AAV MEDIATED APO A1 GENE THERAPY FOR ATHEROSCLEROSIS

动物研究已经提供了令人信服的证据，支持JD；-C和载脂蛋白A I(apo A-I)的直接抗动脉粥样硬化作用。虽然低密度脂蛋白胆固醇水平通常与冠状动脉疾病的风险增加有关，但载脂蛋白A-I基因Milano突变的人类携带者的显著之处在于，尽管高密度脂蛋白胆固醇水平极低，但他们不会患血管疾病，并且有祖先的长寿史。这些观察结果表明，与野生型apo A-I相比，apo A-I/Milano，apo A-I/wt。尽管静脉注射这些rHDL颗粒的结果提供了它们抗动脉粥样硬化有效性的证据，但重复静脉注射的必要性仍然是一个实际限制。因此，体细胞基因治疗诱导载脂蛋白A-I的体内产生为利用载脂蛋白A-I的抗动脉粥样硬化作用提供了一种替代途径。近年来，重组腺相关病毒(RAAV)载体介导的转基因在体内的长期表达已被证实。因此，在本项目中，我们将探索利用含有apo A-I/wt和apo A-I/Milano基因的重组腺相关病毒载体进行基因治疗的可行性和生物学效果，以预防载脂蛋白E缺陷小鼠模型的动脉粥样硬化和盆腔损伤的内膜增生。使用小鼠动脉损伤模型的研究将扩展到大型动物模型，以检验局部基因传递对猪冠状动脉球囊过度拉伸和支架过度膨胀损伤后冠状动脉管腔狭窄的影响。载脂蛋白A-I/wt/载脂蛋白A-I/Milano基因治疗在猪血管损伤模型中的应用可能为在人类中进行载脂蛋白A-I基因治疗的可行性提供所需的临床前研究。其具体目的是：1)分析体内注射编码apo A-I/wt和apo A-I/Milano基因的rAAV载体后小鼠的转基因表达，2)rAAV介导的apo A-I/wt和apo A-I/Milano基因转移对载脂蛋白E缺陷小鼠颈动脉损伤反应的影响，3)评价用编码人apo A-I/wt和apo A-I/Milano基因的rAAV载体转导巨噬细胞进行基因转移的可行性和有效性，4)评价局部rAAV介导的载脂蛋白A-I/wt和载脂蛋白A-I/Milano基因转移对猪冠状动脉球囊过度拉伸和支架过度膨胀损伤后管腔狭窄反应的影响。

Animal studies have provided compelling evidence in favor of direct anti- atherogenic effects pf JD;-C and apolipoprotein A I (apo A-I). While low HDL-C levels are often associated with an increased risk of coronary artery diseases, human carriers of the Milano mutation in the apo A-I gene are remarkable in that despite extremely low levels of HDL-C, they enjoy freedom from vascular disease and have an ancestral history of longevity. These observations have suggested that the apo A-I/Milano, compared to the wild type apo A-I, apo A-I/wt. While the results of intravenous administration of these rHDL particles have provided evidence of their anti- atherogenic efficacy, the need for repeated intravenous administration remains a practical limitation. Therefore, somatic gene therapy to induce in vivo production of apo A-I offers an alternative approach for utilizing the anti-atherogenic effects of apo A-I. Recently, long-term in vivo expression of transgenes delivered by recombinant adeno-associated virus (rAAV) vectors have been demonstrated. Therefore, in this project we will explore the feasibility and biologic efficacy of gene therapy utilizing rAAV vectors containing the apo A-I/wt and apo A-I/Milano genes in preventing atherosclerosis and pot-injury intimal hyperplasia in the apo E-deficient mouse model. Studies using mouse model of arterial injury will be extended to a large animal model to examine the effects of local gene delivery on the coronary arterial luminal narrowing following balloon overstretch and stent over-inflation injury in swine. The application of gene therapy with apo A- I/wt/apo A-I/Milano gene in the swine vascular injury model may provide the type of preclinical studies needed to demonstrate the feasibility of apo A-I gene therapy in humans. The specific aims are: 1) Analysis of transgene expression in mice following in vivo administration of rAAV vectors encoding apo A-I/wt and apo A-I/Milano genes, 2) Effect of rAAV- mediated transfer of apo A-I/wt and apo A-I/Milano genes on arterial response to carotid injury in apo E-deficient mice, 3) Assessment of feasibility and efficacy of gene transfer using macrophages transduced with rAAV vectors encoding human apo A-I/wt and apo A-I/Milano, and 4) Evaluation of the effects of local rAAV mediated transfer of human apo A- I/wt and apo A-I/Milano genes on coronary arterial luminal narrowing response to balloon-overstretch and stent-overinflation injury in swine.