Suprachoroidal nonviral gene transfer of engineered VEGF antagonists

项目摘要 眼部基因治疗具有持续表达抗血管生成蛋白的巨大潜力 视网膜/脉络膜血管疾病和遗传性视网膜患者突变基因的替换 堕落的人。病毒载体可有效转导光感受器和视网膜色素上皮 (RPE)细胞注射到视网膜下间隙，但不是注射到玻璃体腔中。视网膜下 注射需要玻璃体切割术，这带来了威胁视力的小而重大的并发症风险。病毒式传播 载体诱导免疫反应，可导致旁观者对视网膜细胞的损害，并可能减少 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，也使 对基因货物的运载能力很大，但由于低效的转基因而受到限制。我们已经开发出 一种新的方法，脉络膜上非病毒基因转移，它提供了广泛、持久、强大的 脉络膜上腔注射基因工程菌后光感受器和RPE细胞中的转基因表达 可生物降解的聚合物纳米颗粒。我们建议对聚合物结构和纳米粒子进行优化 配方、表达质粒特性和启动子，以最大限度地提高水平、地形范围和 转基因在视网膜和RPE细胞中表达的持续时间。脉络膜上腔注射是在 门诊部设置，可比视网膜下注射更安全。这种纳米粒子将用于非病毒性疾病 脉络膜上血管内皮细胞生长因子(VEGF)165的基因转移，以确定是否有 与在转基因小鼠中高水平表达VEGF165的结果进行比较，并建立新的 大鼠和兔视网膜/脉络膜血管疾病模型的建立。同时，我们将创造一种新的生物工程 通过高亲和力和高亲和力拮抗多种血管内皮生长因子家族配体而阻断血管生成的蛋白质 这是由新设计的载体在高水平上表达的。在脉络膜上注射我们的新的 非病毒抗血管内皮生长因子载体，我们将测量蛋白表达水平、表达持续时间和治疗 在已建立的视网膜/脉络膜血管疾病模型和我们发现的新疾病模型中的疗效 发展。这些研究解决了高度流行的眼病中一个主要的未得到满足的需求，方法是开发一种 非侵入性方法实现对血管内皮生长因子家族成员的强有力的、长期的抑制。此外， 可实现非病毒基因传递技术和新型治疗性抗血管生成蛋白的创建 可能也适用于许多其他疾病。

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.