Predictable re-programming of mammalian cells remains one of the major challenges for applied biological design. This proposal seeks to design well-defined single copy human artificial chromosomes (HACs) to rigorously address robust, stable and regulated expression of multiple transgenes and complex genetic circuits for cell-based therapeutics, diagnostics and screening. Programmable HACs will enhance our ability to explore cells through rigorously controlled composition at higher throughput. To enable this vision, we will address questions around disease sensing and therapeutic development. Can we add expression cassettes with diverse regulation and avoid cross-talk? Can we add several partly homologous coding sequences to different sites and avoid recombination? Can we achieve consistent regulation of transcriptional reporters and proteins? Our goal is to generate precisely programmable HACs for cell-based screening and therapeutics. We will focus our efforts on the single copy 750kB HAC which contains a human centromere, does not undergo multimerization and can be assembled and delivered from yeast to human cells. We will build vehicles for expression of multiple genes relevant to inflammation and demonstrate utility for cell-based sensing and screening. The Specific Aims are: To use the HAC to program inducible expression of multiple therapeutic proteins in response to inflammation. Our therapeutic concept is that to treat certain relapsing-remitting autoimmune diseases, it would be ideal to express several anti-cytokine antibodies, but only when they are needed. We will insert gene expression cassettes designed to induce therapeutic antibodies upon inflammation into the HAC. We will test regulated gene expression and the need for chromatin boundaries. We will insert genes and regulatory elements at multiple sites within the HAC and determine the degree to which the HAC filler DNA is inert. Together, these experiments will establish a standard HAC for further engineering to manufacture living therapeutic cells. To build a HAC-based sensor cell line for screening associated with innate immune response. The ultimate application is to screen for drugs that might change the regulated expression or localization of several genes/proteins with a desired profile of effects. We will use the HAC to create cell lines for reliable and reproducible cell-based assays. A multi-gene visual reporter system that registers innate immune response pathways suppressed by viral genes will be constructed and tested for response to candidate small molecules. These results will establish the HAC as a tool for biosensing and drug screening.

The proposed work has broad implications for development of therapeutics and diagnostics. The work will lay a foundation for therapeutic cells that can sense and respond to specific disease states such as inflammation through production of novel therapeutic molecules. Cells generated from this work will allow for more rigorous approaches to drug discovery as well.