(543f) Programmable Promoter Editing for Precise Control of Transgene Expression

Subtle changes in gene expression can direct cells to distinct cellular states. However, identifying dosage-sensitive transgenes requires fine-tuned control which remains a challenge. Tools such as small molecule inducible promoters exhibit limitations including bimodality, whereas toggle switches or recombinase-based systems are often limited to ON/OFF binary expression. A tool that generates fine-scale change in unimodal setpoints could reveal how levels of transgenes map to phenotypes as well as support control of levels in therapeutic contexts for precision cell and gene therapy.

To support dosage control, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression. Expression from DIAL promoters increases upon excision of spacers between the binding sites of a synthetic zinc finger transcriptional activator (ZFa) and a core promoter. Different combinations of inputs of ZFa and recombinase lead to different output levels of the target gene. By nesting varying numbers and lengths of spacers, DIAL generates a tunable range of multiple unimodal setpoints from a single promoter. The setpoints and fold change can further be tuned by choice of core promoter and ZFa. At high levels of ZFa, DIAL outputs are stable setpoints that are robust to variation in input levels.

Transmitting transient events into heritable states supports event recording and stable direction of cell trajectories. With the advantage of DNA level editing, we show that the change in setpoint level from transient recombinase expression persists over weeks and is inherited with cell division. Through small-molecule control of ZFa and recombinases, DIAL supports external, temporally defined, and user-guided control of transgene expression level. By inclusion of tetO binding sites, we show that the DIAL framework is extensible to other transcription factors.

For the broadest impact across research and therapeutics, genetic control systems need to perform in multiple contexts. Using lentiviral delivery, we demonstrate that DIAL changes setpoints in primary cells like mouse embryonic fibroblasts and in human iPSCs. We also show that DIAL can regulate physiologically-relevant transgenes. A key use case of DIAL is harnessing the stable inherited promoter states to facilitate mapping of transgene levels to phenotypes. The ability of the same starting construct for multiple levels allows control over batch-to-batch differences in noisy systems like primary cells and production of viruses of variable titers. To this end, using a testbed of primary fibroblasts, we demonstrate that DIAL generates distinct levels of functional transgenes resulting in difference in proliferation and conversion to induced motor neurons.

Overall, the DIAL framework opens new opportunities for tailoring transgene expression and improving the predictability and performance of gene circuits across diverse applications including cellular therapies and regenerative medicine. Control via a single promoter offers the scalability needed generate multiple setpoints from libraries of transgenes and inherently controls for bias of clonal founder lines for a single transgene. Thus, DIAL may be used to identify and control transgenes with subtle dose-dependent effects on cellular states. DIAL could also be used to change expression at intermediate timepoints in response to external stimuli to promoter therapeutically advantageous transformations. Finally, we anticipate that the extensible framework offered by DIAL will support expansion to novel synthetic promoter systems and broadly improve precision cell engineering.