Gene Delivery Stents

项目摘要 在过去的十年中，支架血管成形术对改善治疗结果产生了重大影响， 冠心病我们的团队开创了一个概念，即比可能的更大的治疗益处 与目前的裸金属支架（BMS）或药物洗脱支架（DES）相比， 从支架的裸露金属表面进行治疗。本提案将解决以下问题：（1） 足够高剂量的基因载体被装载到支架上2)转基因表达能持续很长时间吗 足以达到治疗效果吗第一个问题现在通过我们对一个 三组分合成复合物，其包括合成放大器构造（AC），从而实现更高的 剂量。延长的表达，将通过我们使用辅助依赖性腺病毒（HD-Ad）来解决。 可以证明转基因在体内表达数年。 目标1.为了研究新型放大器结构（AC）的合成， HD-Ad附着于支架的裸金属表面。整个复合物由以下组成：1）水溶性 二膦酸盐（PABT），其与支架表面具有不可逆结合; 2）可水解的连接体（HL），其可与支架表面结合; 与Ad反应（通过巯基化学）以将Ad拴系到支架上;和3）放大器构建体（AC）。这 目的将集中在新的AC合成构建体，以增加HD-Ad剂量。 目标2.为了在细胞培养中研究和建模，从钢表面局部递送HD-Ad， 评估新型合成AC的位点特异性效率和治疗潜力。我们将创建HD- Ad载体：对于报告基因HD-Ad细胞培养研究，我们将创建绿色荧光蛋白（GFP）载体 在巨细胞病毒启动子（CMV）的控制下。我们还将创建一个HD-Ad luciferease（LUC） 具有CMV启动子的构建体将在体外进行评估，并且也用于Aim 3体内研究，用于光学 影像学研究我们的治疗载体将包括大鼠诱导型一氧化氮合酶（iNOS）， 动脉平滑肌细胞特异性SM 22 α启动子，既靶向平滑肌细胞，又最小化 免疫因子对治疗性转基因活性的功效和持续时间的影响。 目标3：为了在体内研究AC增强的HD-Ad局部递送的效率和功效， 支架表面。这些研究将研究剂量范围和治疗剂量反应能力 我们的三个组成部分复杂的方法，从支架表面基因输送。记者研究交付 LUCHD-Ad将使用支架输送的体内LUC光学成像研究转基因活性随时间的变化。一旦 具有最大体内递送报道分子HD-Ad能力的主要AC制剂（re.的上限 剂量范围）已经表征，将进行治疗研究，调查持续的转基因 使用我们的3种成分递送iNOSHD-Ad，随着时间的推移表达和抗支架内再狭窄功效 具有最大AC HD-Ad递送能力的复合体。

PROJECT SUMMARY Stent angioplasty over the last decade has made a major impact on improved therapeutic outcomes for coronary disease. Our group has pioneered the concept that an even greater therapeutic benefit than possible with current bare metal stents (BMS) or drug eluting stents (DES) could be achieved with stent-mediated gene therapy from the bare metal surfaces of stents. This proposal will address the following questions: 1) Can a high enough dose of a gene vector be loaded onto a stent? 2) Can transgene expression be sustained long enough to achieve a therapeutic benefit? The first question is now addressed through our investigations of a three component synthetic complex that includes a synthetic amplifier construct (AC) thereby enabling higher dosing. Prolonged expression, will be addressed through our use of Helper Dependent Adenoviruses (HD-Ad) that can demonstrate transgene expression in vivo for several years. Aim 1. To investigate the syntheses of novel Amplifier Constructs (AC) for attaching higher levels of HD-Ad to the bare metal surfaces of stents. The overall complex complex consists of: 1) A water soluble bisphosphonate (PABT) with irreversible binding to stent surfaces; 2) A hydrolysable linker (HL) that can be reacted with Ad (via sulfyhydryl chemistry) for tethering Ad to stents; and 3) An amplifier construct (AC). This Aim will focus on novel AC synthetic constructs to increase HD-Ad doses. Aim 2. To investigate and model in cell culture, local delivery of HD-Ad from steel surfaces, in order to assess the site specific efficiency and therapeutic potential of novel synthetic ACs. We will create HD- Ad vectors: For reporter HD-Ad cell culture studies, we will create a green fluorescent protein (GFP) vector under the control of the cytomegalovirus promoter (CMV). We will also create a HD-Ad luciferease (LUC) construct with a CMV promoter to be evaluated in vitro and also used in the Aim 3 in vivo studies for optical imaging studies. Our therapeutic vector will include rat inducible nitric oxide synthase (iNOS) with the rat arterial smooth muscle cell specific SM22alpha promoter, to both target smooth muscle cells and to minimize the effects of immune factors on both the efficacy and duration of therapeutic transgene activity. Aim 3. To investigate in vivo the efficiency and efficacy of AC enhanced local delivery of HD-Ad from stent surfaces. These investigations will study the dose ranging and therapeutic dose response capabilities of our three component complex approach to gene delivery from stent surfaces. Reporter studies delivering LUCHD-Ad will investigate transgene activity over time using in vivo LUC optical imaging of stent delivery. Once the lead AC formulation with the greatest in vivo delivery capacity of reporter HD-Ad (re. the upper limits of the dose range) has been characterized, therapeutic studies will be carried out investigating sustained transgene expression and anti-instent restenosis efficacy over time with iNOSHD-Ad delivery using our 3 component complex with maximal AC HD-Ad delivery capacity.