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: 9069937
• 负责人: Jean-Philippe Pellois
• 金额: $ 27.58万
• 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 2019-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9069937
• 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; Health; Knowledge; Label; Lead; Learning; Libraries; Life; 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; Structure; Structure-Activity Relationship; System; Therapeutic; Tumor Suppressor Proteins; Variant; Work; analog; base; biophysical analysis; cancer cell; chemical property; cytotoxic; cytotoxicity; design; dimer; disulfide bond; human disease; improved; in vivo; knock-down; late endosome; macromolecule; prototype; repaired; research study; therapeutic development; tool; trafficking

 DESCRIPTION (provided by applicant): 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.

 描述（由申请人提供）：可以在活细胞内递送细胞不可渗透的生物制剂的试剂有可能极大地改善人类疾病的治疗，并使一般的医学研究受益。将肽、蛋白质或siRNA递送到细胞中可以例如用于将肿瘤抑制因子重新引入癌细胞中或通过RNA干扰敲低致病基因。然而，将大分子递送到细胞中的方法是低效的，并且这一瓶颈极大地限制了基于蛋白质或RNA的疗法的发展。在过去的十年里，细胞穿透肽（CPP）由于其携带大分子货物进入细胞的能力而引起了人们的极大兴趣。使用CPP的一个主要障碍是，虽然它们能够通过内吞作用进入细胞，但CPP和货物被保留在内体中，大大限制了它们的有用性。我们最近发现了一种特定的CPP衍生物，荧光标记的TAT（dfTAT）的二硫键二聚体，以惊人的高效率逃逸内体的能力。因此，我们进一步确定dfTAT可以轻松地将蛋白质递送到活细胞中。值得注意的是，由该试剂介导的内体逃逸不是细胞毒性的。因此，dfTAT是一种非常有前途的递送剂，其拥有有效和安全的细胞渗透的秘密。本提案的目的是建立dfTAT介导的内体逃逸的机制，并确定该活性所需的分子和细胞特征。我们将确定 dfTAT内体的结构和分子决定因素通过建立迫切需要的结构-活性关系而逃逸。此外，我们将确定内体渗漏的触发因素并确定促成这一过程的细胞因素。所提出的研究的基本原理是，所获得的机理知识将允许设计改进的内体溶解试剂，其可以最佳地并入治疗相关的药物递送系统中。这反过来将极大地促进基于蛋白质和RNA的治疗方法的开发，并使研究人员和患者受益。