Tumor-Targeted Delivery of Synthetic DNA Payloads By Engineered Bacteria

能够侵入人体细胞的细菌为基于细胞的治疗提供了一种很有前途的技术，也可用于提供DNA递送途径，有可能使这些细菌成为基因治疗、基于DNA的疫苗和体内基因组工程的载体。过去的工作已经证明，模块化的DNA结构可以用来改造大肠杆菌和沙门氏菌，使其靶向并进入癌细胞系和肿瘤，并特异性地传递蛋白质有效载荷（例如毒素）。拟议中的项目将不仅仅是蛋白质的传递，还将开发一个平台，利用肿瘤靶向细菌来控制DNA有效载荷的传递，然后在宿主细胞中表达。我们预计这将成为基于细菌细胞的治疗和哺乳动物合成生物学和合成基因组学的DNA转移的核心技术。这个跨学科项目将结合基于dna的工程和合成表达设计的基本合成生物学方法（Tom Ellis）和细胞内宿主-微生物相互作用的尖端生物医学研究（Teresa Thurston, Ramesh Wigneshweraraj）。它由一家令人兴奋的伦敦工业合作伙伴Prokarium共同赞助，并利用他们在细菌细胞治疗方面的专业知识和商业知识。这项研究将建立在该公司和伦敦生物铸造厂之间现有的合成生物学项目的基础上，但现在将其引入DNA传递的新方向。在计划中的实验工作中，学生将设计、构建和测试模块化DNA程序，当相关的大肠杆菌和沙门氏菌菌株进入目标细胞时，这些程序将触发这些细菌的分泌机制和结合。该项目的一个核心目标将是使用非致病性沙门氏菌菌株作为载体，将编码真核基因的DNA注入癌细胞系。这一过程将通过荧光成像和单分子显微镜跟踪DNA表达和位置来优化和测量。进一步的工作将着眼于测试可以转移的DNA长度的极限。细菌之间的结合通常用于一次性转移数百万个DNA碱基，因此这为将大块合成DNA转移到目标细胞提供了一条途径。

Bacteria capable of invasion into human cells offer a promising technology for cell-based therapies and could also be used to provide a route for DNA delivery, potentially enabling these bacteria to be a vector for gene therapies, DNA-based vaccines and in vivo genome engineering. Past work has demonstrated how modular DNA constructs can be used to engineer E. coli and Salmonella to target and enter cancer cell lines and tumours and specifically deliver protein payloads (e.g. toxins). The proposed project will go beyond delivery of just proteins and develop a platform for using tumor-targeting bacteria for the controlled delivery of DNA payloads that are then expressed in the host cell. We anticipate that this can become a core technology for both bacterial cell-based therapies and for DNA transfer for mammalian synthetic biology and synthetic genomics.This interdisciplinary project will bring together the foundational synthetic biology methods of DNA-based engineering and design of synthetic expression (Tom Ellis) with cutting-edge biomedical research of intracellular host-microbe interactions (Teresa Thurston, Ramesh Wigneshweraraj). It is co-sponsored by an exciting London-based industrial partner, Prokarium, and leverages their expertise and commercial knowledge of bacterial cell therapies. The research will build on an existing synthetic biology project between the company and the London BioFoundry, but now take this in the new direction of DNA delivery.The planned experimental work will see the student design, construct and test modular DNA programs that trigger secretion mechanisms and conjugation in relevant E. coli and Salmonella strains when these bacteria enter target cells. A core aim of this project will be to use non-pathogenic Salmonella strains as a vehicle to inject DNA encoding a eukaryotic gene into a cancer cell line. This process will be optimised and measured by tracking DNA expression and location by fluorescent imaging and single-molecule microscopy. Further work will look to test the limit of the lengths of DNA that can be transferred. Conjugation between bacteria is regularly used to transfer millions of bases of DNA in one go, and so this offers promise as a route to transfer big sections of synthetic DNA into target cells.