Nanoparticles based mRNA delivery for treatment of cystic fibrosis

摘要： 囊性纤维化（CF）是由编码CFTR的基因中≥1200个已知突变中的任何一个引起的，CFTR是一种离子通道。 转运蛋白，但原则上是递送功能性CFTR表达的单一基因治疗剂 先前的尝试已经使用腺相关病毒或脂质体递送系统来递送 CFTR DNA。虽然其中一些达到了I-IIB期临床试验，但它们未能产生一致的， 有效改善患者预后。我们开发了一种新的基于脂质纳米颗粒（LNP）的系统， 传递mRNA，并发现它成功地恢复了55%的正常CFTR介导的氯离子流出， CFTR缺陷小鼠的鼻上皮。为了开发和应用这些新发现，本项目的长期目标是 该项目旨在克服两个关键的剩余生物障碍，这些障碍限制了所有基因治疗载体进入 肺：1）CF的厚的、粘性的气道/肺上皮粘液，其阻碍基因携带者到达气道 细胞，和2）由细胞摄取后遗传物质的胞质生物利用度不足，由于内体 诱捕克服这两个障碍将需要相反的颗粒特性：粘膜渗透 主要通过用粘膜惰性聚合物稳定颗粒来实现， 遗传有效载荷依赖于粒子的不稳定性。迫切需要开发足够稳定的纳米颗粒 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， 粘膜惰性LNP表面。我们发现，用天然胆固醇类似物替代胆固醇， 与临床上成功的含胆固醇的LNP相比，细胞内基因递送增加了200倍。我们 证明基因转移的这一重大改进是通过修饰增强型LNP（eLNP）核心而实现的。 结构和贩运。我们提出的eLNP核心结构的修改将使拆卸成为可能， 内体逃逸和mRNA的胞质递送，同时保持其粘膜惰性表面性质。我们 翻译项目的目标是分析CF肺内的LNP结构和细胞内运输。我们 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， 2）测试和优化改变的核心结构的能力， 增强人CF痰液和现有技术小鼠模型中的粘液渗透，和3）评估 在CF大鼠中通过反复雾化持续经粘膜转染CFTR mRNA，包括 毒理学分析这些研究将显著推进转化治疗，使长期的 恢复CFTR功能以停止或逆转模型CF疾病进展。我们的新方法改变了内部 通过胆固醇的小的结构改性，而不是改性LNP表面涂层或阳离子 与先前研究中的脂质一样。我们的发现将推动翻译新的非病毒载体载体， 穿过厚厚的粘稠粘液和内体屏障，传递有效治疗CF的基因。

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.