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 糖尿病性视网膜病是工业化世界失明的主要原因，并且具有全球流行率 估计有9500万人（1）。增殖性糖尿病性视网膜病（PDR）和糖尿病黄斑水肿（DME） 起源于持续升高的葡萄糖水平，导致微血管缺血，视网膜新血管化 （RNV）和血管泄漏（2，3）。旨在隔离免费血管内皮的抗体和治疗剂 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， 需要每月的玻璃体内注射以维持缓解（4-7）。重复注射风险 炎症，感染和眼出血（9）。另一种方法是使用RNA干扰（RNAI） 沉默致病蛋白的表达。 siRNA修改的最新进步（10）和 多年来，FDA批准了第三种siRNA治疗方法，证明了RNAi的新潜力， 尽管像其他抗VEGF疗法一样，动作的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提供的细胞内RNA疗法的可行性 抑制新血管形成并扩大基本上相对于治疗效力的持续时间 当前治疗。我们最近证明了玻璃体内施用的融合多孔的有效性 硅纳米颗粒（F-PSINPS），用于在DL-Alpha-aminoadipic（DL-AAA）兔中递送VEGF-SIRNA RNV的模型。该项目旨在严格测试和优化该系统以提高功效。在AIM 1中，我们 将评估两种siRNA加载到纳米颗粒中的方法：硅酸钙冷凝和移植 环状硅烷。这些系统将被优化，以用于加载能力，封装效率和体外释放 动力学。 F-PSINP系统的Fusogenic-lipid膜膜涂层将通过挤出和 溶剂交换方法。候选制剂的特征是光谱，DLS和冷冻的特征 EM方法。细胞摄取和作用持续时间将在体外使用RT-QPCR和流式细胞仪进行验证。 然后，最有希望的制剂将通过使用VEGF-SIRNA和 Ang-2-SiRNA有效载荷在RNV的DL-AAA模型中（11，12）。 F-psinps将作为单剂量给出 使用荧光素血管造影仪监测6个月以改变血管泄漏。这些结果将 针对商业可用的抗体疗法Aflibercept和Faricimab进行基准测试。在AIM 3中，F- 在AIM 2中测试的PSIN​​P配方将与吊坠表面肽共轭，以检验以下假设。 选择性细胞靶向可能会大大提高功效。靶向和内在化肽IRGD 将用于这些研究。 IRGD目前正在临床开发中，以改善化学治疗摄取 肿瘤，并因其靶向细胞表面整合蛋白和神经蛋白1的能力而被选为 在新血管形成中特征过表达。这个提议的项目代表了第一步 通过临床和科学效用，开发和测试了一个新型的眼内siRNA传递平台。