EFFICIENT NUCLEAR DELIVERY VECTOR FOR MACROMOLECULES

大分子的高效核传递载体

• 批准号: 6021317
• 负责人: ROBERT A DUBIN
• 金额: $ 9.31万
• 依托单位: PROFILE DIAGNOSTIC SCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-10 至 2000-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6021317
• 关键词: biotin; chimeric proteins; gene targeting; herpes simplex virus 1; histidine; macromolecule; molecular cloning; plasmids; protein purification; protein structure; technology /technique development; tissue /cell culture; transfection /expression vector; virus protein

The ability to efficiently introduce functional, exogenous macromolecules into living cells is severely restricted by the plasma membrane. The development of vectors to overcome this barrier, such as disabled virus, cationic lipids, and direct injection, currently offer the potential for advances in therapeutic intervention and basic genetic research. We propose to develop a novel, flexible, generic, and highly efficient protein and nucleic acid delivery system based upon VP22, a structural protein of the herpes simplex virus 1. VP22 exhibits a unique form of intracellular transfer, whereby it is capable of efficiently exiting the mammalian cell in which it is synthesized, diffusing locally, entering a neighboring cell, and translocating to the recipient cell nucleus. Significantly, fusion proteins between VP22 and a second polypeptide chain retain functional characteristics of both proteins. We propose to develop a specifically modified version of VP22 that will be capable of delivering functional nucleic acid or protein to the nucleus of cells. Our modified VP22 protein will be prepared and incubated in vitro with tagged protein or nucleic acid. Based upon the extremely high affinity between modified VP22 and the tag, modified VP22 will form a tightly bound complex with the tagged macromolecule. Presentation of this complex to naive cells will result in complex uptake and nuclear translocation through the action of VP22; the delivered DNA or protein will retain biological activity. In this Phase I study we will construct, express, and characterize the modified VP22 and demonstrate its ability to efficiently deliver functional DNA and protein into tissue culture cells. Subsequent Phase II research will enhance the system for increased flexibility and explore the use of modified VP22 for the delivery of protein and DNA into tissues of the intact mouse. PROPOSED COMMERCIAL APPLICATION This research is directed towards developing a generalized technology for easily and efficiently introducing functional protein and nucleic acid to the nucleus of living cells. Potential applications include delivery vector for therapeutic intervention, gene therapy, and basic research.

将功能性外源大分子有效引入活细胞的能力受到质膜的严重限制。 克服这一障碍的载体的发展，如失活病毒，阳离子脂质和直接注射，目前提供了潜在的治疗干预和基础遗传研究的进展。 我们建议开发一种新的，灵活的，通用的，高效的蛋白质和核酸的基础上，VP 22，单纯疱疹病毒1的结构蛋白的递送系统。 VP 22表现出独特的细胞内转移形式，由此它能够有效地离开其合成的哺乳动物细胞，局部扩散，进入邻近细胞，并易位到受体细胞核。 值得注意的是，VP 22和第二多肽链之间的融合蛋白保留了两种蛋白质的功能特征。 我们建议开发一种特异性修饰的VP 22，它将能够将功能性核酸或蛋白质递送到细胞核。 我们的修饰的VP 22蛋白将被制备并在体外与标记的蛋白或核酸一起孵育。 基于修饰的VP 22和标签之间的极高亲和力，修饰的VP 22将与标记的大分子形成紧密结合的复合物。 将该复合物呈递给幼稚细胞将通过VP 22的作用导致复合物摄取和核转位;递送的DNA或蛋白质将保留生物活性。 在这个I期研究中，我们将构建、表达和表征修饰的VP 22，并证明其有效地将功能性DNA和蛋白质递送到组织培养细胞中的能力。 随后的第二阶段研究将增强系统的灵活性，并探索使用修饰的VP 22将蛋白质和DNA递送到完整小鼠的组织中。本研究的目的是开发一种通用技术，用于将功能性蛋白质和核酸简单有效地导入活细胞的细胞核。潜在的应用包括用于治疗干预、基因治疗和基础研究的递送载体。