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的直接抗动脉粥样硬化作用；-C和载脂蛋白A-I（载脂蛋白A-I）虽然低HDL-C水平通常与冠状动脉疾病的风险增加有关，但载脂蛋白a - 1基因米兰突变的人类携带者值得注意的是，尽管HDL-C水平极低，但他们没有血管疾病，并且具有长寿的祖先历史。这些观察结果表明，与野生型apo A-I相比，米兰型apo A-I/wt。虽然静脉注射这些rHDL颗粒的结果已经提供了其抗动脉粥样硬化功效的证据，但需要反复静脉注射仍然是一个实际的限制。因此，体细胞基因治疗诱导体内产生载脂蛋白A-I为利用载脂蛋白A-I的抗动脉粥样硬化作用提供了另一种方法。最近，由重组腺相关病毒（rAAV）载体传递的转基因在体内的长期表达已经得到证实。因此，在本项目中，我们将探讨载脂蛋白A-I/wt和载脂蛋白A-I/Milano基因的rAAV载体在载脂蛋白e缺陷小鼠模型中预防动脉粥样硬化和盆腔损伤性内膜增生的可行性和生物学效果。利用小鼠动脉损伤模型的研究将扩展到大型动物模型，以研究局部基因传递对猪球囊过度拉伸和支架过度充气损伤后冠状动脉管腔狭窄的影响。在猪血管损伤模型中应用载脂蛋白A-I/ wt/载脂蛋白A-I/Milano基因进行基因治疗，可能为证明载脂蛋白A-I基因治疗在人类中的可行性提供必要的临床前研究类型。具体目标是：1)分析编码载脂蛋白A-I/wt和载脂蛋白A-I/Milano基因的rAAV载体在体内给药后小鼠的转基因表达；2)rAAV介导的载脂蛋白A-I/wt和载脂蛋白A-I/Milano基因转移对载脂蛋白e缺陷小鼠颈动脉损伤的影响；3)利用编码人载脂蛋白A-I/wt和载脂蛋白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评估。4)评价rAAV介导的人载脂蛋白A-I/ wt和载脂蛋白A-I/Milano基因在猪冠状动脉管腔狭窄对球囊过度拉伸和支架过度膨胀损伤的影响。

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.