Nanoparticles based mRNA delivery for treatment of cystic fibrosis

摘要： 囊性纤维化(CF)是由编码≥基因的任何已知突变引起的 转运蛋白，但原则上，是一种能够表达功能性CFTR的单一基因治疗剂 可以治疗。以前的尝试是使用腺相关病毒或脂质体递送系统来传递 CFTRDNA。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、 对患者预后的有效改善。我们开发了一种新型的基于脂质纳米颗粒(LNP)的系统来 发现它成功地恢复了高达55%的正常CFTR介导的氯外流。 Cftr基因缺陷小鼠的鼻黏膜上皮。为了开发和应用这些新发现，这一长期目标是 项目是克服两个关键的剩余生物障碍，这两个障碍限制了所有基因治疗载体进入 肺：1)厚厚、粘稠的呼吸道/肺上皮样纤维粘液，阻碍基因携带者到达呼吸道 细胞，以及2)由于内体，遗传物质被细胞摄取后在胞内的生物利用度不足 诱捕。克服这两个障碍将需要相反的颗粒特性：粘膜穿透 主要通过用粘液惰性聚合物稳定颗粒来实现，而细胞内的生物利用度 基因有效载荷依赖于粒子的不稳定。迫切需要开发出足够稳定的纳米颗粒 穿过CF粘液并到达呼吸道细胞，但足够不稳定，以促进遗传物质的内体逃逸 在细胞摄取之后。我们将满足这些标准，通过改变LNP核心的稳定性，同时保持 粘液惰性的LNP表面。我们发现，用天然的类似物替代胆固醇可以改善 与临床上成功的含胆固醇LNP相比，细胞内基因传递增加了200倍。我们 假设基因转移的这种重大改进是通过对增强型LNP(ELNPs)核心的修饰来实现的 结构和贩运。我们提议的对eLNPs核心结构的修改将使得能够拆卸， 胞体逃逸和胞浆递送信使核糖核酸，同时保持其粘液惰性表面特性。我们的 翻译项目的目标是分析LNP的结构和在CF肺内的细胞内运输。我们的 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 ELNPs的转移和增强的基因传递，2)测试和优化改变的核心结构的能力 增强人类CF痰和最新小鼠模型的粘液开放能力，以及3)评估 经反复雾化吸入CFTRmRNA在CF大鼠体内的持续黏膜转染法 毒物分析。这些研究将极大地推进翻译治疗，使长期 CFTR功能的恢复，以阻止或逆转模型CF疾病的进展。我们的新方法改变了内部 通过胆固醇的微小结构修饰，而不是修饰LNP表面涂层或阳离子来实现颗粒稳定性 和以前的研究一样，脂类。我们的发现将推动转化新的非病毒载体，这些载体可以 穿越粘稠的粘液和内体屏障，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.