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

项目总结 我们建议开发一种非手术的、空间精确的、超声增强的输送方法 大分子进入视网膜。对这种分娩方法的需求源于最近成功的流行 用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 在全世界范围内，并经常导致失明和失明。大多数视网膜疾病是无法治愈的，但最近的进展 在基因治疗方面已经恢复了许多人的视力和希望。在动物研究中，基因传递到眼睛也 帮助揭示了视网膜疾病的机制。然而，在临床和动物研究中，分娩 基因对眼睛的影响是具有挑战性的。目前，遗传物质的运送需要由一名 玻璃体-视网膜外科医生进入视网膜下间隙进行遗传物质注射。这个过程是 技术上困难，可能导致严重的并发症。在这里，我们将开发一种新的方法 不需要眼部手术或眼内注射基因的大分子和基因输送到视网膜 材料。在这种名为增强型经视网膜超声传输(Etude)的方法中，我们结合了我们的 在聚焦超声基因输送和视网膜疾病方面的经验。在Etude中，聚焦超声(FUS) 以高精度定位于视网膜的一小部分。然后，临床批准的微泡造影剂 通过外周静脉注射。在大脑中，这样的造影剂对超声波产生反应，并产生温和的影响 毛细血管内腔的压力。这种压力会打开血脑屏障(Bbb)的紧密连接。 并允许直径达~20纳米的分子自由通过。这场BBB开幕式将持续几天 数小时，以前曾用于运送小分子、蛋白质和病毒载体。视网膜 含有一种类似的血管系统，称为血-视网膜屏障(BRB)。我们假设BRB和BBB 在超声造影剂存在的情况下，与FUS的反应类似，并将类似地实现部位特异性 将分子输送到眼睛。为了能够将高精度、高安全性的基因输送到眼睛，我们建议 使用一种创新的超声空间靶向方法。我们将使用高频超声波并记录 视网膜内微泡造影剂的超声回声。我们预计目标是视网膜~300微米 小鼠视网膜厚度的大小区域。在整个项目中，我们将为 并定量表征其效率、空间精确度以及基因中任何潜在的组织损伤 静脉应用病毒载体的传递，并使细胞类型特异性基因传递到视网膜神经节细胞 (研资局)。如果成功，我们将实现一种安全的、非手术的、特定部位的基因和大分子输送 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.