Nanoparticles based mRNA delivery for treatment of cystic fibrosis

基于纳米颗粒的 mRNA 递送治疗囊性纤维化

基本信息

批准号：
9898461
负责人：
Gaurav Sahay
金额：
$ 66.67万
依托单位：
OREGON STATE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-04-01 至 2024-02-29
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9898461
关键词：
Aerosols Apical Atomic Force Microscopy Biological Biological Availability Bronchoalveolar Lavage Fluid Carrier Proteins Cations Cells Characteristics Chemicals Chloride Channels Chlorides Cholesterol Clinical Clinical Trials Complex Confocal Microscopy Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cystic Fibrosis sputum DNA Dehydration Disease Disease Progression Dose Electrons Engineering Epithelial Epithelium Exposure to Gene Delivery Gene Expression Gene Therapy Agent Gene Transduction Agent Gene Transfer Genes Genetic Materials Goals Half-Life Histology Human In Vitro Inflammatory Inhalation Investigation Ions Knockout Mice Label Lipids Liquid substance Location Lung Lung diseases Measures Mediating Messenger RNA Modeling Modification Morphology Movement Mucous Membrane Mucous body substance Mus Mutation Nasal Epithelium Nebulizer Non-Viral Vector Nucleic Acids Obstruction Particle Size Patient-Focused Outcomes Patients Penetration Pharmaceutical Preparations Phase Polymers Process Production Property Pulmonary Fibrosis Pulmonary Pathology RNA Rattus Recovery Safety Sputum Structure Surface Surface Properties System Testing Therapeutic Effect Thick Tissues Toxic effect Toxicology Transfection Translations Variant Viral X ray diffraction analysis aerosolized analog base cholesterol analog cystic fibrosis mucus cystic fibrosis patients cytokine disease phenotype epithelial Na+ channel gene correction gene replacement gene therapy genetic payload genotyped patients improved in vivo lipid nanoparticle liposomal delivery loss of function mRNA delivery mouse model nanocarrier nanoparticle next generation novel novel strategies overexpression particle prevent protein expression protein function restoration success surface coating trafficking uptake

项目摘要

ABSTRACT: Cystic fibrosis (CF) is conferred by any of ≥1200 known mutations in the gene encoding the CFTR, an ion transporter protein, but in principle, a single gene therapy agent that delivers expression of functional CFTR could treat. Prior attempts to do so have used adeno-associated viral or liposomal delivery systems to deliver CFTR DNA. Although some of these reached Phase I-IIB clinical trials, they failed to produce consistent, impactful improvement of patient outcomes. We developed a novel lipid nanoparticle (LNP)-based system to deliver mRNA, and found it successfully restored up to 55% of normal CFTR-mediated chloride efflux in the nasal epithelium of CFTR-deficient mice. To exploit and apply these novel discoveries, the long-term goal of this project is to overcome two key remaining biological barriers that limit entry of all gene therapy vectors into the lung: 1) the thick, sticky airway/lung epithelial mucus of CF that impedes gene carriers from reaching airway cells, and 2) inadequate cytosolic bioavailability of genetic material after uptake by cells, due to endosomal entrapment. Overcoming these two obstacles will require opposing particle characteristics: mucosal penetration is achievable primarily by stabilizing particles with a muco-inert polymer, whereas intracellular bioavailability of genetic payloads relies on particle destabilization. There is urgent need to develop nanoparticles stable enough to cross CF mucus and reach airway cells, yet labile enough to facilitate endosomal escape of genetic material following cellular uptake. We will meet these criteria, by altering the stability of the LNP core, while maintaining a muco-inert LNP surface. We found that replacing cholesterol with its naturally-occurring analogues improves intracellular gene delivery by 200-fold, vs. that seen with a clinically successful cholesterol-containing LNP. We posit that this major improvement in gene transfer occur via modifications in the enhanced LNP (eLNPs) core structure and trafficking. Modifications in eLNPs' core structure as we propose will enable disassembly, endosomal escape and cytosolic delivery of mRNA, while maintaining their muco-inert surface properties. Our translational project's goal is to analyze LNP structure and intracellular trafficking within the CF lung. Our objectives are thus 1) elucidate and optimize the structural features of nanocarriers that drive endosomal trafficking of eLNPs and enhanced gene delivery, 2) test and optimize the ability of altered core structures to enhance mucopenetration across human CF sputum and state of art mice models, and 3) assess safety of sustained transmucosal transfection of CFTR mRNA via repeated aerosolization in CF rats, including toxicological analyses. These studies will significantly advance translational therapy, enabling long-term restoration of CFTR function to halt or reverse model CF disease progression. Our novel approach alters internal particle stability via small structural modifications of cholesterol, rather modifying LNP surface coating or cationic lipid as in prior studies. Our discoveries will propel toward translation new non-viral vector carriers that can traverse the thick sticky mucus and endosomal barriers to deliver genes for efficacious CF treatment.

抽象的：囊性纤维化 (CF) 是由编码 CFTR 的基因中任何 ≥1200 个已知突变引起的，CFTR 是一种离子转运蛋白，但原则上是一种可表达功能性 CFTR 的单一基因治疗剂之前的尝试是使用腺相关病毒或脂质体递送系统来递送。尽管其中一些达到了 I-IIB 期临床试验，但它们未能产生一致的、我们开发了一种基于脂质纳米颗粒（LNP）的新型系统来有效改善患者的治疗效果。递送 mRNA，发现它成功恢复了正常 CFTR 介导的氯离子流出量的 55% 开发和应用这些新发现是本研究的长期目标。该项目旨在克服限制所有基因治疗载体进入市场的两个关键的生物障碍肺：1) 浓稠、粘稠的气道/肺上皮粘液 CF，阻碍基因携带者到达气道细胞，以及 2) 由于内体的原因，细胞摄取后遗传物质的胞质生物利用度不足克服这两个障碍将需要相反的颗粒特性：粘膜渗透。主要通过用粘液惰性聚合物稳定颗粒来实现，而细胞内生物利用度遗传有效载荷依赖于粒子不稳定，迫切需要开发足够稳定的纳米粒子。穿过CF粘液并到达气道细胞，但足够不稳定以促进遗传物质的内体逃逸在细胞摄取后，我们将通过改变 LNP 核心的稳定性来满足这些标准，同时保持不变。我们发现，用天然存在的类似物替代胆固醇可以改善粘液惰性 LNP 表面。与临床上成功的含胆固醇 LNP 相比，细胞内基因传递提高了 200 倍。假设基因转移的这一重大改进是通过增强 LNP (eLNP) 核心的修改而实现的我们建议的 eLNP 核心结构的修改将能够实现拆卸、 mRNA 的内体逃逸和胞质传递，同时保持其粘膜惰性表面特性。转化项目的目标是分析 CF 肺内的 LNP 结构和细胞内运输。因此，目标是1）阐明和优化驱动内体的纳米载体的结构特征 eLNP 的运输和增强的基因传递，2) 测试和优化改变的核心结构的能力增强对人类 CF 痰和最先进小鼠模型的粘液渗透性，以及 3) 评估通过反复雾化对 CF 大鼠进行持续跨粘膜转染 CFTR mRNA，包括这些研究将显着推进转化治疗，从而实现长期治疗。恢复 CFTR 功能以阻止或逆转模型 CF 疾病进展。通过胆固醇的小结构修饰（而不是修饰 LNP 表面涂层或阳离子）来提高颗粒稳定性正如之前的研究一样，我们的发现将推动新的非病毒载体载体的转化。穿过厚厚的粘液和内体屏障，传递有效治疗 CF 的基因。