Project Summary/Abstract. Synthetic biology transformed our ability to rationally reprogram cells and use such engineered living organisms, instead of small molecule drugs or biologics, as novel therapeutics. However, living therapeutics proliferate and release their engineered genetic information into natural biomes through horizontal gene transfer. Consequently, the widespread use of engineered living therapeutics necessitates the development of efficient biocontainment technologies that not only prevent the unwanted proliferation of cells but also eliminate the release of genetic information (transgenes) from such genetically modified organisms (GMOs). The overarching goal of my proposal is to solve these challenges and develop the first microbial host for programmable drug delivery that simultaneously provides tight biocontainment, prevents transgene release ─ horizontal gene transfer ─ into wild organisms, and offers increased stability for long-term drug production. The PI recently demonstrated that engineering the genetic code of living cells provides a tight, potentially unbreakable genetic firewall that eliminates horizontal gene transfer and links the survival of cells to the presence of small molecules not available without human supplementation. However, these early experiments also revealed significant barriers in front of the clinical translation of this technology. This project will overcome these barriers and generate a bacterial host for controlled drug production that prevents transgene release and viral predation while offering strict biocontainment without escape from human therapeutic doses. This goal will be achieved through 3 specific aims: 1) The construction of a broadly virus-resistant microbial host that prevents transgene release by generating and characterizing multiple artificial genetic codes. 2) The creation of a tightly biocontained microbial host that utilizes a safe, food-supplement-based genetic biocontainment system. Finally, in Aim 3, the PI will combine these developments into a microbial living therapeutic host and demonstrate in a proof-of-concept experiment that this host enables stable, long-term therapeutic enzyme production inside the GI tract. In summary, this work will create a technology and microbial host capable of addressing a wide range of unmet needs in therapeutics development and de-risk the use of microbial GMOs for clinical translation, with potentially broad impact on diseases ranging from autoimmune and metabolic disorders to cancer. The proposed research and career development plan will be conducted in the lab of Dr. George M. Church at Harvard Medical School, and the PI, Dr. Akos Nyerges, will receive extensive training in proteomics, the use of animal models, and host-virus interaction analyses during the K99 phase from an expert advisory team. The career development plan and the outstanding scientific environment of Harvard will enable the PI to achieve the scientific goals of this ...