VP22 MEDIATED DELIVERY OF PROTEIN AND DNA INTO CELLS

该提案概述了VP22作为蛋白质和DNA分子向培养细胞的递送载体的发展。第一阶段的建议证明了蛋白质介导的权力，通过表达和纯化蛋白质融合与Escherichia peptide。杆菌这些融合蛋白跨生物膜转运，在细胞质和细胞核中积累。最近描述了一种新的易位蛋白VP22，其相对于双足肽具有两个显著的优点：1）VP22可以易位较大的肽，以及2）VP22在细胞亚群中的表达导致蛋白质在整个培养物中的扩散。在II期期间，将构建用于表达和纯化VP22融合蛋白的原核和真核载体。VP22融合体将用于开发可以调节基因表达的系统。将开发与DNA相互作用的VP22融合物，并用于研究VP22将寡核苷酸和质粒DNA递送至培养物中的细胞的能力。这项革命性的技术具有广泛的应用，可以将蛋白质和DNA输送到无法用现有技术转染的细胞中，并将为研究所有细胞类型中的基因功能提供新技术。拟议的商业应用：将构建原核表达载体和真核表达载体，使研究人员能够表达和纯化VP22融合蛋白。将构建VP22融合体以开发诱导型基因表达系统并将单链抗体递送至细胞内靶标。将开发基于VP22的试剂，用于将寡核苷酸和质粒DNA递送至当前转染技术难以处理的组织培养细胞。

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.