL-1β介导的β细胞功能障碍、NO的产生和Fas介导的细胞凋亡。因此，与其他抗凋亡药物不同的是，抑制核因子-kB能够同时防止胰岛功能障碍和细胞凋亡。此外，我们一直在开发利用阳离子多肽转导结构域将治疗性蛋白输送到胰岛的方法。我们已经证明，在培养和胰岛分离过程中，多肽介导的核因子-kB抑制物的转导也能够阻止β细胞功能障碍。因此，本建议的重点是进一步评估通过肽介导的蛋白转导和基因转移传递的核因子-kB抑制剂的使用，以提高胰岛在培养中的活力，并在移植到同基因和同种异基因小鼠受体后改善其存活率。将对两种不同类型的核因子-kB抑制剂进行评估， IKB阻断基础因子和细胞因子刺激的NF-kB活性和IKB激酶的抑制剂IKK，以防止IKB的磷酸化和随后泛素介导的降解。这些研究的成功完成应该确定通过多肽和基因介导的转导抑制胰岛中的核因子-kB是否能够改善移植前和移植后的胰岛存活率。

DESCRIPTION (provided by applicant): Insulin-dependent diabetes mellitus (IDDM) is characterized by destruction of the insulin-producing pancreatic beta-cell. Allogeneic islet transplantation has the potential to effectively treat and possibly cure diabetes it successful. However, several factors effect the success of islet transplantation including the large number of islets need for transplantation, the viability of the transplanted islets, and regulation of the allo and autoimmune responses to the transplanted cells. One approach to facilitate islet transplantation is through the genetic modification of islets to express anti-apoptotic or immunoregulatory agents. We have been examining the ability to modify murine and human islets by gene transfer with different viral vectors including adenovirus, lentiviruses, different serotypes of AAV, HSV and non-viral vectors in order to identify the appropriate vector for clinical application. We also have examined the ability to inhibit IL-1beta mediated islet dysfunction and Fas mediated apoptosis in culture through gene transfer of IL-1 inhibitors and anti-apoptotic genes. However, our preliminary results suggest that the most effective method for protection of islets in culture is mediated by adenoviral gene transfer of IkB, an inhibitor of NF-kB. Inhibition of NF-kB activity in murine and human islets in culture completely blocked IL-1beta mediated beta cell dysfunction, induction of NO production and Fas-mediated apoptosis. Thus NF-kB inhibition, unlike other anti-apoptotic agents, is able to prevent both islet dysfunction and apoptosis. In addition, we have been developing approaches to deliver therapeutic proteins to islets using cationic peptide transduction domains. We have demonstrated that peptide-mediated transduction of an NF-kB inhibitor is also able to block beta cell dysfunction in culture and during islet isolation. Thus the focus of this proposal is to evaluate further the use of NF-kB inhibitors, delivered by peptide mediated protein transduction and by gene transfer, to improve the viability of islets in culture and to improve their survival following transplantation into both syngeneic and allogeneic murine recipients. Two different types of NF-kB inhibitors will be evaluated, IkB that blocks both basal and cytokine stimulated NF-kB activity and inhibitors of the IkB kinase, IKK, that prevent phosphorylation of IkB and subsequent ubiquitin mediated degradation. The successful completion of the proposed studies should determine if NF-kB inhibition in islets by peptide and gene mediated transduction is able to improve islet viability prior to and post-transplantation.