Inhibition of NF-KB to Facilitate Islet Transplantation

描述（由申请方提供）：胰岛素依赖型糖尿病（IDDM）的特征是产生胰岛素的胰腺β细胞遭到破坏。同种异体胰岛移植有可能有效地治疗糖尿病，并可能治愈糖尿病。然而，有几个因素影响胰岛移植的成功，包括移植所需的大量胰岛、移植胰岛的活力以及对移植细胞的同种异体和自身免疫应答的调节。促进胰岛移植的一种方法是通过对胰岛进行遗传修饰以表达抗凋亡剂或免疫调节剂。我们已经检查了通过用不同的病毒载体（包括腺病毒、慢病毒、不同血清型的AAV、HSV和非病毒载体）进行基因转移来修饰鼠和人胰岛的能力，以鉴定用于修饰胰岛的合适载体。 临床应用。我们还检测了通过IL-1抑制剂和抗凋亡基因的基因转移抑制IL-1 β介导的胰岛功能障碍和Fas介导的细胞凋亡的能力。然而，我们的初步研究结果表明，最有效的方法，在培养中的胰岛保护介导的腺病毒基因转移的IkB，NF-κ B的抑制剂。在培养的鼠和人胰岛中抑制NF-kB活性完全阻断了IL-1 β介导的β细胞功能障碍、诱导NO产生和Fas介导的细胞凋亡。因此，与其他抗凋亡剂不同，NF-kB抑制能够预防胰岛功能障碍和凋亡。此外，我们一直在开发使用阳离子肽转导结构域将治疗性蛋白质递送至胰岛的方法。我们已经证明，肽介导的NF-kB抑制剂的转导也能够阻断培养和胰岛分离过程中的β细胞功能障碍。因此，该建议的重点是进一步评估使用NF-kB抑制剂，通过肽介导的蛋白转导和基因转移，以提高培养的胰岛的活力，并提高其移植到同基因和同种异体小鼠受体后的存活率。将评价两种不同类型的NF-κ B抑制剂， 阻断基础和细胞因子刺激的NF-kB活性的IkB和防止IkB磷酸化和随后的泛素介导的降解的IkB激酶IKK的抑制剂。所提出的研究的成功完成应确定通过肽和基因介导的转导在胰岛中的NF-κ B抑制是否能够改善移植前后的胰岛活力。

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.