Virus-inspired nanoparticles for mucus penetrating gene delivery

项目摘要 虽然粘液屏障提供了天然的保护免受病原体的侵害，并允许营养物质通过， 囊性纤维化（CF）等疾病中的内稳态的破坏导致粘液分泌异常， 功能失调的清除机制因此，晚期CF患者的大多数发病率和死亡率 肺部疾病部分是由于粘液微环境的改变。在CF中，局部粘液环境不 只会促进慢性细菌感染的发展，但它是高度浓缩和粘稠的， 阻止治疗剂有效穿透屏障以矫正受影响的上皮，或 感染.为了改善药物的递送和疗效，关键是增强药物的渗透性 穿过粘液屏障目前的策略集中在亲水性、净中性电荷聚合物上， 改善运输并最小化与粘液的相互作用。然而，目前的技术可能是免疫原性的， 重复给药后，可能表现出次优的细胞摄取。此外，目前还不清楚目前的技术 实现了最大的运输，研究仅限于少数可测试的配方， 均匀的表面化学性质，这可能不是粘液渗透的最佳界面。使用细菌 我们已经从随机的噬菌体的大量组合中鉴定出噬菌体呈递肽， 肽（107-109），其是粘液惰性的并且促进通过粘液屏障的转运。 根据这一发现，该提案的目标是开发模拟粘液的壳聚糖纳米颗粒， 穿透噬菌体并递送靶向缺陷CFTR突变的CRISPR/Cas9以处理细胞培养物 和囊性纤维化的动物模型。我们假设我们的壳聚糖纳米颗粒将实现有效的 CRISPR/Cas9核酸的复合，以及这些纳米颗粒与粘液惰性纳米颗粒的功能化 肽将成功地克服粘液屏障以有效地将基因递送到患病的上皮细胞中。 我们进一步提出穿透肽的组成和分布会改变转运行为 在粘液中。为了验证这些假设，本工作的具体目标将集中在以下几个方面：（1）开发 壳聚糖CRISPR/Cas9复合物包被粘液穿透肽;（2）验证CRISPR/Cas9复合物的快速转运， 这些纳米粒子通过粒子跟踪显微镜和进行诱变研究，以解剖氨基 负责促进粘液渗透的酸;和（3）确认缺陷型的递送和基因校正。 CF支气管上皮细胞系和CF动物模型。这项工作是创新的，因为 模仿粘液穿透噬菌体，我们将开发出一类新的合成壳聚糖， 纳米颗粒能够靶向基因组编辑用于治疗。拟议工作的意义在于 细菌病毒为设计新的基因靶向递送系统提供了灵感，并且从这项工作中， 我们可以开始了解物理化学性质对运输的影响，因此，将提供 全面的设计原则，以开发更有效的基因和药物输送通过粘液屏障。

Project Summary While mucus barriers provide natural protection from pathogens and allow for passage of nutrients, loss of homeostasis in diseases such as cystic fibrosis (CF) results in abnormal mucus secretions with dysfunctional clearance mechanisms. As a result, most morbidity and mortality in CF patients from advanced lung disease is in part due to the altered mucus microenvironment. In CF, the local mucus environment not only promotes the development of chronic bacterial infections, but it is hyperconcentrated and viscous, preventing effective penetration of therapeutics through the barrier to correct the affected epithelia or infections. To improve delivery and efficacy of therapeutics, it is critical to enhance therapeutic penetration through the mucus barrier. Current strategies have focused on hydrophilic, net-neutral charge polymers to improve transport and minimize interactions with mucus. However, current technology may be immunogenic after repeated dosing and may demonstrate suboptimal cellular uptake. Also, it is unclear if current technology achieves maximum transport, and studies have been limited to a small number of testable formulations with uniform surface chemistries, which may not be optimal interfaces for mucus penetration. Using bacterial viruses, i.e. bacteriophage, we have identified phage-presenting peptides from a large combination of random peptides (107-109) that are mucus-inert and facilitate transport through the mucus barrier. From this finding, the objective of this proposal is to develop chitosan nanoparticles that mimic mucus- penetrating bacteriophage and deliver CRISPR/Cas9 targeting defective CFTR mutations to treat cell culture and animal models of cystic fibrosis. We hypothesize that our chitosan nanoparticles will achieve effective complexation of CRISPR/Cas9 nucleic acids, and functionalization of these nanoparticles with mucus-inert peptides will successfully overcome the mucus barrier for effective gene delivery into the diseased epithelia. We further propose that the composition and distribution of penetrating peptides will change transport behavior in mucus. To test these hypotheses, the specific aims of this work will focus on the following: (1) develop chitosan CRISPR/Cas9 complexes coated with mucus penetrating peptides; (2) validate rapid transport of these nanoparticles by particle tracking microscopy and perform mutagenesis studies to dissect the amino acids responsible for facilitating mucus penetration; and (3) confirm delivery and gene correction of defective CF bronchial epithelium cell lines and animal model of CF. The proposed work is innovative because by mimicking mucus-penetrating bacteriophage, we will have developed a new class of synthetic chitosan nanoparticles capable of targeted genomic editing for therapy. The significance of the proposed work is bacterial viruses provided the inspiration for design of new gene targeting delivery systems, and from this work, we can begin to understand physicochemical properties impacts transport, and thus, will provide comprehensive design principles to develop more effective gene and drug delivery through mucus barriers.