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Virus-inspired nanoparticles for mucus penetrating gene delivery

受病毒启发的纳米粒子用于粘液穿透基因传递

基本信息

批准号：
9921468
负责人：
Debadyuti Ghosh
金额：
$ 44.77万
依托单位：
UNIVERSITY OF TEXAS AT AUSTIN
依托单位国家：
美国
项目类别：
财政年份：
2017
资助国家：
美国
起止时间：
2017-08-15 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9921468
关键词：
Address Affect Affinity Amino Acids Animal Model Antibodies Bacterial Infections Bacteriophages Behavior Breathing CRISPR/Cas technology Cell Culture Techniques Cell Line Charge Chemicals Chemistry Chitosan Chronic Chronic Obstructive Airway Disease Complex Cystic Fibrosis Cystic Fibrosis Transmembrane Conductance Regulator Cystic Fibrosis sputum DNA Data Development Diffuse Diffusion Disease Disease model Dose Drug Delivery Systems Drug Targeting Electrostatics Engineering Environment Epithelial Epithelium Epitopes Formulation Gene Delivery Gene Targeting Genes Goals HIV Health Homeostasis Human Impairment Infection Knowledge Lung diseases Microscopy Mission Modeling Morbidity - disease rate Mucins Mucociliary Clearance Mucous Membrane Mucous body substance Mutagenesis Mutation Nucleic Acids Nutrient Oligonucleotides Patient-Focused Outcomes Patients Penetration Peptides Permeability Phage Display Plasmids Polymers Property Public Health Role Saline Sampling Small Interfering RNA Spatial Distribution Surface Synthetic Genes System Technology Testing Therapeutic Treatment Efficacy United States National Institutes of Health Virus Work base biological systems bronchial epithelium cystic fibrosis mucus cystic fibrosis patients design ethylene glycol gene correction gene therapy genome editing hydrophilicity immunogenic immunogenicity improved in vivo in vivo Model innovation insight mortality nanocomplexes nanoparticle nanoscale novel nucleic acid-based therapeutics particle pathogen prevent reconstitution screening self assembly small molecule targeted delivery therapeutic genome editing tool uptake vector

项目摘要

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.

项目摘要虽然粘液屏障提供了对病原体的天然保护，并允许营养物质通过，但损失囊性纤维化(CF)等疾病的动态平衡导致异常粘液分泌功能失调的清除机制。因此，晚期CF患者的发病率和死亡率最高肺部疾病的部分原因是粘液微环境的改变。在CF中，局部粘液环境不是只会促进慢性细菌感染的发展，但它是高度浓缩和粘性的，阻止治疗药物有效地穿透屏障以纠正受影响的上皮细胞或感染。为了改善治疗药物的传递和疗效，提高治疗渗透率是至关重要的。通过粘液屏障。目前的策略主要集中在亲水性、净中性电荷聚合物上改善运输，最大限度地减少与粘液的相互作用。然而，目前的技术可能具有免疫原性。在重复给药后，可能会显示出次佳的细胞摄取。此外，目前还不清楚目前的技术是否实现了最大限度的传输，研究仅限于少量的可测试配方均匀的表面化学，这可能不是粘液渗透的最佳界面。使用细菌病毒，即噬菌体，我们已经从大量的随机组合中鉴定出噬菌体提呈肽多肽(107-109)是粘液惰性的，有助于通过粘液屏障运输。根据这一发现，这项提议的目标是开发模仿粘液的壳聚糖纳米颗粒- 穿透噬菌体并靶向CFTR缺陷突变的CRISPR/Cas9治疗细胞培养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达到有效的 CRISPR/Cas9核酸的络合及其与粘液惰性物的功能化多肽将成功地克服粘液屏障，有效地将基因输送到病变的上皮细胞。我们进一步提出，穿透性多肽的组成和分布将改变转运行为在粘液中。为了验证这些假设，这项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复合体粘液穿透性多肽；(2)验证快速转运这些纳米粒子通过粒子跟踪显微镜进行突变研究，以解剖氨基负责促进粘液渗透的酸；以及(3)确认缺陷的交付和基因纠正慢性支气管炎的支气管上皮细胞株及动物模型。拟议的工作具有创新性，因为通过模仿粘液穿透噬菌体，我们将开发出一种新的合成壳聚糖能够进行靶向基因组编辑以用于治疗的纳米颗粒。拟议工作的意义在于细菌病毒为设计新的基因靶向传递系统提供了灵感，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，从而提供全面的设计原则，通过粘液屏障开发更有效的基因和药物输送。