Oxidation Resistant ApoA1 Gene Delivery Stents

抗氧化性载脂蛋白A1基因载体支架研究进展 支架血管成形术显著改善了冠状动脉疾病的预后。药物洗脱支架 (DES)显著降低了支架内再狭窄(ISR)的发生率。然而，ISR仍然是一个问题， 尤其是对高危患者，如糖尿病患者。这项提案将研究基因输送支架 (GDS)以解决这些未得到满足的需求。本项目对GDS的治疗策略是基于预防 载脂蛋白A1(ApoA1)氧化。该计划将研究apo-A1基因结构，该基因结构具有4个 被苯丙氨酸(4WF)取代的色氨酸，既能抵抗氧化，又能实现apoA1功能 用来反向运输胆固醇。4WF apo-A1 AAV2基因GDS将在一个具有良好特征的、严重的 用高胆固醇血症糖尿病猪(HDS)建立糖尿病动脉粥样硬化性疾病模型。HDS模型 在支架血管成形术之前，需要20周的时间，导致猪患上晚期动脉粥样硬化和 糖尿病；因此，拟议的研究设计需要五年的项目期。的中心假说 我们认为AAV2编码4WF apoA1的GDS既能抑制ISR的病理生理作用，又能抑制ISR的发病。 减轻HDS中涉及动脉粥样硬化形成的氧化机制。 目的1：利用2型AAV(GFP， 野生型apoA1和4WF apoA1)，并研究GDS在HDS冠状动脉中的局部递送机制。 苏巴伊姆1a。构建载体，配制AAV连接试剂。编码CMV启动子的AAV2 绿色荧光蛋白(GFP)或野生型人apoA1已经被构建、放大和 用于我们在健康猪身上的可行性研究；4WF apo-A1基因构建正在进行中。AAV链接 所使用的系统将用聚烯丙基胺-双膦酸盐处理支架表面，该聚烯丙基胺-双膦酸盐具有与 将蛋白G与硫醇反应结合，然后与抗AAV2抗体亲和结合，进行载体连接。 苏巴伊姆1b.一周的AAV2 apoA1和4WF研究将在体内检查以下组：组1- 对照组，裸金属支架；第2组-AAV2-GFP；第3组-AAV2-apoA1(野生型)；第4组-AAV2-4WF apoA1 (抗氧化)。终点包括载脂蛋白A1的表达、炎症、两个动脉壁的差异 氧化载脂蛋白A1和氧化氨基酸的形成，以及载体的生物分布。 目的2：进行一项治疗研究，评估GDS的疗效。研究设计为：第1组， HDS冠脉中的裸金属支架(对照组)或2组，野生型AAV2-apoA1和组3，AAV2-4WF apoA1 动脉。终点将包括抗ISR疗效、抑制apoA1氧化、再内皮化和 减轻局部动脉粥样硬化的病理生理学。 预期的结果将验证GDS的治疗用途，并将证明 抗氧化4WF载脂蛋白A1亚型在预防支架术后病理生理、促进再狭窄中的作用 内皮化，以及减轻影响再狭窄和动脉粥样硬化的氧化机制。

Summary: Oxidation Resistant apoA1 Gene Delivery Stents Stent angioplasty has led to dramatic improvements in outcomes for coronary artery disease. Drug eluting stents (DES) have sharply reduced the incidence of in-stent restenosis (ISR). Nevertheless, ISR remains a problem, especially for high risk patients, such as those with diabetes. This proposal will investigate gene delivery stents (GDS) to address these unmet needs. The therapeutic strategy for GDS in this project is based on prevention of oxidation of apolipoprotein A1 (apoA1). The program will investigate an apo-A1 gene construct that has 4 tryptophans substituted with phenylalanine (4WF), that both resists oxidation and enables apoA1 functionality for reverse cholesterol transport. A 4WF apo-A1 AAV2 gene GDS will be studied in a well characterized, severe model of diabetic atherosclerotic disease by using hypercholesterolemic diabetic swine (HDS). The HDS model requires 20 weeks to develop, prior to stent angioplasty, resulting in pigs with advanced atherosclerosis and diabetes; thus a five year project period is requested for the proposed study design. The central hypothesis of this proposal is that GDS with AAV2 encoding 4WF apoA1 will both inhibit the pathophysiology of ISR and mitigate oxidative mechanisms involved with atherogenesis in HDS. Aim 1: To formulate and characterize AAV stent-delivery components utilizing Type 2 AAV (GFP, wild type apoA1, and 4WF apoA1), and study GDS local delivery mechanisms in HDS coronary arteries. Subaim 1a. Construct vectors, and formulate AAV linker reagents. AAV2 with a CMV promoter encoding either green fluorescent protein (GFP) or wild-type human apoA1 have already been constructed, scaled up and used in our feasibility studies in healthy pigs; 4WF apo-A1 gene constructs are underway. The AAV linking system to be used will treat the stent surfaces with polyallylamine-bisphosphonate that has conjugation sites to attach Protein G with thiol reactions, followed by affinity binding of anti-AAV2 antibody for vector attachment. Subaim 1b. One week AAV2 apoA1 & 4WF studies will examine in vivo the following groups: Group 1 - control, bare metal stents; group 2 - AAV2-GFP; group 3 - AAV2-apoA1 (wild type); group 4 - AAV2-4WF apoA1 (oxidation resistant). The endpoints will include apoA1 expression, inflammation, differences in both arterial wall oxidized apoA1 and oxidized amino acid formation, and vector biodistribution. Aim 2: Perform a therapeutic study evaluating the efficacy of GDS. The study design will be: Group 1, bare metal stent (controls) or Group 2, wild type AAV2-apoA1 and Group 3, AAV2-4WF apoA1, in HDS coronary arteries. Endpoints will include anti-ISR efficacy, inhibition of apoA1 oxidation, re-endothelialization, and mitigation of regional atherosclerotic pathophysiology. The expected results will validate the therapeutic use of GDS, and will also demonstrate the superiority of the oxidant resistant 4WF apoA1 isoform in preventing post-stent pathophysiology, promoting re- endothelialization, and mitigating oxidative mechanisms that impact both restenosis and atherosclerosis.