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）的表达可以抑制内膜增生的发展，防止CAV的发展。本文所述的建议将验证能够转移人类iNOS基因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的假设。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，我们将进行以下具体目的：1)确定几种含有人类iNOS基因（Ad-iNOS, AAV-iNOS, Lt-iNOS）的病毒载体（腺病毒，腺相关病毒和慢病毒）及其相应的对照载体对大鼠心脏移植动脉硬化模型的抑制效果；2)测定Ad-iNOS、AAV-iNOS和Lt- iNOS及其对照载体对大鼠模型的毒性；3)确定理想病毒载体（Ad-iNOS， AAV-iNOS或Lt-iNOS）及其各自的控制载体在猪慢性排斥模型中的功效和毒性，如特异性目标#2所确定的；4)确定在心室辅助装置支持下的人类心脏中的毒性。在本提案中，我们计划解决几个关键问题，包括1)慢性iNOS表达对心肌收缩性的影响；2)慢性iNOS表达是否会诱导心肌细胞凋亡；3) iNOS表达是否会诱发急性细胞排斥反应；4)急性细胞排斥反应期间iNOS表达对整体心功能有什么影响？ 5)在大型慢性排斥动物模型中，iNOS能否以安全有效的方式递送，从而预防CAV ？我们计划使用体外和体内模型来解决这些问题。在完成本提案中概述的具体目标后，我们的目标是利用从这些实验中获得的数据作为指导方针，根据该指导方针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 and prevent the development of CAV. The proposal described herein will test the hypothesis that viral vectors capable of transferring the human iNOS gene 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 carry out the following Specific Aims: 1) to determine the efficacy of several viral vectors (adenoviral, adeno-associated virus, and lentivirus) containing the human iNOS gene (Ad-iNOS, AAV-iNOS, Lt-iNOS) and their respective control vectors for suppression of cardiac transplant arteriosclerosis in the rat model; 2) to determine toxicity of Ad-iNOS, AAV-iNOS, and Lt- iNOS and their respective control vectors in rat models; 3) to determine efficacy and toxicity of the ideal viral vector (Ad-iNOS, AAV-iNOS, or Lt-iNOS) and its respective control vector as determined in Specific Aim #2, in a porcine model of chronic rejection and 4) to determine the toxicity in human hearts that are being supported with ventricular assist devices. In this proposal we plan to address several key questions including 1) what effect will chronic iNOS expression have on myocardial contractility; 2) will chronic iNOS expression induce cardiac myocyte apoptosis; 3) will iNOS expression induce acute cellular rejection; 4) what effect will iNOS expression during an episode of acute cellular rejection have on global cardiac function and 5) can iNOS be delivered in a safe and efficacious manner such that CAV can be prevented in a large animal model of chronic rejection? We plan to address these questions using both in vitro and in vivo models. Upon completion of the specific alms outlined whin this proposal our goal will be utilize the data obtained from these experiment 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.