Mechanisms and optimization of endosomal escape for cell delivery applications

项目摘要/摘要 细胞递送的内体逃逸机制及其优化 应用 能够将不透细胞的生物制剂输送到活细胞内的试剂有可能 极大地改善人类疾病的治疗，并造福于 将军。例如，将多肽、蛋白质或siRNAs送入细胞可用于 将抑癌基因重新引入癌细胞或敲除致病基因 通过RNA干扰。然而，将大分子输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的发展 治疗。在过去的十年中，细胞穿透肽(CPPs)产生了大量的 热情是因为它们有能力将大分子货物带入细胞。一位少校 使用CPPs的障碍在于，虽然它们能够通过内吞进入细胞，但CPPs 而货物被留在内体中，极大地限制了它们的用途。我们最近做了 发现了一种特定的CPP衍生物的能力，一种荧光的二硫键二聚体 标记的TAT(DfTAT)，以惊人的高效率逃离内小体。 因此，我们进一步证实了dftat可以将蛋白质输送到活细胞中。 轻而易举地。值得注意的是，这种药物介导的内体逃逸并不是 细胞毒性。因此，DfTAT是一种非常有希望的递送代理，它掌握着这个秘密 到有效和安全的细胞渗透。这项提案的目标是建立 DfTAT介导的内体逃逸机制及分子生物学鉴定 本练习所需的蜂窝功能。我们将鉴定其结构和分子。 通过建立急需的结构-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 确定对这一过程有贡献的细胞因素。建议的理由是 研究表明，获得的机械知识将使改进的设计成为可能 能以最佳方式并入治疗相关的内溶试剂 药物输送系统。这反过来又应该极大地促进蛋白质的发展和 基于RNA的疗法，使研究人员和患者受益。

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.