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.

项目概要/摘要 标题：细胞递送的内体逃逸机制和优化 应用 能够在活细胞内传递细胞不可渗透的生物制剂的药物具有潜力 大大提高人类疾病的治疗水平，有利于医学研究 一般的。例如，将肽、蛋白质或 siRNA 递送到细胞中可用于 将肿瘤抑制剂重新引入癌细胞或敲除致病基因 通过RNA干扰。然而，将大分子递送到细胞中的方法是 效率低下，这一瓶颈极大地限制了基于蛋白质或RNA的开发 疗法。在过去的十年中，细胞穿透肽（CPP）产生了大量的 由于它们能够将大分子货物携带到细胞中而受到人们的热情。一个专业 使用 CPP 的障碍在于，虽然它们能够通过内吞作用进入细胞，但 CPP 货物被保留在核内体中，极大地限制了它们的用途。我们最近有 发现了一种特定的 CPP 衍生物（一种荧光二硫键二聚体）的能力 标记的 TAT (dfTAT)，以惊人的高效率逃逸内体。 因此，我们进一步确定 dfTAT 可以将蛋白质递送到活细胞中 轻松地。值得注意的是，该药物介导的内体逃逸不是 细胞毒性。因此，dfTAT 是一种非常有前途的递送代理，它掌握着秘密 有效且安全的细胞渗透。该提案的目标是建立 dfTAT 介导的内体逃逸机制并鉴定分子和 此活动所需的蜂窝功能。我们将确定结构和分子 通过建立急需的结构来确定 dfTAT 内体逃逸的决定因素 活动关系。此外，我们将确定内体渗漏的触发因素和 建立有助于这一过程的细胞因素。拟议的理由 研究表明，获得的机械知识将允许设计改进的 可以最佳地融入治疗相关的内体溶解试剂 药物输送系统。这反过来又会极大地促进蛋白质和 基于 RNA 的疗法使研究人员和患者受益。