VP22 MEDIATED DELIVERY OF PROTEIN AND DNA INTO CELLS

本提案概述了VP22作为蛋白质和DNA分子在培养细胞中的传递载体的发展。I期研究通过表达和纯化大肠杆菌中触角肽的蛋白融合体，证明了蛋白介导递送的能力。这些融合蛋白在生物膜上易位，在细胞质和细胞核中积累。最近发现了一种新的易位蛋白VP22，与Antennapedia肽相比，它有两个显著的优势：1)VP22可以易位较大的肽；2)VP22在细胞亚群中的表达导致该蛋白在整个培养物中扩散。在第二阶段，将构建表达和纯化VP22融合蛋白的原核和真核载体。VP22的融合将用于开发能够调节基因表达的系统。与DNA相互作用的VP22融合物将被开发并用于研究VP22将寡核苷酸和质粒DNA传递到培养细胞的能力。这项革命性的技术在将蛋白质和DNA传递到当前技术无法转染的细胞方面具有广泛的应用，并将为研究所有细胞类型的基因功能提供新技术。建议的商业应用：将构建原核和真核载体，使研究人员能够表达和纯化VP22融合蛋白。VP22融合物将构建一个可诱导的基因表达系统，并将单链抗体传递到细胞内靶点。基于VP22的试剂将被开发用于将寡核苷酸和质粒DNA递送到组织培养细胞中，这些细胞对当前的转染技术是难以接受的。

This proposal outlines the development of VP22 as a delivery vehicle for protein and DNA molecules to cells in culture. The Phase I proposal demonstrated the power of protein-mediated delivery by expressing and purifying protein fusions with the Antennapedia peptide from E. coli. These fusion proteins were translocated across biological membranes, accumulating in the cytoplasm and nucleus. A new translocating protein, VP22, was recently described and has two significant advantages over the Antennapedia peptide: 1) VP22 can translocate larger peptides and 2) expression of VP22 in a subpopulation of cells results in spread of the protein throughout the entire culture. During the Phase II period, prokaryotic and eukaryotic vectors for the expression and purification of VP22 fusion proteins will be constructed. VP22 fusions will be used to develop systems that can regulate gene expression. VP22 fusions that interact with DNA will be developed and used to investigate the ability of VP22 to deliver oligonucleotides and plasmid DNA to cells in culture. This revolutionary technology has broad applications for the delivery of proteins and DNA to cells that cannot be transfected with current technologies and will provide new techniques to study gene function in all cell types. PROPOSED COMMERCIAL APPLICATIONS: Prokaryotic and eukaryotic vectors will be constructed to allow researchers to express and purify VP22 fusion proteins. VP22 fusions will be constructed to develop an inducible gene expression system and to deliver single chain antibodies to intracellular targets. Reagents based on VP22 will be developed for the delivery of oligonucleotides and plasmid DNA to tissue culture cells that are refractory to current transfection technologies.