Cell-penetrating peptide adaptors for intracellular cargo delivery

用于细胞内货物递送的细胞穿透肽接头

• 批准号: 10653590
• 负责人: JONATHAN L MCMURRY
• 金额: $ 40.65万
• 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 2026-04-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10653590
• 关键词: Adaptor Signaling Protein; Address; Affinity; Architecture; Binding; Binding Sites; Biochemical; Biological Assay; Biology; Calcium; Calmodulin; Cell Fractionation; Cell Surface Receptors; Cell membrane; Cells; Charge; Chimeric Proteins; Complex; Confocal Microscopy; Coupled; Coupling; Cytoplasm; Destinations; Development; Dissociation; Doctor of Philosophy; Dose; Endosomes; Engineering; Failure; Fluorescence; Future; Generations; Goals; Human; Kinetics; Light; Link; Location; Mammalian Cell; Mediating; Mentors; Methods; Nucleic Acids; Participant; Penetration; Peptides; Preparation; Proteins; Research; Research Personnel; Research Training; Rest; Sampling; System; Technology; Testing; Therapeutic; Time; Training; Underrepresented Populations; design; experience; experimental study; improved; innovation; macromolecule; member; movie; next generation; peptide drug; programs; prototype; recruit; success; tat Protein; technology platform; tool; undergraduate student

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.

项目概要 代表重要治疗线索的生物分子常常会失败，因为它们无法达到目标， 通常是因为无法穿过细胞膜并到达适当的亚细胞目的地。细胞- 长期以来，穿透肽（CPP）在克服这些失败方面有着巨大的希望。他们有能力 介导与其偶联的分子对质膜的渗透，从而允许递送 “货物”到细胞内部，这是一种潜在的变革性平台技术，可以实现一系列特定的 应用程序。然而，CPP 疗法的发展一直令人失望，因为传统的 CPP- 货物分子大部分仍被困在内体中而不是到达细胞质。最大的技术 CPP 疗法开发的障碍是未能逃离内体——我们的技术解决了这个问题 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附着货物 给 CPP。我们的原型 CPP 接头融合蛋白 TAT-钙调蛋白 (TAT-CaM) 由细胞穿透性 来自HIV转录反式激活子和人钙调蛋白的部分。 TAT-CaM 结合 CaM 结合位点 (CBS) 含有钙的情况下具有 nM 亲和力的货物，但在钙不存在的情况下，亲和力可以忽略不计。因为哺乳动物 细胞通常维持较低的静息钙浓度，一旦货物与 CPP 适配器分离 在细胞内部，将货物释放到细胞质或其他亚细胞目的地。 该 R15 AREA 更新申请描述了阐明机制、动力学和其他基本原理的努力 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 使用它们有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应性强的工具，可用于递送包括核酸在内的多种大分子 酸，有可能促进新一代创新疗法和研究工具的开发。