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.

项目概要： RNA疗法代表了一类新的现代医学，针对的是不可成药的靶点。 基于纳米颗粒的平台仍然是基于 RNA 的药物临床试验中最先进的平台。然而， 缺乏对纳米粒子的细胞运输和内体逃逸的机制认识 成为高效细胞内递送的主要障碍。纳米粒子通过高度进入细胞 流向溶酶体进行降解的动态内吞途径。本研究的目的是 1) 确定脂质纳米颗粒 (LNP) 的细胞进入途径和后续行程 通过使用最先进的显微镜技术将信使 RNA (mRNA) 传递到细胞内 与不同的内吞作用标记物和/或选定运输途径的抑制剂组合2) 利用基于 CRISPR/Cas9 和/或 shRNA 的技术剖析内体逃逸的生产位点 针对内体蛋白的文库，将纳米粒子引导至早期、回收、晚期 或溶酶体递送途径。内吞运输关键步骤的破坏将触发释放 来自囊泡限制的纳米颗粒并揭示内体逃逸的活性位点 3) 识别 生物活性脂质可改善从生产性内吞区室的逃逸。这些脂质是 根据其影响细胞膜动力学、细胞信号传导和 富集到内/溶酶体系统中，可以触发内体逃逸。在这些研究中 定制 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识别改善的触发因素 胞质传递。我们使用超分辨率显微镜的初步研究揭示了 LNP 以非常高的时空分辨率传递核酸。我们利用基因改造细胞 能够查明 LNP 介导的 mRNA 传递的关键阶段。新型生物活性脂质可以 改善 mRNA 的细胞内递送也已被鉴定，并正在对其进行研究 突破内体屏障的能力。我们的目标是解锁载体介导的机制 细胞内递送，解开内体逃逸的生产位点并鉴定可以的生物活性脂质 实现细胞内 mRNA 传递，这将在未来实现治疗药物的高效生产 用于治疗各种破坏性疾病的蛋白质。我们的长远计划是打牢基础 基础，从而能够进一步开发和优化新型纳米颗粒以克服 细胞屏障并达到药物靶点。

项目成果

Boosting Intracellular Delivery of Lipid Nanoparticle-Encapsulated mRNA.

促进脂质纳米粒子封装的 mRNA 的细胞内递送。

• 发表时间: 2017-09-13
• 影响因子: 10.8
• 作者: Patel, Siddharth;Ashwanikumar, N;Robinson, Emily;DuRoss, Allison;Sun, Conroy;Murphy;Mihai, Cosmin;Almarsson, Örn;Sahay, Gaurav
• 通讯作者: Sahay, Gaurav

Advances in intracellular delivery through supramolecular self-assembly of oligonucleotides and peptides.

通过寡核苷酸和肽的超分子自组装进行细胞内递送的进展。

• 发表时间: 2019
• 影响因子: 12.4
• 作者: Kim, Jeonghwan;Narayana, Ashwanikumar;Patel, Siddharth;Sahay, Gaurav
• 通讯作者: Sahay, Gaurav