Cell-Penetrating Peptide-Adaptors for Intracellular Cargo Delivery

用于细胞内货物输送的细胞穿透肽适配器

• 批准号: 9813250
• 负责人: JONATHAN L MCMURRY
• 金额: $ 39.56万
• 依托单位: KENNESAW STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2022-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9813250
• 关键词: Adaptor Signaling Protein; Address; Affinity; Apoptosis; Binding; Binding Sites; Biological Assay; Biology; Biomedical Research; Calcium; Calmodulin; Cell membrane; Cells; Chimeric Proteins; Clathrin; Complex; Confocal Microscopy; Coupled; Coupling; Cytoplasm; Destinations; Development; Doctor of Philosophy; Endocytosis; Endosomes; Environment; Eukaryotic Cell; Failure; Gene Expression; Generations; Genetic Transcription; Goals; HIV; Human; Human Papillomavirus; Kinetics; Label; Malignant neoplasm of cervix uteri; Mammalian Cell; Mediating; Membrane; Mentors; Messenger RNA; Methods; Mitochondria; Nucleic Acid Binding; Nucleic Acids; Oligonucleotides; Participant; Penetration; Peptides; Plasmids; Preparation; Problem Solving; Proteins; Research; Research Training; Rest; Small Interfering RNA; Speed; Students; System; Technology; Testing; Therapeutic; Therapeutic Human Experimentation; Training; Trans-Activators; Transfection; Underrepresented Groups; base; cancer cell; experience; innovation; interest; knock-down; macromolecule; member; nucleic acid binding protein; peptide drug; programs; prototype; recruit; senescence; stem; success; tool; trafficking; undergraduate student

PROJECT SUMMARY Biomolecules that represent important therapeutic leads are often impractical because they cannot reach their targets, most commonly because of failure to cross cell membranes and reach appropriate subcellular destinations. Cell-penetrating peptides (CPPs) hold great promise for overcoming these failures. CPPs 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 and develop methods to use our CPP-adaptors to deliver nucleic acids such as siRNA for research and therapeutic purposes. We will also utilize them to deliver cargo molecules that will demonstrate the broad utility of our system and its advantages over transfection, namely efficiency, speed and tunability. 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 therapeutics and research tools.

项目摘要 代表重要治疗线索的生物分子通常是不切实际的，因为它们不能到达 它们的目标，最常见的是因为未能穿过细胞膜并到达适当的亚细胞， 目的地.细胞穿透肽（CPP）为克服这些失败提供了巨大的希望。cpp是 能够介导与它们偶联的分子穿透质膜， 将“货物”运送到细胞内部，这是一种潜在的变革性平台技术，可以实现一系列 具体应用。然而，CPP治疗剂的开发一直令人失望，因为 传统的CPP-货物分子大部分保持被捕获在内体中，而不是到达细胞质。的 开发CPP治疗剂的最大技术障碍是未能从核内体逃逸-我们的 技术解决了这个问题。 我们的创新方法是使用高亲和力但可逆的非共价偶联将货物连接到 CPP。我们的原型CPP-接头融合蛋白，TAT-CaM，由细胞穿透 部分来自HIV转录反式激活因子和人钙调蛋白。TAT-CaM结合CaM结合位点（CBS） 含有在钙存在下具有nM亲和力但在钙不存在下可忽略不计的货物。因为哺乳动物 细胞通常维持低的静息钙浓度，一旦钙转运蛋白从CPP-接头上解离， 在细胞内，释放货物到细胞质或其他亚细胞目的地。 这个R15区域更新应用程序描述了努力阐明机制，动力学和其他基本 CPP生物学的问题，并开发使用我们的CPP接头递送核酸如siRNA的方法 用于研究和治疗目的我们还将利用它们来运送货物分子， 证明了我们的系统的广泛实用性及其相对于转染的优势，即效率、速度和 可调性这些努力的成功将证明，我们的战略是一个适应性强的工具，可以提供广泛的 包括核酸在内的一系列大分子，有可能开发出新一代 治疗和研究工具。