E. COLI BASED VECTORS FOR GENE DELIVERY TO HUMAN CELLS

描述：(申请人的描述)本提案的总体目标 是开发一种以大肠杆菌为基础的载体系统，用于功能性地递送 大规模的基因组转基因进入人类细胞。基因疗法为人类带来巨大的希望 遗传疾病和癌症治疗中的生物医学科学。首字母 病毒和脂质体为基础的载体系统的结果令人鼓舞，但 受限于可以传递的相对较短的DNA长度， 由此产生的基因表达的可变水平和持续时间。提供大型 基于基因组DNA的转基因，具有足够的周围基因组序列 不仅包括目的基因，还包括内源启动子、内含子 和基本的顺式作用元素，将显示更准确的时空 与构建的cDNAs进行比较。人类基因组计划的努力 已经制作了包含人类基因的大型测序BAC和PAC克隆 周围的基因组DNA很容易获得。然而，缺乏适当的交付 方法阻碍了使用这些宝贵资源的基因治疗研究。因此， 以下三个具体目标旨在开发基于 能够提供大型基因组克隆的基因治疗载体。1)一个 可诱导同源重组系统将适用于大肠杆菌DH10B 允许对BAC进行工程设计。大肠杆菌DH10B重组缺陷株 (RecA-)，使其成为稳定克隆大型完整人的首选载体 基因组DNA进入BAC。提供几种受控制的重组蛋白 依赖于阿拉伯糖的启动子的结果是可诱导的同源 重组系统。2)大肠杆菌DH10B将有能力入侵 哺乳动物细胞，并通过表达Versinia来传递大的BAC 假结核侵袭素基因与细胞壁缺陷 合成(DAPA)。3)人类功能着丝粒DNA序列将是 被设计到这些BAC上，为转移的DNA提供有丝分裂的稳定性 在细胞分裂中。人类着丝粒的阿尔法卫星DNA已被证明 当被引入人类细胞时形成新的着丝粒，创造出人类 人工染色体(HAC)。然而，这些第一代HAC包括 大大重排的DNA，使它们作为基因表达载体的用途有限。 改造大型BAC以包含人类着丝粒DNA和 然后将HAC引入人类细胞，将克服许多限制 以前的HAC载体。这些研究建议开发以大肠杆菌为基础的HAC 包含新的基因治疗方法的人类基因治疗载体 传递，准确的基因表达，以及有丝分裂的稳定性。

DESCRIPTION: (applicant's description) The overall objective of this proposal is to develop an E. coli based vector system for the functional delivery of large genomic transgenes into human cells. Gene therapy holds great promise for the biomedical sciences in the treatment of genetic disease and cancer. Initial results with viral and liposome-based vector systems have been encouraging, but are limited by the relatively short length of DNA that can be delivered, with resultant variable levels and duration of gene expression. Delivering large genomic DNA-based transgenes, with sufficient surrounding genomic sequences to include not only the gene of interest but also endogenous promoters, introns and essential cis-acting elements, will display more accurate spatio-temporal expression compared to cDNA constructs. The efforts of the Human Genome Project have made large sequenced BAC and PAC clones containing human genes and surrounding genomic DNA readily available. However, lack of a suitable delivery method has hindered gene therapy studies using these valuable resources. Thus, the following three specific aims are designed to develop novel E. coli based gene therapy vectors capable of delivering large genomic clones. 1) An inducible homologous recombination system will be adapted to the E. coli DH10B to permit engineering of BACs. E. coli strain DH10B is recombination deficient (RecA-), making it the preferred vector for stably cloning large intact human genomic DNA into BACs. Providing several recombination proteins under control of an arabinose-depending promoter results in an inducible homologous recombination system. 2) E. coli DH10B will be made competent to invade mammalian cells and deliver large BACs, by expressing the Versinia pseudotuberculosis invasin gene and creating a deficiency in cell wall synthesis (dapA). 3)Human functional centromere DNA sequences will be engineered onto these BACs to provide mitotic stability to the transferred DNA in dividing cells. The human centromeric alpha satellite DNA has been shown to form de novo centromeres when introduced into human cells, creating human artificial chromosomes (HACs). However, these first generation HACs consist of greatly rearranged DNA, making them of limited use as gene expression vectors. The ability to engineer large BACs to contain human centromeric DNA and introduce then into human cells as HACs will overcome many of the limitations of previous HAC vectors. These studies propose to develop E. coli-based HAC vectors for human gene therapy that encompass novel approaches for gene delivery, accurate gene expression, and mitotic stability.