iNOS Gene Therapy to Prevent Allograft Vasculopathy

iNOS 基因疗法预防同种异体移植血管病变

• 批准号: 7139403
• 负责人: TIMOTHY R BILLIAR
• 金额: $ 21.6万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-29 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7139403
• 关键词: Adenoviridae; apoptosis; biotechnology; cardiac myocytes; cardiovascular disorder; cooperative study; cytotoxicity; gene delivery system; gene expression; gene therapy; heart; heart contraction; homologous transplantation; immunocytochemistry; laboratory rat; nitric oxide synthase; nonhuman therapy evaluation; swine; transfection /expression vector; transplant rejection; vascular endothelium; vascular smooth muscle

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.

根据器官共享联合网络(UNOS)的最新数据，心脏移植物血管病(CAV)是移植后第一年死亡的主要原因，第三年后每年占所有死亡人数的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资助期已经证明，由巨细胞病毒启动子驱动的iNOS转基因可以成功地预防CAV的发展，而iNOS在心肌中的普遍表达会降低心肌的收缩能力。本文描述的方案将检验这样一个假设，即能够将人iNOS基因特异性转移到心脏血管组织的病毒载体可以在不对心肌细胞造成过度毒性的情况下抑制CAV的发展。此外，这项建议将作为临床前基础，为未来预防人类CAV的近期临床试验奠定基础。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iNOS表达在大鼠模型中抑制CAV的有效性和毒性；3)确定血管特异性iNOS载体在猪慢性排斥模型中的有效性和毒性；以及4)在人类身上进行初步的毒性研究。在这项建议中实施的基因转移策略将使用第三代腺病毒载体-无胆腺病毒，它将限制转基因表达的免疫原性降至最低。在这项建议中，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特异性的iNOS过表达对心肌收缩性能的影响；2)iNOS过表达诱导心肌细胞凋亡；3)iNOS过表达对急性细胞排斥反应的影响；4)iNOS过表达在啮齿动物模型中抑制CAV；5)iNOS安全有效地应用于预防慢性排斥反应的大型动物模型中的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，通过 开展以诱导型一氧化氮合酶为基础的治疗性预防CAV的安全有效的基因治疗试验。