Cell-penetrating peptide adaptors for intracellular cargo delivery

项目总结 代表重要治疗线索的生物分子往往因为无法达到目标而失败， 通常是因为无法穿过细胞膜并到达适当的亚细胞目的地。牢房- 穿透性多肽(CPP)长期以来一直被认为是克服这些失败的巨大希望。他们有能力 通过它们所偶联的分子调节质膜的穿透，从而允许传递 这是一种潜在的变革性平台技术，可以实现一系列特定的 申请。然而，CPP疗法的发展一直令人失望，因为传统的CPP- 货物分子主要被困在内体中，而不是到达细胞质。最大的技术 CPP疗法发展的障碍是无法逃脱内体--我们的技术解决了这个问题 有问题。我们的创新方法是使用高亲和力但可逆的非共价偶联来连接货物 致CPP。我们的CPP-Adaptor融合蛋白TAT-CaM(TAT-CaM)由穿透细胞组成 部分来自HIV转录反式激活因子和人钙调素。Tat-Cam结合CaM结合位点(CBS) 在有钙的情况下含有与NM有亲和力的货物，但在没有钙的情况下可以忽略不计。因为哺乳动物 细胞通常保持较低的静息钙浓度，一旦货物从CPP-适配器上解离 在细胞内，将货物释放到细胞质或其他亚细胞目的地。 此R15区域更新应用描述了为阐明机理、动力学和其他基本原理所做的努力 CPP生物学问题，设计适配器和货物的下一代改进，并开发方法以 使用它们来高效地运送用于研究和治疗目的的货物。这些努力的成功将 验证我们的策略是一种适合于输送包括核酸在内的多种大分子的工具 酸，潜在地使新一代创新疗法和研究工具的开发成为可能。

PROJECT SUMMARY Biomolecules that represent important therapeutic leads often fail because they cannot reach their targets, commonly because of inability to cross cell membranes and reach appropriate subcellular destinations. Cell- penetrating peptides (CPPs) have long held great promise for overcoming these failures. They are capable of mediating penetration of the plasma membrane by molecules to which they are coupled, allowing delivery of ‘cargos’ to cell interiors, a potentially transformative platform technology that can enable an array of specific applications. Nevertheless, development of CPP therapeutics has been disappointing because traditional CPP- cargo molecules largely remain trapped in endosomes rather than reach the cytoplasm. The largest technical hurdle to development of CPP therapeutics is failure to escape from endosomes – our technology solves this problem. Our innovative approach is the use of high affinity but reversible noncovalent coupling to attach cargos to CPPs. Our prototype CPP-adaptor fusion protein, TAT-Calmodulin (TAT-CaM), consists of the cell penetrating moiety from HIV transactivator of transcription and human calmodulin. TAT-CaM binds CaM binding-site (CBS) containing cargos with nM affinity in the presence of calcium but negligibly in its absence. Because mammalian cells typically maintain low resting concentrations of calcium, cargos dissociate from the CPP-adaptor once inside the cell, releasing cargo to the cytoplasm or other subcellular destination. This R15 AREA renewal application describes efforts to elucidate the mechanisms, kinetics and other basic issues of CPP biology, engineer next-generation improvements in adaptors and cargos and develop methods to use them to efficiently deliver cargos for research and therapeutic purposes. Success in these endeavors will validate that our strategy is an adaptable tool for delivery of a wide array of macromolecules including nucleic acids, potentially enabling the development of a new generation of innovative therapeutics and research tools.