Acoustically targeted, high-resolution, site-specific, transretinal delivery of macromolecules

项目摘要 我们建议开发一种非手术的，空间精确的，超声增强的方法， 大分子到视网膜。对这种交付方法的需要源于最近成功的普及 通过基因疗法治疗视网膜疾病。遗传性视网膜疾病影响数百万人 在世界范围内，这通常会导致视力丧失和失明。大多数视网膜疾病是无法治愈的，但最近的进展 基因治疗已经恢复了许多人的视力和希望。在动物研究中，将基因输送到眼睛也 帮助揭示了视网膜疾病的机制然而，在临床和动物研究中， 基因对眼睛的影响是一个挑战。目前，遗传物质的递送需要由外科医生进行手术。 玻璃体视网膜外科医生进入视网膜下腔注射遗传物质。这个过程是 技术上很困难，可能导致严重的并发症。在这里，我们将开发一种新的方法， 大分子和基因递送到视网膜，不需要眼睛手术或眼内注射遗传物质。 材料在这种称为增强型经视网膜超声输送（ETUDE）的方法中，我们将联合收割机与我们的 在聚焦超声基因传递和视网膜疾病方面的经验。在ETUDE中，聚焦超声（FUS） 以高精度瞄准视网膜的小区域。然后，临床批准的微泡造影剂 通过外周静脉注射在大脑中，这种造影剂对超声有反应，并产生轻微的 毛细血管内腔的压力。这种压力然后打开血脑屏障（BBB）中的紧密连接 并且允许直径高达~20纳米的分子自由通过。这个BBB开幕式持续了几个 小时，以前曾用于递送小分子、蛋白质和病毒载体。视网膜 包含类似的血管系统，称为血视网膜屏障（BRB）。我们假设BRB和BBB 在存在超声造影剂的情况下与FUS类似地反应， 将分子输送到眼睛。为了实现高精度，高安全性的基因递送到眼睛，我们建议 使用一种创新的超声波空间定位方法。我们会用高频超声波 视网膜中微泡造影剂的超声回声。我们预计目标视网膜~300微米 在小鼠中跨越视网膜厚度的大小区域。在整个项目中，我们将制定安全协议， ETUDE，并定量表征其效率，空间精度，和任何潜在的组织损伤基因 递送静脉内施用的病毒载体，并且能够将细胞类型特异性基因递送至视网膜神经节细胞 （研资局）。如果成功的话，我们将实现一种安全的、非手术的、位点特异性的基因和大分子传递 到特定的视网膜细胞类型。

Project summary We propose to develop a method of non-surgical, spatially precise, ultrasound-enhanced delivery of macromolecules to the retina. The need for such delivery methods stems from the prevalence of recent success in the treatment of retinal disorders by gene therapy. Inherited retinal diseases affect several million people worldwide and often result in vision loss and blindness. Most retinal disorders are incurable, but recent advances in gene therapy have restored vision and hope to many. In animal research, gene delivery to the eye has also helped uncover mechanisms of retinal disease. However, in both the clinic and animal research, delivery of genes to the eye is challenging. Currently, the delivery of genetic material requires surgery performed by a vitreo-retinal surgeon to access the subretinal space for the injection of genetic material. This process is technically difficult and can result in serious complications. Here, we will develop a new method of macromolecule and gene delivery to the retina that does not require eye surgery or intraocular injection of genetic material. In this method called, Enhanced Transretinal Ultrasound Delivery (ETUDE), we combine our experience in focused-ultrasound gene delivery and retinal disorders. In ETUDE, focused ultrasound (FUS) is targeted with high precision to a small region of retina. Then, a clinically-approved microbubble contrast agent is injected through a peripheral vein. In the brain, such a contrast agent responds to ultrasound and exerts mild pressure on interior lumen of capillaries. This pressure then opens the tight junctions in blood-brain barrier (BBB) and allows for free passage of molecules up to ~20 nanometers in diameter. This BBB opening lasts for several hours and has was previously used for delivery of small molecules, proteins, and viral vectors. The retina contains a similar vasculature referred to as a blood-retinal barrier (BRB). We hypothesize that BRB and BBB react similarly to the FUS in presence of an ultrasound contrast agent and will similarly enable site-specific delivery of molecules to the eye. To enable high-precision, high-safety gene delivery to the eye we propose to use an innovative method of spatial targeting of ultrasound. We will use high-frequency ultrasound and record the ultrasound echo of the microbubble contrast agent in the retina. We expect to target retinal ~300-micron sized regions spanning the retinal thickness in mice. Throughout this project, we will develop safe protocols for ETUDE, and quantitatively characterize its efficiency, spatial precision, and any potential tissue damage in gene delivery of intravenously applied viral vectors, and enable cell-type specific gene delivery to retinal ganglion cells (RGC). If successful, we will have enabled a safe, non-surgical, site-specific, gene and macromolecule delivery to specific retinal cell-types.