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 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.然而， lack of mechanistic insights into the cellular trafficking and endosomal escape of nanoparticles has become a major hurdle for efficient intracellular delivery.纳米粒子通过高度进入细胞 dynamic endocytic pathways that are routed towards lysosomes for degradation.本研究的目的是 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 或溶酶体递送途径。 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.这些脂质是 selected based on their properties of influencing cell membrane dynamics, cell signaling and enrichment into the endo/lysosomal system that can trigger endosomal escape.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 microscopy (3D-MM) will be employed to visualize endosomal escape and identify triggers that improve 胞质传递。 Our preliminary investigation using super-resolution microscopy reveals transport of LNP delivered nucleic acids with very high spatiotemporal resolution.我们利用基因改造细胞 were able to pinpoint key stages of LNP mediated mRNA delivery.新型生物活性脂质可以 improve intracellular delivery of mRNA have also been identified and are being interrogated for their 突破内体屏障的能力。我们的目标是解锁载体介导的机制 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 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 foundation, which enables further development and optimization of novel nanoparticles to overcome 细胞屏障并达到药物靶点。

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