Salmonella-to-Eukaryotic Cell Multi-task Gene Delivery

描述(申请人提供)：本R03提案的目的是进行初步实验：1.开发从沙门氏菌到真核宿主细胞的基因转移系统；2.检查沙门氏菌的性质和影响基因转移过程的宿主细胞因素；以及3.比较接合与其他基于沙门氏菌的DNA转移系统。第一个目标来自我们最近发现的大肠杆菌到哺乳动物细胞的接合DNA转移。为大肠杆菌系统开发的方法学将适用于沙门氏菌的供体菌株，以便最终利用沙门氏菌有机体特有的细菌-宿主细胞相互作用。对于非侵入性的大肠杆菌K12供体，严格的控制措施最终排除了DNA转移的非结合机制，如转化和细菌吞噬，包括在环境中使用Dnasel和内吞作用的抑制剂。对于沙门氏菌系统，假设有不止一种DNA传递机制是有效的，因为沙门氏菌有多种与宿主细胞相互作用的方式。这种输送机制可以包括细胞内(“胞浆内”)细菌输送、细胞外细菌输送和液泡内细菌输送。在每个蜂窝环境中，不同的过程都可能起作用。第二个目标将有助于确定影响这些事件的细菌和宿主细胞属性。这种方法将表征转移机制，同时提供改进DNA传递作为治疗工具的方法。第三个目标将使用这些定义因素来区分沙门氏菌和大肠杆菌的细菌接合转移，并将接合转移与其他基于沙门氏菌的DNA转移系统进行比较。这些研究旨在开发减毒沙门氏菌菌株，用于DNA疫苗方法、纠正性基因治疗、免疫治疗以及某些类型癌症的预防和治疗。

DESCRIPTION (provided by applicant): The aims of this R03 proposal are to perform the initial experiments: 1. to develop a system of gene transfer from Salmonella to eukaryotic host cells; 2. to examine the Salmonella properties and host cell factors that affect gene transfer processes; and 3. compare conjugation with other Salmonella-based DNA transfer systems. The first aim emerges from our recent discovery of E. coli-to-mammalian cell conjugative DNA transfer. The methodology developed for the E. coli system will be adapted to donor strains of Salmonella, in order to ultimately capitalize on bacterial-host cell interactions that are unique to the Salmonella organism. With the noninvasive E. coli K12 donors, non-conjugative mechanisms of DNA transfer such as transformation and bacterial engulfment were conclusively ruled out by rigorous controls, including the use of Dnasel in the milieu and inhibitors of endocytosis. With the Salmonella system, the hypothesis is that more than one DNA delivery mechanism will be operative, since Salmonella has multiple ways of interacting with host cells. Such delivery mechanisms could involve intracellular ("intracytoplasmic") bacterial delivery, extracellular bacterial delivery, and intravacuole bacterial delivery. In each cellular locale, distinct processes can be at work. The second aim will help define the bacterial and host cell properties that impact these events. This approach will characterize the transfer mechanisms while providing ways to improve DNA delivery as a therapeutic tool. The third aim will use those defining factors to distinguish bacterial conjugative transfer by Salmonella from that of E. coli and to compare conjugative transfer with the other Salmonella-based DNA transfer systems. These studies are intended to develop attenuated Salmonella strains for DNA delivery for DNA vaccine approaches, corrective gene therapy, immunotherapy, and the prevention and treatment of certain kinds of cancer.