Adenoviral/lentiviral vectors for genetic modification

用于基因修饰的腺病毒/慢病毒载体

• 批准号: 6661537
• 负责人: CHRISTOPHER G.A. MCGREGOR
• 金额: $ 30.7万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-09-01 至 2003-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6661537
• 关键词: angiogenesis; blood vessel disorder; coronary vessels; enzyme therapy; gene therapy; heart transplantation; homologous transplantation; isolation perfusion; laboratory rat; nitric oxide; nitric oxide synthase; nonhuman therapy evaluation; postoperative complications; swine; transfection /expression vector

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.

这项建议的长期目标是实现靶向、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)亚型的生物相关基因，在小动物和大动物模型的同种异体移植后使冠状动脉血管反应性正常化，减少心脏移植物血管病(CAV)。这一建议的中心假设是，利用独特的移植环境，可以实现心脏或心肌血管的靶向、高效和持久的基因转导，而心脏移植后冠状动脉血管松弛的异常和CAV的发展可以通过与内皮或诱导型一氧化氮合酶(eNOS或iNOS)的有效心脏转导来减少。CAV是心脏移植后患者长期存活的最大障碍。基因疗法可能特别适用于移植的环境，因为在移植到接受者体内之前，供体器官是唯一可以在体外进行基因修改的器官。成功的临床基因在植入受体前进行体外基因修饰的主要挑战。成功的临床基因治疗的主要挑战包括需要最佳的载体和递送系统来将遗传物质转移到体内的组织中。最近，首次证明了将基因传递到心脏的物理和化学条件允许将载体选择性地靶向心肌冠状动脉的中层。使用常温灌流系统，供者心脏在移植前通常的保存期可以转变为最佳的基因输送时期，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存活的情况下，转变为最佳的基因传递时期。本方案的第一个目的是通过对常温灌流系统进行化学修饰，确定最合适的载体(腺病毒或慢病毒)和递送系统，以在异位大鼠心脏移植模型中高效、靶向和持久地传递基因。第二个目的是利用特定目的1中最合适的载体和相同的递送系统，研究内皮型一氧化氮合酶或诱导型一氧化氮合酶基因转移到供体心脏的冠脉血管或心肌对异位猪心脏移植后冠脉血管反应性和冠脉血流速度的影响。一氧化氮合酶将被作为选择基因进行研究，因为降低一氧化氮的生物利用度是接受CAV的血管的特征。血管内皮依赖的松弛异常先于CAV，因此，成功的NOS基因转移可能会对疾病的早期阶段产生影响。在移植心脏的基因转移技术能够进入临床之前，需要在大型动物模型上获得满意的经验。从生理学和病理学的角度来看，猪模型特别适合，也是未来临床异种移植的首选动物。成功地实现了这一建议的特定目标，为临床基因治疗避免心脏移植后CAV开辟了可能性。这些技术的应用对非同种异体动脉硬化的改善也有意义。