Intracellular RNA Nanoparticle Therapeutics to Treat Retinal Neovascularization

细胞内 RNA 纳米颗粒治疗视网膜新生血管

• 批准号: 10717749
• 负责人: William R. Freeman
• 金额: $ 42.34万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2027-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10717749
• 关键词: Acids; Adverse effects; Affect; Age related macular degeneration; Amines; Animal Diseases; Animal Model; Antibodies; Antibody Therapy; Bacterial Infections; Benchmarking; Biological Assay; Biotinylation; Blindness; Blood Vessels; Calcium; Cell Culture Techniques; Cell Line; Cell surface; Cells; Characteristics; Charge; Chemistry; Clinical; Cryoelectron Microscopy; Data; Daunorubicin; Development; Dexamethasone; Diabetic Retinopathy; Diffusion; Disease; Disease remission; Dose; Drug Delivery Systems; Drug Kinetics; Effectiveness; Emerging Technologies; Encapsulated; Endosomes; Enzyme-Linked Immunosorbent Assay; Extravasation; Eye Hemorrhage; Eye diseases; Flow Cytometry; Fluorescein; Fluorescein Angiography; Formulation; Frequencies; Glucose; Goals; Half-Life; Homeostasis; Image; Immunohistochemistry; In Vitro; Industrialization; Infection; Inflammation; Injections; Injury; Integrins; Ischemia; Kinetics; Label; Lipids; Measurable; Measures; Membrane Lipids; Methodology; Methods; Modeling; Modification; Monitor; Nature; Neuropilin-1; Oryctolagus cuniculus; Outcome; Pathogenicity; Penetration; Peptides; Periodicity; Persons; Pharmaceutical Preparations; Pharmacotherapy; Physical condensation; Porosity; Prevalence; Protein Biosynthesis; Proteins; RNA; RNA Interference; RNA Interference Therapy; RNA Sequences; RNA delivery; Research; Retina; Retinal Neovascularization; Risk; Safety; Silanes; Silicates; Silicon; Sirolimus; Site; Small Interfering RNA; Solvents; Specificity; Structure; Subgroup; Surface; System; Technology; Testing; Therapeutic; Therapeutic Agents; Therapeutic antibodies; Time; Tissues; Treatment Efficacy; United States Food and Drug Administration; Vascular Endothelial Growth Factors; Western Blotting; animal tissue; bevacizumab; biological systems; biomaterial compatibility; cellular targeting; clinical development; cytotoxicity; delivery vehicle; design; diabetic; geographic atrophy; improved; in vivo; interest; intravitreal injection; knock-down; macular edema; nanoparticle; nanosized; neovascular; neovascularization; novel; novel therapeutics; overexpression; particle; physical property; proliferative diabetic retinopathy; randomized, clinical trials; retinal toxicity; side effect; standard of care; therapeutic RNA; therapeutic nanoparticles; therapy duration; tumor; uptake

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.

抽象的 糖尿病视网膜病变是工业化国家失明的主要原因，并且在全球范围内患病率高达 估计有 9500 万人 (1)。增殖性糖尿病视网膜病变（PDR）和糖尿病性黄斑水肿（DME） 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)。旨在隔离游离血管内皮的抗体和疗法 生长因子 (VEGF) 是当前的护理标准 (4-8)。由于抗 VEGF 疗法的半衰期短， 需要每月进行一次玻璃体内注射以维持缓解(4-7)。重复注射有眼内风险 炎症、感染和眼部出血 (9)。另一种方法是使用 RNA 干扰 (RNAi) 沉默致病蛋白的表达。 siRNA 修饰的最新进展 (10) 和 FDA 批准了多年来第三种 siRNA 治疗药物，证明了 RNAi 的新潜力， 尽管与其他抗 VEGF 疗法一样，作用持续时间是一个关键限制。我们建议评估 通过玻璃体内注射纳米颗粒载体进行细胞内 RNA 治疗的可行性 相对于其他药物，抑制新血管形成并显着延长治疗效果的持续时间 目前的治疗方法。我们最近证明了玻璃体内注射融合多孔的有效性 硅纳米颗粒 (F-pSiNPs) 用于 DL-α-氨基己二酸 (DL-AAA) 兔子体内的 VEGF-siRNA 递送 RNV 模型。该项目旨在严格测试和优化该系统以延长功效。在目标 1 中，我们 将评估两种将 siRNA 加载到纳米颗粒中的方法：硅酸钙缩合和接枝 环状硅烷。这些系统将针对负载能力、封装效率和体外释放进行优化 动力学。 F-pSiNP 系统的融合脂质膜涂层将通过挤出和 溶剂交换法。候选配方将通过光谱、DLS 和冷冻表征 电磁法。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。 然后，最有希望的制剂将使用 VEGF-siRNA 在 Aim 2 中通过玻璃体内注射进行测试， RNV DL-AAA 模型中的 Ang-2-siRNA 有效负载 (11, 12)。 F-pSiNPs 将作为单剂量给药，并且 使用荧光素血管造影术监测血管渗漏变化 6 个月。然后这些结果将 以市售抗体疗法阿柏西普和法里西单抗为基准。在目标 3 中，F- 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： 选择性细胞靶向可以显着提高疗效。靶向和内化肽 iRGD 将用于这些研究。 iRGD 目前正在进行临床开发，以提高化疗药物的吸收率 肿瘤，并因其靶向细胞表面整合素和神经毡蛋白-1 的能力而被选中，这 在新血管形成中特征性地过度表达。这个拟议的项目代表了第一步 开发和测试具有临床和科学实用性的眼内 siRNA 递送的新型平台。