Mechanisms and optimization of endosomal escape for cell delivery applications

 描述(由申请人提供)：能够在活细胞内提供细胞不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，并总体上有利于医学研究。例如，将多肽、蛋白质或siRNAs输送到细胞中可以用来将肿瘤抑制因子重新引入癌细胞，或者通过RNA干扰来敲除致病基因。然而，将大分子输送到细胞内的方法效率低下，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(CPPs)因其携带大分子货物进入细胞的能力而引起了人们的极大热情。使用CPPs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CPPs和货物保留在内吞体内，极大地限制了它们的用途。我们最近发现了一种特殊的CPP衍生物，一种荧光标记的TAT的二硫键二聚体(DfTAT)，能够以惊人的高效率逃脱内体。因此，我们进一步证实了dfTAT可以非常容易地将蛋白质输送到活细胞中。值得注意的是，这种药物介导的内体逃逸不具有细胞毒性。因此，dfTAT是一种非常有前途的递送剂，它掌握着有效和安全的细胞渗透的秘密。这项建议的目的是建立dfTAT介导的内体逃逸的机制，并确定这一活动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急需的结构-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。拟议研究的基本原理是，所获得的机制知识将使改进的内溶试剂的设计成为可能，这些试剂可以以最佳方式并入与治疗相关的药物输送系统。这反过来将极大地促进基于蛋白质和RNA的疗法的发展，并使研究人员和患者受益。

 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.