iNOS Gene Therapy to Prevent Allograft Vasculopathy

根据器官共享联合网络（UNOS）的最新数据，同种异体心脏移植血管病变（CAV）是移植一年后死亡的主要原因，占移植三年后每年所有死亡人数的25%。我们实验室的初步研究表明，诱导型一氧化氮合酶（iNOS; NOS2）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资助期内已经表明，由巨细胞病毒启动子驱动的iNOS转基因可以成功地阻止CAV的发展，而心肌中iNOS的普遍表达会降低心肌收缩力。本文所述的建议将验证一种假设，即能够将人类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，该建议将作为未来预防人类CAV的近期临床试验的临床前基础。为了达到这些目的，我们将1)确定含有人类iNOS基因的腺病毒载体及其相应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)在大鼠模型中测定血管特异性表达iNOS对CAV的抑制作用和毒性；3)研究血管特异性iNOS载体在猪慢性排斥反应模型中的作用和毒性；4)对人体进行初步毒性研究。本方案中所进行的基因转移策略将采用第三代腺病毒载体，即无胆腺病毒，这将最大限度地减少限制转基因表达的免疫原性。在本提案中，我们计划解决几个关键问题，包括1)内皮细胞或平滑肌细胞中iNOS特异性过表达对心肌收缩性的影响；2) iNOS过表达诱导心肌细胞凋亡（如果有的话）；3) iNOS过表达对急性细胞排斥反应的影响；4)小鼠模型中iNOS过表达对CAV in的抑制作用；5)在大型慢性排斥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。我们的目标是利用从这些实验中获得的数据作为指导

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.