EFFICIENT NUCLEAR DELIVERY VECTOR FOR MACROMOLECULES

有效地将功能的外源大分子引入活细胞的能力受到质膜的严重限制。目前，为克服这一障碍而开发的载体，如去活病毒、阳离子脂类和直接注射，为治疗干预和基础遗传学研究提供了潜在的进展。基于单纯疱疹病毒1的结构蛋白VP22，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递送系统。VP22具有一种独特的细胞内转移形式，能够有效地离开合成它的哺乳动物细胞，局部扩散，进入邻近细胞，并转移到受体细胞核。值得注意的是，VP22和第二个多肽链之间的融合蛋白保留了这两种蛋白的功能特征。我们建议开发一种特定修饰的VP22版本，它将能够将功能核酸或蛋白质运送到细胞核。我们将制备我们修饰的VP22蛋白，并在体外与标记蛋白或核酸孵育。基于修饰的VP22与标签之间极高的亲和力，修饰的VP22将与标记的大分子形成紧密结合的络合物。将这种复合体呈现给幼稚细胞将通过VP22的作用导致复杂的摄取和核转位；所传递的DNA或蛋白质将保持生物活性。在这项第一阶段的研究中，我们将构建、表达和鉴定修饰的VP22，并证明它能够有效地将功能DNA和蛋白质输送到组织培养细胞中。随后的第二阶段研究将增强该系统的灵活性，并探索使用修改后的VP22将蛋白质和DNA输送到完整小鼠的组织中。这项研究旨在开发一种通用技术，方便有效地将功能蛋白质和核酸引入活细胞的细胞核。潜在的应用包括治疗干预、基因治疗和基础研究的递送载体。

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.