Mechanisms and optimization of endosomal escape for cell delivery applications

细胞递送应用的内体逃逸机制和优化

• 批准号: 9924775
• 负责人: Jean-Philippe Pellois
• 金额: $ 4.39万
• 依托单位: TEXAS A&M AGRILIFE RESEARCH
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-06-01 至 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9924775
• 关键词: Affect; Arginine; Biochemical; Biological; Biological Assay; Cells; Chemicals; Complement; Data; Development; Disease; Drug Delivery Systems; Endocytosis; Endosomes; Extravasation; Foundations; Future; Future Generations; Genes; Hydrophobicity; Knowledge; Label; Lead; Learning; Libraries; Liposomes; Masks; Mediating; Medical Research; Membrane; Methodology; Modeling; Molecular; Nucleic Acids; Organelles; Oxidative Stress; Patients; Penetration; Peptides; Problem Solving; Process; Property; Proteins; RNA; RNA Interference; Reagent; Refractory; Research; Research Personnel; Small Interfering RNA; Structure; Structure-Activity Relationship; System; Therapeutic; Tumor Suppressor Proteins; Variant; analog; base; biophysical analysis; cancer cell; chemical property; cytotoxicity; design; dimer; disulfide bond; experimental study; human disease; improved; in vivo; knock-down; late endosome; macromolecule; nucleic acid-based therapeutics; prototype; repaired; therapeutic development; tool; trafficking

PROJECT SUMMARY/ABSTRACT Title: Mechanisms and optimization of endosomal escape for cell delivery applications Agents that can deliver cell-impermeable biologics inside live cells have the potential to greatly improve the treatment of human diseases and benefit medical research in general. The delivery of peptides, proteins, or siRNAs into cells can for instance be used to reintroduce tumor-suppressors into cancer cells or to knock down disease-causing genes by RNA interference. Yet, methodologies that deliver macromolecules into cells are inefficient and this bottleneck has greatly limited the development of protein or RNA-based therapies. Over the past decade, cell-penetrating peptides (CPPs) have generated a lot of enthusiasm because of their ability to carry macromolecular cargos into cells. A major obstacle to the use of CPPs is that, while they are able to enter cells by endocytosis, CPPs and cargo are retained in endosomes, greatly limiting their usefulness. We have recently uncovered the ability of a specific CPP derivative, a disulfide-bonded dimer of fluorescently labeled TAT (dfTAT), to escape endosomes with astonishingly high efficiency. Consequently, we have further established that dfTAT can deliver proteins into live cells with great ease. Remarkably, the endosomal escape mediated by this agent is not cytotoxic. dfTAT is therefore an extremely promising delivery agents that holds the secret to effective and safe cellular penetration. The objectives of this proposal are to establish the mechanisms of dfTAT-mediated endosomal escape and to identify molecular and cellular features required for this activity. We will identify the structural and molecular determinants of dfTAT endosomal escape by establishing critically needed structure- activity relationships. In addition, we will identify the triggers of endosomal leakage and establish the cellular factors that contribute to this process. The rationale for the proposed research is that the mechanistic knowledge gained will permit the design of improved endosomolytic reagents that can be optimally incorporated into therapeutically relevant drug delivery systems. This should in turn greatly facilitate the development of protein and RNA-based therapeutics and benefit researchers as well as patients.

项目摘要/摘要 标题：内体逃生的机理和优化用于细胞输送 申请 可以在活细胞内传递可侵蚀细胞的生物制剂的药物具有潜力 大大改善人类疾病的治疗并受益于医学研究 一般的。例如 将肿瘤抑制剂重新引入癌细胞或击倒引起疾病的基因 通过RNA干扰。然而，将大分子传递到细胞中的方法是 效率低下，这种瓶颈极大地限制了蛋白质或基于RNA的发展 疗法。在过去的十年中，细胞穿透肽（CPP）产生了很多 热情是因为它们能够将大分子陈代携带到细胞中。专业 使用CPP的障碍是，尽管它们能够通过内吞作用，但CPP可以进入细胞 货物保留在内体中，极大地限制了它们的实用性。我们最近有 发现了特定的CPP衍生物，荧光的二硫键二聚体的能力 标记为TAT（DFTAT），以惊人的效率逃脱内体。 因此，我们进一步确定DFTAT可以将蛋白质输送到活细胞中 轻松。值得注意的是，该试剂介导的内体逃生不是 细胞毒性。因此，DFTAT是一个非常有前途的送货代理商，持有秘密 有效且安全的细胞穿透。该提议的目标是建立 DFTAT介导的内体逃生的机制，并确定分子和 此活动所需的细胞特征。我们将确定结构和分子 DFTAT内体逃生的决定因素通过建立急需的结构 - 活动关系。此外，我们将确定内体泄漏的触发器和 建立有助于此过程的细胞因素。提议的理由 研究是获得的机械知识将允许设计改进 可以最佳地纳入治疗相关的内溶胶试剂 药物输送系统。反过来，这应该大大促进蛋白质和 基于RNA的治疗剂和受益研究人员和患者。