Optimizing heterologous pathways in mammalian cells via competitive splicing

The application of synthetic biology in mammalian cells offers great promise for developing new tools for exploring biology and novel therapeutics to treat human disease. However, the dearth of tools for regulating gene expression quantitatively limits the phenotypic landscape that can be explored with engineered genetic circuits. This challenge is particularly evident with efforts to engineer metabolite production, for which the relative expression level of enzymes can impact yield of the final product or result in the formation of unwanted or toxic byproducts. Similarly, from a developmental perspective, nuances in expression level among multiple genes often lead to drastic changes in cell fate. I address this technological gap by engineering alternative 3’ splice site selection as a mechanism for regulating and optimizing the relative expression level of multiple genes from a single, polycistronic operon. In doing so, not only have I develop a novel strategy for modulating gene expression, but I will also gain significant insight into the rules that govern RNA splicing in mammalian cells. This technology will specifically be applied to engineer essential amino acid production in human cells and to improve the efficiency of induced pluripotent stem cell conversion during cellular reprogramming, demonstrating the far-reaching capabilities and impact of this modular platform.