Suprachoroidal nonviral gene transfer of engineered VEGF antagonists

项目摘要 眼部基因治疗在持续表达抗血管生成蛋白， 视网膜/脉络膜血管疾病，也用于替换遗传性视网膜病变患者的突变基因， 退化病毒载体提供光感受器和视网膜色素上皮细胞的有效转导 (RPE)当注射到视网膜下间隙时，但当注射到玻璃体腔中时，则不是。视网膜下 注射需要玻璃体切除术，这带来了威胁视力的并发症的小但显著的风险。病毒 载体诱导免疫应答，该免疫应答可引起对视网膜细胞的旁观者损伤， 转基因表达的水平和持续时间。非病毒基因转移的免疫原性较低， 大的遗传货物的携带能力，但受到低效转染的限制。我们已经开发 一种新的方法，脉络膜上的非病毒基因转移，它提供了广泛的，长期的，强大的， 脉络膜上腔注射工程化的人视网膜色素上皮细胞和感光细胞中的转基因表达 可生物降解的聚合物纳米颗粒。我们建议优化聚合物结构和纳米粒子 制剂、表达质粒特征和启动子，以最大化水平、拓扑程度和 视网膜和RPE细胞中转基因表达的持续时间。脉络膜上注射是在 门诊诊所设置，可以比视网膜下注射更安全。纳米颗粒将用于非病毒 血管内皮生长因子（VEGF）165的脉络膜上基因转移，以确定其作用是否 与转基因小鼠中VEGF 165高水平表达的结果相当， 大鼠和兔视网膜/脉络膜血管疾病模型。与此同时，我们将创造一种新的生物工程 通过以高亲和力拮抗广泛的VEGF家族配体来阻断血管生成的蛋白质， 其通过新工程化的载体以高水平表达。在脉络膜上注射我们的新的 在非病毒抗VEGF载体中，我们将测量蛋白质表达水平、表达持续时间和治疗效果。 在已建立的视网膜/脉络膜血管疾病模型和我们 开发.这些研究解决了高度流行的眼病中一个主要的未满足的需求， 这是一种非侵入性的方法，可以实现VEGF家族成员的稳健、长期抑制。此夕h 创造一种非病毒基因传递技术和一种新的治疗性抗血管生成蛋白 也可以应用于许多其他疾病。

Project Summary Ocular gene therapy has great potential for sustained expression of anti-angiogenic proteins for retinal/choroidal vascular diseases and also for replacement of mutant genes in patients with inherited retinal degenerations. Viral vectors provide efficient transduction of photoreceptors and retinal pigmented epithelial (RPE) cells when injected into the subretinal space, but not when injected into the vitreous cavity. Subretinal injections require vitrectomy, which carries small but significant risks of vision-threatening complications. Viral vectors induce an immune response which can cause bystander damage to retinal cells and potentially reduce the level and duration of transgene expression. Nonviral gene transfer is less immunogenic and also enables a large carrying capacity for genetic cargos, but has been limited by inefficient transfection. We have developed a new approach, suprachoroidal nonviral gene transfer, which provides widespread, prolonged, robust transgene expression in photoreceptors and RPE cells after suprachoroidal injection of engineered biodegradable polymeric nanoparticles. We propose to optimize the polymer structure and nanoparticle formulation, expression plasmid characteristics, and promoter to maximize the level, topographical extent, and duration of transgene expression in the retina and RPE cells. Suprachoroidal injections are performed in an outpatient clinic setting and can be safer than subretinal injections. The nanoparticles will be used for nonviral suprachoroidal gene transfer of vascular endothelial growth factor (VEGF)165, to determine if effects are comparable to those seen with high level expression of VEGF165 in transgenic mice and to establish new models of retinal/choroidal vascular disease in rats and rabbits. In parallel, we will create a new bioengineered protein that blocks angiogenesis by antagonizing a broad range of VEGF family ligands with high affinity and that is expressed at high levels by the newly engineered vector. After suprachoroidal injection of our new nonviral anti-VEGF vector, we will measure protein expression level, duration of expression, and therapeutic efficacy in established models of retinal/choroidal vascular disease and in the new disease models that we develop. These studies address a major unmet need in highly prevalent eye diseases by developing a noninvasive approach to achieve robust, long term suppression of VEGF family members. Further, the creation of an enabling nonviral gene delivery technology and a novel therapeutic anti-angiogenesis protein could be applicable to many other diseases as well.