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 就是开发一个E基于大肠杆菌的载体系统，用于功能性递送 大的基因组转基因到人类细胞中。基因治疗对人类有很大的希望， 治疗遗传疾病和癌症的生物医学科学。初始 基于病毒和脂质体的载体系统的结果是令人鼓舞的，但是 由于DNA的长度相对较短， 由此产生的基因表达的可变水平和持续时间。提供大量 基于基因组DNA的转基因，具有足够的周围基因组序列， 不仅包括感兴趣的基因，而且包括内源启动子、内含子 和必要的顺式作用元件，将显示更准确的时空 与cDNA构建体相比的表达。人类基因组计划的努力 已经制造了含有人类基因的大规模测序BAC和PAC克隆， 周围的基因组DNA。然而，缺乏合适的交付 方法阻碍了利用这些宝贵资源的基因治疗研究。因此，在本发明中， 以下三个具体目标是设计开发新的E.基于大肠杆菌的 能够递送大基因组克隆的基因治疗载体。1)一个 诱导型同源重组系统将适应E.杆菌dh 10 b 以允许BAC的工程化。E.大肠杆菌DH 10 B是重组缺陷型 （RecA-），使其成为用于稳定克隆大的完整的人 基因组DNA转化为BAC。在控制下提供几种重组蛋白 依赖于阿拉伯糖的启动子导致诱导型同源 重组系统2)E.大肠杆菌DH 10 B将有能力侵入 哺乳动物细胞，并通过表达Versinia 假结核病侵袭基因并造成细胞壁缺陷 合成（达帕）。3)人类功能性着丝粒DNA序列将是 工程化到这些BAC上，为转移的DNA提供有丝分裂稳定性 在分裂的细胞中。人类着丝粒α卫星DNA已被证明 当引入人类细胞时，重新形成着丝粒， 人工染色体（HACs）。然而，这些第一代HAC包括 这使得它们作为基因表达载体的用途有限。 工程化大BAC以包含人着丝粒DNA的能力， 然后将其作为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E.大肠杆菌基HAC 用于人类基因治疗的载体， 递送、准确的基因表达和有丝分裂稳定性。

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.