iNOS Gene Therapy to Prevent Allograft Vasculopathy

根据联合器官共享网络（UNOS）的现有数据，心脏移植物血管病变（CAV）是移植第一年后死亡的主要原因，占第三年后每年死亡人数的25%。我们实验室的初步研究表明，诱导型一氧化氮合酶（iNOS; NOS 2）的表达可以抑制新生内膜增生的发展。此外，我们在之前的PEGT资助期间已经表明，由巨细胞病毒启动子驱动的iNOS转基因可以成功预防CAV的发展，而心肌中iNOS的普遍表达降低了心肌收缩力。本文描述的提案将测试这样的假设：能够将人iNOS基因特异性转移到心脏血管组织的病毒载体可以抑制CAV的发展，而不会对心肌细胞造成过度毒性。此外，该提案将作为近期临床试验的临床前基础，用于未来预防人类CAV。为了达到这些目的，我们将1）确定含有人iNOS基因的腺病毒载体及其各自的对照载体在体外血管平滑肌细胞或内皮细胞中的血管特异性表达; 2）确定血管特异性表达的iNOS在大鼠模型中抑制CAV的功效和毒性; 3）确定血管特异性iNOS载体在猪慢性排斥模型中的功效和毒性;和4）在人中进行初始毒性研究。本提案中进行的基因转移策略将使用第三代腺病毒载体，无肠腺病毒进行，其最大限度地减少了限制转基因表达的免疫原性。本研究拟探讨内皮细胞或平滑肌细胞中特异性诱导型一氧化氮合酶（iNOS）过表达对心肌收缩力的影响、诱导心肌细胞凋亡的作用、iNOS过表达对急性细胞排斥反应的影响、iNOS过表达对CAV的抑制作用、iNOS过表达对心肌细胞凋亡的影响、iNOS过表达对CAV的抑制作用以及iNOS过表达对CAV的抑制作用。5）在慢性排斥反应的大型动物模型中安全有效地递送iNOS以预防CAV。我们计划使用体外和体内模型来解决这些问题。我们的目标是利用这些实验获得的数据作为指导方针， 继续进行安全有效的基于iNOS的基因治疗试验，用于治疗性预防CAV。

Based on current data from the United Network for Organ Sharing (UNOS), cardiac allograft vasculopathy (CAV) is the leading cause of death after the first year of transplantation and accounts for 25% of all deaths annually after the third year. Initial studies from our laboratory have demonstrated that the expression of inducible nitric oxide synthase (iNOS; NOS2) can suppress the development of neointimal hyperplasia. In addition, we have shown in the previous PEGT funding period that an iNOS transgene driven by the cytomegaloviral promoter can successfully prevent the development of CAV, while ubiquitous expression of iNOS in the myocardium declined myocardial contractility. The proposal described herein will test the hypothesis that viral vectors capable of transferring the human iNOS gene specifically to vascular tissue in the heart can suppress the development of CAV without causing undue toxicity to cardiac myocytes. Furthermore, this proposal will serve as the pre-clinical basis for near-term clinical trials for the future prevention of CAV in humans. In order to serve these ends, we will 1) ascertain the vascular-specific expression of adenoviral vectors containing the human iNOS gene and their respective control vectors in vascular smooth muscle cells or endothelial cells in vitro; 2) determine the efficacy and toxicity of vascular-specific expression of iNOS for suppression of CAV in a rat model; 3) determine the efficacy and toxicity of vascular-specific iNOS vector in a porcine chronic rejection model; and 4) carry out an initial toxicity study in humans. The gene transfer strategy carried out in this proposal will be performed with the third generation adenoviral vector, gutless adenovirus, which minimizes immunogenicity that limits the expression of transgene. In this proposal, we plan to address several key issues including 1) that effect of specific over-expression of iNOS in endothelial cells or smooth muscle cells on myocardial contractility; 2) the induction, if any, of apoptosis in cardiac myocytes by iNOS over-expression; 3) the effect of iNOS over-expression on acute cellular rejection; 4) the suppression of CAV in by iNOS over-expression in a rodent model; 5) the safe and efficacious delivery of iNOS to prevent CAV in a large animal model of chronic rejection. We plan to address these issues using both in vitro and in vivo models. Our goal will be to utilize the data obtained from these experiments to serve as a guideline by which to proceed with a safe and efficacious iNOS-based gene therapy trial for the therapeutic prevention of CAV.