Tumor-Targeted Delivery of Synthetic DNA Payloads By Engineered Bacteria

能够侵入人类细胞的细菌为基于细胞的治疗提供了一种有前途的技术，也可用于提供DNA递送途径，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了如何使用模块化DNA构建体来工程化E。大肠杆菌和沙门氏菌靶向并进入癌细胞系和肿瘤，并特异性地递送蛋白质有效载荷（例如毒素）。拟议的项目将超越仅仅递送蛋白质，并开发一个平台，用于使用肿瘤靶向细菌控制递送DNA有效载荷，然后在宿主细胞中表达。我们预计这将成为细菌细胞疗法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。这个跨学科项目将把基于DNA的工程和合成表达设计的基础合成生物学方法（Tom Ellis）与细胞内宿主-微生物相互作用的前沿生物医学研究（Teresa Thurston，Ramesh Wigneshweraraj）结合起来。它由一个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疗法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输送的新方向。计划的实验工作将看到学生设计，构建和测试模块化DNA程序，以触发相关E.大肠杆菌和沙门氏菌菌株时，这些细菌进入靶细胞。该项目的核心目标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，因此这有望成为将合成DNA的大部分转移到靶细胞中的途径。

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.