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 (IDDM)是一种针对胰岛β细胞的自身免疫性疾病 T淋巴细胞介导的朗格汉斯胰岛细胞破坏。而当 胰岛素替代疗法可以纠正高血糖，但不是治愈方法，因为 大多数IDDM患者最终会死于与 不精确的葡萄糖稳态。一种可以恢复紧张血糖的方法 对照是以同种异体胰岛移植或 异种移植物。后者可能不会很快成为临床现实，主要是 出于对人畜共患病的担忧。同种异体移植在道德上是可以接受的，但它们 面临同种异体免疫排斥反应和自身免疫破坏 移植。看起来有三条截然不同的死亡效应通路 对β细胞的破坏负责：1)Fas，2)肿瘤坏死因子 和3)穿孔素/颗粒酶B类似于自身免疫破坏，似乎 Fas、TNFa和穿孔素/颗粒酶B是同种异体移植物重要的死亡效应因子 对小岛的排斥。利用基因工程技术生产胰岛生长抑制因子 这些途径可能促进同种异体胰岛移植，并可能导致 在长期生存中。在非肥胖糖尿病(NOD)小鼠模型中，工程学 胰岛在体外表达多种免疫调节细胞因子和蛋白，导致同种异体胰岛显著延长，但不是无限期延长 胰岛移植存活。长期或无限期的一个重要原因 同种异体移植物存活尚未实现是基因传递的本质 载体，其中大多数是病毒来源的，具有高度免疫原性或对 胰岛细胞。包括人类在内的慢病毒工程的最新进展 免疫缺陷病毒(HIV-1)、猫免疫缺陷(FIV)和马 传染性贫血病毒(EIAV)，已导致载体能够 感染非分裂细胞，体内无免疫原性，可稳定整合 进入宿主细胞。慢病毒载体的这些理想特性包括 基因载体用于体外胰岛，与高度不同的 最近使用的载体的免疫原性、暂时性和毒性 胰岛基因转移策略(腺病毒、单纯疱疹病毒)。虽然以艾滋病毒为基础 慢病毒载体已经被证明很容易感染人类的胰岛， 病毒株的性质是临床上的一个重大障碍。 申请。另一方面，EIAV提供了与HIV相同的特征 并且对人类没有致病性。这项提议的重点是证明 可溶性Fas、肿瘤坏死因子和颗粒酶B拮抗剂对胰岛的保护作用 从细胞凋亡激活培养和进一步发展EIAV慢病毒 系统作为编码血管紧张素转换酶抑制剂的cDNA的有效基因传递载体 Fas、TNFa和穿孔素/颗粒酶B依赖的死亡效应通路 在组合中。这可能是促进同种异体移植的可取方法。 胰岛移植是治疗IDDM的一种可能方法。

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.