Oxidation Resistant ApoA1 Gene Delivery Stents

抗氧化 ApoA1 基因递送支架

基本信息

批准号：
9361961
负责人：
Ilia Fishbein
金额：
$ 85.38万
依托单位：
CHILDREN'S HOSP OF PHILADELPHIA
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-07-15 至 2021-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9361961
关键词：
Address Affinity Amino Acids Angioplasty Animals Antibodies Apolipoprotein A-I Area Arteries Atherosclerosis Binding CMV promoter Chemistry Cholesterol Clinic Complementary DNA Control Groups Coronary Coronary Arteriosclerosis Coronary artery Diabetes Mellitus Family suidae Feasibility Studies Functional disorder G-substrate GTP-Binding Proteins Gene Delivery Genes Green Fluorescent Proteins Human Impairment In Vitro Incidence Inflammation Inflammatory Laboratories Learning Link Metals Modeling Monitor Outcome Outcome Study Oxidants Oxidative Stress Oxides Pathogenesis Pathologic Patient risk Pharmaceutical Preparations Phenylalanine Prevention Process Protein Isoforms Publishing Reaction Reagent Research Design Resistance Risk Site Site-Directed Mutagenesis Stents Sulfhydryl Compounds Surface System Therapeutic Therapeutic Studies Therapeutic Uses Toxic effect Transgenes Tryptophan Vascular Diseases Work adeno-associated viral vector atherogenesis atheroprotective base bisphosphonate design diabetic high risk improved outcome in vivo novel oxidation polyallylamine preclinical study prevent programs restenosis reverse cholesterol transport scale up therapeutic gene transgene expression vector vector biodistribution

项目摘要

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.

摘要：抗氧化 apoA1 基因递送支架支架血管成形术显着改善了冠状动脉疾病的治疗效果。药物洗脱支架（DES）大大降低了支架内再狭窄（ISR）的发生率。尽管如此，ISR 仍然是一个问题，特别是对于高危患者，例如糖尿病患者。该提案将研究基因递送支架（GDS）来解决这些未满足的需求。本项目中 GDS 的治疗策略基于预防载脂蛋白 A1 (apoA1) 的氧化。该计划将研究 apo-A1 基因构建体，该基因构建体具有 4 色氨酸被苯丙氨酸 (4WF) 取代，既能抗氧化又能实现 apoA1 功能用于反向胆固醇转运。 4WF apo-A1 AAV2 基因 GDS 将在一个充分表征的、严重的使用高胆固醇血症糖尿病猪（HDS）建立糖尿病动脉粥样硬化疾病模型。 HDS模型在支架血管成形术之前需要 20 周的时间才能形成，导致猪患有晚期动脉粥样硬化糖尿病;因此，拟议的研究设计需要五年的项目期。中心假设为该提议认为，具有编码 4WF apoA1 的 AAV2 的 GDS 既会抑制 ISR 的病理生理学，减轻 HDS 中与动脉粥样硬化形成相关的氧化机制。目标 1：利用 2 型 AAV（GFP、野生型 apoA1 和 4WF apoA1），并研究 HDS 冠状动脉中的 GDS 局部递送机制。苏拜姆 1a.构建载体并配制 AAV 连接试剂。带有 CMV 启动子编码的 AAV2 绿色荧光蛋白 (GFP) 或野生型人类 apoA1 已被构建、放大并用于我们对健康猪的可行性研究； 4WF apo-A1 基因构建正在进行中。 AAV 链接所使用的系统将用具有缀合位点的聚烯丙胺双膦酸盐处理支架表面通过硫醇反应连接 Protein G，然后通过抗 AAV2 抗体的亲和结合进行载体连接。苏拜姆 1b.一周的 AAV2 apoA1 和 4WF 研究将检查以下组的体内情况：第 1 组 - 控制，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）。终点将包括 apoA1 表达、炎症、两侧动脉壁的差异氧化 apoA1 和氧化氨基酸的形成以及载体生物分布。目标 2：进行评估 GDS 功效的治疗研究。研究设计将是：第 1 组， HDS 冠状动脉中的裸金属支架（对照）或第 2 组，野生型 AAV2-apoA1 和第 3 组，AAV2-4WF apoA1 动脉。终点包括抗 ISR 功效、apoA1 氧化抑制、再内皮化和缓解局部动脉粥样硬化的病理生理学。预期结果将验证 GDS 的治疗用途，也将证明 GDS 的优越性抗氧化剂 4WF apoA1 亚型可预防支架术后病理生理学，促进重新内皮化，并减轻影响再狭窄和动脉粥样硬化的氧化机制。