Mechanisms of Intracellular trafficking and endosomal escape of nanoparticles for mRNA delivery

用于 mRNA 递送的纳米粒子的细胞内运输和内体逃逸机制

• 批准号: 9232538
• 负责人: Gaurav Sahay
• 金额: $ 44.13万
• 依托单位: OREGON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2020-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9232538
• 关键词: Ablation; Active Sites; Artificial nanoparticles; Biogenesis; Brain; Caveolae; Cell membrane; Cells; Cellular biology; Clathrin; Clinical Trials; Clustered Regularly Interspaced Short Palindromic Repeats; Communicable Diseases; Complex; Confocal Microscopy; Custom; Development; Disease; Drug Targeting; Endocytic Vesicle; Endocytosis; Endocytosis Pathway; Endosomes; Engineering; Enhancers; Family; Foundations; Future; Gene Delivery; Gene Expression; Gene Silencing; Genes; Genetic; Goals; Haploid Cells; Imagery; Investigation; Kinetics; Lead; Libraries; Lipids; Lysosomes; Malignant Neoplasms; Mammalian Cell; Measures; Mediating; Messenger RNA; Methods; Microscopy; Modern Medicine; Monitor; Monomeric GTP-Binding Proteins; Neurodegenerative Disorders; Nucleic Acids; Optics; Organelles; Outcome; Pathway interactions; Permeability; Pharmaceutical Preparations; Process; Production; Property; Proteins; RNA; Recycling; Resolution; Route; Signal Transduction; Site; Sorting - Cell Movement; Surface; System; Techniques; Technology; Testing; Therapeutic; Therapeutic Agents; Tracer; Treatment Efficacy; Vesicle; base; cyanine dye 5; improved; inhibitor/antagonist; insight; lipid mediator; lipid transport; nanoparticle; novel; protein expression; reconstruction; screening; small hairpin RNA; small molecule; spatiotemporal; therapeutic protein; tool; trafficking; treatment group; uptake

Project Summary: RNA therapeutics represents a new class of modern medicine for targets considered undruggable. Nanoparticle based platforms remain the most advanced in clinical trials for RNA based drugs. Yet, the lack of mechanistic insights into the cellular trafficking and endosomal escape of nanoparticles has become a major hurdle for efficient intracellular delivery. Nanoparticles enter cells through highly dynamic endocytic pathways that are routed towards lysosomes for degradation. This study aims to 1) Determine the gateways of cellular entry and subsequent itinerary of lipid nanoparticles (LNP) that deliver messenger RNA (mRNA) inside cells through the use of state of the art microscopy techniques in combination with different markers of endocytosis and/or inhibitors of select trafficking pathways 2) Dissect the productive sites for endosomal escape by utilizing an CRISPR/Cas9 and/or shRNA based library targeted against endosomal proteins, that direct nanoparticles towards the early, recycling, late or lysosomal routes of delivery. The disruption of key steps in endocytic trafficking will trigger release of nanoparticles from vesicular confinement and reveal the active sites for endosomal escape 3) Identify bioactive lipids that improve escape from productive endocytic compartments. These lipids were selected based on their properties of influencing cell membrane dynamics, cell signaling and enrichment into the endo/lysosomal system that can trigger endosomal escape. In these studies custom-built 3D stochastic optical reconstruction microscopy (3D-STORM) and 3D multi-resolution microscopy (3D-MM) will be employed to visualize endosomal escape and identify triggers that improve cytosolic delivery. Our preliminary investigation using super-resolution microscopy reveals transport of LNP delivered nucleic acids with very high spatiotemporal resolution. Using genetically altered cells we were able to pinpoint key stages of LNP mediated mRNA delivery. Novel bioactive lipids that can improve intracellular delivery of mRNA have also been identified and are being interrogated for their ability to breach endosomal barriers. Our goal is to unlock the mechanisms of carrier-mediated intracellular delivery, unravel productive sites of endosomal escape and identify bioactive lipids that can enable intracellular mRNA delivery that will, in the future, lead to efficient production of therapeutic proteins for the treatment of various devastating disorders. Our long-term plan is to build a firm basic foundation, which enables further development and optimization of novel nanoparticles to overcome cellular barriers and reach drug targets.

项目摘要： RNA疗法代表了一类新的现代医学，其目标被认为是不可治疗的。 基于纳米颗粒的平台仍然是基于RNA的药物的临床试验中最先进的。然而 由于缺乏对纳米颗粒的细胞运输和内体逃逸的机理认识， 成为有效细胞内递送的主要障碍。纳米颗粒通过高密度的 动态内吞途径，其被路由到溶酶体以进行降解。本研究旨在：（1） 确定脂质纳米颗粒（LNP）进入细胞的通道和随后的行程， 通过使用最先进的显微技术在细胞内传递信使RNA（mRNA） 与内吞作用的不同标记物和/或选择运输途径的抑制剂组合2） 通过利用基于CRISPR/Cas9和/或shRNA的基因工程技术， 针对内体蛋白质的文库，将纳米颗粒导向早期，再循环，晚期 或溶酶体递送途径。内吞运输中关键步骤的中断将触发 从囊泡限制的纳米颗粒，并揭示内体逃逸的活性位点3）识别 改善从生产性内吞区室逃逸的生物活性脂质。这些脂质是 基于它们影响细胞膜动力学、细胞信号传导和细胞增殖的性质进行选择。 富集到内体/溶酶体系统中，其可触发内体逃逸。在这些研究中 定制的3D随机光学重建显微镜（3D-STORM）和3D多分辨率 显微镜（3D-MM）将用于可视化内体逃逸，并确定改善内体逃逸的触发因素。 胞质递送。我们使用超分辨率显微镜进行的初步研究显示， LNP以非常高的时空分辨率递送核酸。使用基因改变的细胞， 能够精确定位LNP介导的mRNA递送的关键阶段。新的生物活性脂质， 也已经鉴定了mRNA改善的细胞内递送，并正在研究其 突破内体屏障的能力。我们的目标是解开载体介导的 细胞内递送，解开内体逃逸的产生位点，并鉴定可 使细胞内mRNA的传递，这将在未来导致有效的生产治疗， 用于治疗各种破坏性疾病的蛋白质。我们的长期计划是建立一个坚实的基础， 基金会，这使得进一步开发和优化新的纳米粒子，以克服 细胞屏障和到达药物靶点。

项目成果

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA.

• DOI: 10.1021/acs.nanolett.7b02664
• 发表时间: 2017-09-13
• 期刊: Nano letters
• 影响因子: 10.8
• 作者: Patel S;Ashwanikumar N;Robinson E;DuRoss A;Sun C;Murphy-Benenato KE;Mihai C;Almarsson Ö;Sahay G
• 通讯作者: Sahay G