Anti-apoptotic gene therapy: Islet allotransplantation

描述（由申请人提供） 1型糖尿病，又称胰岛素依赖型糖尿病 胰岛素依赖型糖尿病（IDDM）是一种自身免疫性疾病， T淋巴细胞介导的破坏胰岛细胞。而 胰岛素替代疗法纠正高血糖症，但它不是治愈，因为 大多数胰岛素依赖型糖尿病患者最终死于 不精确的葡萄糖稳态。一种方法可以恢复紧张的血糖 对照是胰岛同种异体移植物形式的β细胞替代， 异种移植后者可能不会很快成为临床现实，主要是 因为担心人畜共患病。同种异体移植在伦理上是可以接受的，但它们 面临同种免疫排斥和自身免疫破坏， 移植似乎有三种不同的死亡效应途径， 负责β细胞破坏：1）Fas，2）肿瘤坏死因子α 3）穿孔素/颗粒酶B。与自身免疫性破坏类似， Fas、TNF α和穿孔素/颗粒酶B是同种异体移植中重要的死亡效应因子 胰岛排斥。胰岛的基因工程以产生 这些途径可以促进同种异体胰岛移植， 长期生存。在非肥胖糖尿病（NOD）小鼠模型中， 胰岛离体表达多种免疫调节细胞因子和蛋白质 导致显著的，但不是无限期的， 胰岛移植存活率。长期或无限期的一个重要原因 移植物存活率尚未实现是基因递送的性质 载体，其中大多数是病毒来源的，并且具有高度免疫原性或毒性， 胰岛细胞包括人类在内的慢病毒的最新工程 免疫缺陷病毒（HIV-1）、猫免疫缺陷病毒（FIV）和马免疫缺陷病毒（ 传染性贫血病毒（EIAV），已经导致能够 感染非分裂细胞，在体内是非免疫原性的，且可以稳定整合 进入宿主细胞慢病毒载体的这些期望的特征， 用于离体胰岛的基因递送载体，与高表达载体相反， 最近使用载体的免疫原性、短暂性和毒性 胰岛基因转移策略（腺病毒，单纯疱疹）。虽然艾滋病毒 已经证明慢病毒载体容易感染人胰岛， 病毒株的性质是临床应用的重大障碍。 应用.另一方面，EIAV具有与HIV相同的特征 并且对人类没有致病性。本提案的重点是证明 可溶性Fas、TNF和颗粒酶B拮抗剂对胰岛有保护作用， 从凋亡激活培养并进一步开发EIAV慢病毒 系统作为编码抑制剂的cDNA的有效基因递送载体， Fas、TNF α和穿孔素/颗粒酶B依赖性死亡效应子途径，单独或 结合起来。这可能是促进同种异体移植的理想方法。 胰岛移植作为治疗胰岛素依赖型糖尿病可能性。

DESCRIPTION (provided by applicant) Type 1 diabetes mellitus, also termed insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease that specifically targets the pancreatic beta cells of the islets of Langerhans in a T-lymphocyte-mediated destruction. While insulin replacement therapy corrects the hyperglycemia, it is not a cure, since most IDDM patients eventually succumb to the complications associated with imprecise glucose homeostasis. One approach that can restore tight glycemic control is the replacement of beta cells in the form of islet allografts or xenografts. The latter may not become a clinical reality anytime soon primarily because of concerns for zoonoses. Allografts are ethically acceptable, yet they face both alloimmune rejection as well as autoimmune destruction following transplantation. It appears that three distinct death effector pathways are responsible for beta cell destruction: 1) Fas, 2) tumor necrosis factor alpha and 3) perforin/granzyme B. Similar to autoimmune destruction, it appears that Fas, TNFa and perforin/granzyme B are important death effectors in allograft rejection of islets. Genetic engineering of islets to produce inhibitors of these pathways may facilitate allogeneic islet transplantation and may result in long-term survival. In the non-obese diabetic (NOD) mouse model, engineering islets ex vivo to express a variety of immunoregulatory cytokines and proteins has resulted in significant, but not indefinite, prolongation of allogeneic islet transplant survival. One important reason why long-term or indefinite allograft survival has not been achieved is the nature of the gene delivery vectors, most of which are of viral origin and highly immunogenic or toxic to islet cells. Recent engineering of lentiviruses including human immunodeficiency virus (HIV-1), feline immunodeficiency (FIV) and equine infectious anemia viruses (EIAV), has resulted in vectors that are able to infect non-dividing cells, are non-immunogenic in vivo and can stably integrate into the host cell. These desirable characteristics of lentiviral vectors as gene delivery vehicles for islets ex vivo, are in contrast to the highly immunogenic, transient and toxic nature of vectors that have been recently used in islet gene transfer strategies (adenovirus, herpes simplex). While HIV-based lentiviral vectors have been demonstrated to readily infect human islets, the nature of the virus strain is a significant impediment for clinical applications. EIAV, on the other hand, offers the same characteristics as HIV and is not pathogenic in humans. The focus of this proposal is to demonstrate that soluble antagonists of Fas, TNF and granzyme B can protect islets in culture from apoptosis activation and to further develop the EIAV lentiviral system as an efficient gene delivery vector of cDNAs encoding inhibitors of Fas, TNFa and perforin/granzyme B-dependent death effector pathways, alone or in combinations. This may be a desirable approach to facilitate allogeneic islet transplantation as a possible therapy for IDDM.