Adenoviral/lentiviral vectors for genetic modification

本研究的长期目标是实现有针对性、高效和持久的基因转移到移植心脏，并利用一氧化氮合酶（NOS）的生物学相关基因，在小型和大型动物模型中实现同种异体移植后冠状动脉血管反应性正常化，减少心脏同种异体移植血管病变（CAV）。本研究的中心假设是，利用独特的移植环境，可以实现心脏血管或心肌的靶向、有效和持久的基因转导，并且可以通过内皮或诱导的NOS （eNOS或iNOS）有效的心脏转导来减少心脏移植后冠状动脉松弛异常和CAV的发展。CAV是心脏移植术后患者长期生存的最大障碍。基因治疗可能特别适用于移植的设置，因为供体器官在植入受体之前是唯一可用于体外遗传修饰的。在植入受体之前成功进行体外基因改造的主要挑战。成功的临床基因治疗面临的主要挑战包括需要最佳载体和传递系统将遗传物质转移到体内组织。最近，基因传递到心脏的物理和化学条件第一次被证明允许载体选择性地靶向心肌冠状动脉的介质。使用恒温灌注系统，供体心脏在移植前的通常储存期可以转化为最佳基因传递期，而不会影响移植物的生存能力。第一个目标是在不影响移植物生存能力的情况下转化为最佳基因传递期。本研究的第一个目的是确定最合适的载体（腺病毒或慢病毒）和递送系统，利用化学修饰的恒温灌注系统，在异位大鼠心脏移植模型中高效、靶向和持久地递送基因。第二个目的是利用Specific aim 1中最合适的载体和相同的递送系统，研究将eNOS或iNOS基因转移到供体心脏冠状血管或心肌对异位猪心脏移植后冠状动脉血管反应性和CAV的影响。NOS将作为首选基因进行研究，因为一氧化氮生物利用度降低是发生CAV的血管的特征。异常内皮依赖性松弛先于CAV，因此成功的NOS基因转移可能会影响疾病的早期阶段。在移植心脏的基因转移技术进入临床之前，需要在大型动物模型中取得令人满意的经验。从生理学和病理学的角度来看，猪模型是特别合适的，也是未来临床异种移植的首选动物。成功实现这一建议的具体目标开辟了临床基因治疗的潜力，以避免心脏移植后CAV。这些技术在非同种异体移植动脉硬化治疗中的应用也具有重要意义。

The broad long-term objectives of this proposal are to achieve targeted, efficient and durable gene transfer to the transplanted heart and, using biologically relevant genes for isoforms of nitric oxide synthase (NOS), to normalize coronary arterial vasoreactivity and reduce cardiac allograft vasculopathy (CAV) after allotransplantation in both a small and large animal model. The central hypotheses of this proposal are that, using the unique transplant setting, targeted, efficient and durable gene transduction of blood vessels of the heart or the myocardium can be achieved and that abnormalities in coronary vascular relaxation and the development of CAV after heart transplantation can be reduced by efficient cardiac transduction with endothelial or inducible NOS (eNOS or iNOS). CAV is the greatest obstacle to long-term patient survival after cardiac transplantation. Gene therapy may be particularly applicable in the setting of transplantation as the donor organ is uniquely available for genetic modification ex vivo prior to implantation into a recipient. Major challenges to successful clinical gene genetic modification ex vivo prior to implantation into a recipient. Major challenges to successful clinical gene therapy include the need for optimal vectors and delivery systems to transfer genetic material to tissues in vivo. Recently, for the first time, physical and chemical conditions of gene delivery to the heart have been shown to allow selective targeting of the vector to the media of the coronary arteries of the myocardium. Using a normothermic perfusion system, the usual period of storage of the donor heart before transplantation can be transformed into a period for optimal gene delivery without affecting the viability of the graft. The first aim of transformed into a period for optimal gene delivery without affecting the viability of the graft. The first aim of the current proposal is to identify the most suitable vector (adenovirus or lentivirus) and delivery system using chemical modification of a normothermic perfusion system for efficient, targeted and durable gene delivery in the heterotopic rat heart transplant model. The second aim is to study, using the most suitable vector and same delivery system from Specific Aim 1, the effects of eNOS or iNOS gene transfer to either the coronary vasculature or myocardium of the donor heart on coronary arterial vascular reactivity and on CAV after heterotopic pig heart allotransplantation. NOS will be studied as the gene of choice as reduced nitric oxide bioavailability is characteristic of vessels undergoing CAV. Abnormal endothelium dependent relaxation precedes CAV, so that successful NOS gene transfer might be expected to impact on the early stages of the disease. Before gene transfer technology to the transplanted heart can progress to the clinic, satisfactory experience in a large animal model will be necessary. The porcine model is particular suitable from a physiological and pathological viewpoint and is also the animal of choice for future clinical xenotransplantation. Success in achieving the specific aims of this proposal opens up the potential for clinical gene therapy to avoid CAV after heart transplantation. Implications also exist for the application of these techniques for the modification of non-allograft arteriosclerosis.