Intracellular RNA Nanoparticle Therapeutics to Treat Retinal Neovascularization

摘要 糖尿病性视网膜病变是工业化国家致盲的主要原因， 估计有9500万人（1）。糖尿病性视网膜病变（PDR）和糖尿病性黄斑水肿（DME） 起源于持续升高的葡萄糖水平，导致微血管缺血、视网膜新生血管形成 (RNV)和血管渗漏（2，3）。设计用于隔离游离血管内皮细胞的抗体和治疗剂 生长因子（VEGF）是目前的护理标准（4-8）。由于抗VEGF疗法的半衰期短， 需要每月玻璃体内注射以维持缓解（4-7）。重复注射眼内风险 炎症、感染和眼出血（9）。另一种方法是使用RNA干扰（RNAi） 来抑制致病蛋白的表达。siRNA修饰的最新进展（10）和 FDA批准的第三个siRNA治疗在许多年内证明了RNAi的新潜力， 尽管与其它抗VEGF疗法一样，作用持续时间是关键限制。我们建议评估 通过玻璃体内注射纳米颗粒载体递送细胞内RNA治疗剂以 抑制新血管形成并延长治疗效果的持续时间， 目前的治疗。我们最近证明了玻璃体内注射融合多孔 用于在DL-α-氨基己二酸（DL-AAA）兔中递送VEGF-siRNA的硅纳米颗粒（F-pSiNPs） RNV模型该项目旨在严格测试和优化该系统，以延长疗效。目标1： 将评估两种将siRNA装载到纳米颗粒中的方法：硅酸钙缩合和 环状硅烷。这些系统将优化负载能力，包封效率和体外释放 动力学F-pSiNP系统的融合脂质膜涂层将使用挤出和 溶剂交换法候选制剂将通过光谱、DLS和低温表征。 EM方法。将使用RT-qPCR和流式细胞术在体外验证细胞摄取和作用持续时间。 最有希望的制剂然后将在Aim 2中通过玻璃体内注射使用VEGF-siRNA和 RNV的DL-AAA模型中的Ang-2-siRNA有效载荷（11，12）。F-pSiNP将作为单次剂量给予， 使用荧光素血管造影术监测6个月的血管渗漏变化。这些结果将 以市售的抗体治疗剂阿柏西普和法瑞单抗为基准。在目标3中，F 在目标2中测试的pSiNP制剂将与悬垂表面肽缀合以测试以下假设： 选择性细胞靶向可显著提高功效。靶向和内化肽iRGD 将用于这些研究。iRGD目前正处于临床开发中，以改善患者的化疗吸收。 肿瘤，并选择其靶向细胞表面整合素和神经纤毛蛋白-1的能力，这是 在新血管形成中特征性地过表达。这一拟议的项目是第一步， 开发和测试用于眼内siRNA递送的具有临床和科学效用的新平台。

ABSTRACT Diabetic retinopathy is a leading cause of blindness in the industrialized world and has a global prevalence of an estimated 95 million people (1). Proliferative diabetic retinopathy (PDR) and diabetic macular edema (DME) originate from persistently elevated glucose levels leading to microvascular ischemia, retinal neovascularization (RNV), and vascular leakage (2, 3). Antibodies and therapeutics designed to sequester free vascular endothelial growth factor (VEGF) are the current standard of care (4-8). Due to the short half-life of anti-VEGF therapies, monthly intravitreal injections are needed to maintain remission (4-7). Repeat injections risk intraocular inflammation, infection, and ocular hemorrhage (9). An alternative approach is to use RNA interference (RNAi) to silence the expression of the pathogenic proteins. The recent advancements in siRNA modifications (10) and FDA approval of the third siRNA therapeutic in as many years demonstrate the renewed potential of RNAi, though like other anti-VEGF therapies the duration of action is a key limitation. We propose to evaluate the feasibility of intracellular RNA therapeutics delivered by intravitreal injection of a nanoparticle carrier to inhibit neovascularization and extend the duration of therapeutic efficacy substantially relative to current treatments. We recently demonstrated the effectiveness of intravitreally administered fusogenic porous silicon nanoparticles (F-pSiNPs) for VEGF-siRNA delivery in a DL-alpha-aminoadipic acid (DL-AAA) rabbit model of RNV. This project aims to rigorously test and optimize this system for extended efficacy. In Aim 1, we will evaluate two methods of siRNA loading into the nanoparticles: calcium silicate condensation and grafting of cyclic silanes. These systems will be optimized for loading capacity, encapsulation efficiency, and in vitro release kinetics. The fusogenic-lipid membrane coating of the F-pSiNP system will be optimized using extrusion and solvent exchange methods. The candidate formulations will be characterized by spectroscopic, DLS, and Cryo- EM methods. Cellular uptake and duration of action will be validated in vitro using RT-qPCR and flow cytometry. The most promising formulations will then be tested by intravitreal injection in Aim 2 using VEGF-siRNA and Ang-2-siRNA payloads in the DL-AAA model of RNV (11, 12). F-pSiNPs will be given as a single dose and monitored for 6 months for changes to vascular leakage using fluorescein angiography. These results will then be benchmarked against commercially available antibody therapeutics aflibercept and faricimab. In Aim 3, F- pSiNP formulations tested in Aim 2 will be conjugated with pendent surface peptides to test the hypothesis that selective cellular targeting may dramatically improve efficacy. The targeting and internalization peptide iRGD will be used for these studies. iRGD is currently in clinical development to improve chemotherapeutic uptake in tumors, and was selected for its ability to target cell surface integrins and neuropilin-1, which are characteristically overexpressed in neovascularization. This proposed project represents a first step in developing and testing a novel platform for intraocular siRNA delivery with both clinical and scientific utility.