Oxidation Resistant ApoA1 Gene Delivery Stents

抗氧化 ApoA1 基因递送支架

• 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
• 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

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.

综述：抗氧化apoA 1基因递送支架 支架成形术已导致冠状动脉疾病结局的显著改善。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， 特别是对于高风险患者，例如糖尿病患者。这项提案将研究基因输送支架 (GDS)来满足这些未满足的需求。本项目中GDS的治疗策略是基于预防 载脂蛋白A1（apoA 1）的氧化。该计划将研究一个apo-A1基因构建体， 用苯丙氨酸（4 WF）取代的色氨酸，既抗氧化又能使apoA 1功能化 胆固醇逆向运输的关键4 WF apo-A1 AAV 2基因GDS将在一个充分表征的，严重的， 采用高胆固醇血症糖尿病猪（HDS）建立糖尿病动脉粥样硬化模型。HDS模型 在支架血管成形术之前，需要20周的时间来发展，导致猪患有晚期动脉粥样硬化， 糖尿病;因此，拟定的研究设计需要5年的项目期。的中心假设 该建议是具有编码4 WF apoA 1的AAV 2的GDS将抑制ISR的病理生理学， 减轻HDS中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apoA 1和4 WF apoA 1），并研究HDS冠状动脉中的GDS局部递送机制。 Subaim 1a.构建载体并配制腺相关病毒接头试剂。具有CMV启动子的AAV 2，其编码 绿色荧光蛋白（GFP）或野生型人apoA 1已经被构建、放大并 用于我们在健康猪中的可行性研究; 4 WF apo-A1基因构建正在进行中。AAV连接 使用的系统将用聚烯丙基胺-二膦酸盐处理支架表面， 用硫醇反应连接蛋白G，然后亲和结合抗AAV 2抗体用于载体连接。 Subaim 1b.为期一周的AAV 2 apoA 1和4 WF研究将在体内检查以下组：第1组- 对照，裸金属支架;组2 -AAV 2-GFP;组3 -AAV 2-apoA 1（野生型）;组4 -AAV 2 - 4 WF apoA 1 （抗氧化）。终点将包括apoA 1表达、炎症、两个动脉壁的差异 氧化的apoA 1和氧化的氨基酸形成，以及载体生物分布。 目的2：进行一项评价GDS疗效的治疗研究。研究设计为：第1组， 裸金属支架（对照）或组2野生型AAV 2-apoA 1和组3 AAV 2 - 4 WF apoA 1，在HDS冠状动脉中 动脉终点将包括抗ISR功效、抑制apoA 1氧化、再内皮化和 减轻局部动脉粥样硬化病理生理学。 预期的结果将验证GDS的治疗用途，也将证明GDS的优越性。 抗氧化剂4 WF apoA 1亚型在预防支架后病理生理学，促进再狭窄， 内皮化和减轻影响再狭窄和动脉粥样硬化的氧化机制。