Gene Delivery in Retinal Diseases

描述(申请人提供)：在治疗视网膜和视网膜色素上皮(RPE)致盲疾病中，将治疗性结构输送到后眼是一个持续存在的问题。为了迎接这一挑战，我们的长期目标是深入了解将基因输送到RPE的细胞外和细胞内贩运。在过去的资助期间，我们证明了报告基因在小鼠视网膜色素上皮细胞中的高表达。最近，我们开发了新的纳米颗粒，由于其特定的尺寸、核心和外壳设计，比以前的纳米颗粒具有许多功能优势。在我们的初步数据中，我们表征了我们的新颗粒的药代动力学。我们发现，它们可以到达视网膜的后段，并有效地转染视网膜细胞，使它们成为实际基因治疗的理想材料。中心假说-透明质酸包被纳米粒(HA-NPs)可以运送其他非病毒方法所不能的货物：1.它们通过亲和力和粘附性穿透组织间隙。2.它们通过脂筏摄取有效地穿透细胞膜。3.在抑制剂的辅助下，它们避免了细胞内和胞内外源DNA宿主的天然防御。4. 它们可以通过微管有效地输送到细胞核。我们的研究将展示HA-NPs如何克服非病毒基因治疗的这些经典障碍：我们将了解这四个关键特征的机制。这些HA-NPs(以及对其传递机制的了解，以及如何操纵它们以优化传递)很可能在基因治疗中有效，而其他非病毒基因治疗方法可能无法做到这一点。对领域的影响：这项研究应该改进基因传递，使患者能够用眼药水自我治疗。使用局部滴眼液将基因治疗药物输送到视网膜后段和RPE是变革性的。

DESCRIPTION (provided by applicant): Delivery of therapeutic constructs to the posterior eye is an ongoing problem in treating blinding diseases of the retina and retinal pigmented epithelium (RPE). To meet this challenge, our long- term goal is to gain insights into extra- and intracellular trafficking in gene delivery to the RPE. In the past grant period, we demonstrated high in vivo expression of reporter genes in mouse RPE following ocular electroporation with plasmid DNA. Recently, we developed new nanoparticles that have many functional advantages over predecessors due to their specific sizing, core, and shell design. In our preliminary data, we characterized the pharmacokinetics of our new particles. We showed that they reach the posterior segment and transfect retinal cells efficiently, making them ideal for practical gene therapy. Central hypotheses-Hyaluronan coated nanoparticles (HA-NPs) can deliver cargos that other nonviral approaches cannot: 1. They penetrate through interstitial spaces through avidity and adhesiveness. 2. They penetrate cell membranes efficiently through lipid-raft uptake. 3. With aid of inhibitors, they avoid intracellular and cytosolic foreign-DNA host innate defenses. 4. They can be efficiently transported to the nucleus on microtubules. Our studies will show how the HA-NPs overcome these classic barriers to nonviral gene therapy: We will learn the mechanisms of these four key features. These HA-NPs (and knowledge of their mechanism of delivery, and how to manipulate them for optimized delivery) are likely to be effective in gene therapy where other nonviral gene therapy approaches might not. Impact on Field: This research should improve gene delivery to the point that a patient can self- treat with eye drops. The use of topical eye drops to deliver gene therapy agents to the posterior segment and the RPE is transformative.