Multiplexing Engineered Receptors for Multiparametric Evaluation of Environmental Ligands

Engineered
cell-based therapies comprise a promising, emerging
biomedical technology. Broad utilization of this strategy will require
new approaches for implementing sophisticated functional programs,
such as sensing and responding to the environment in a defined fashion.
Toward this goal, we investigated whether our self-contained receptor
and signal transduction system (MESA) could be multiplexed to evaluate
extracellular cues, with a focus on elucidating principles governing
the integration of such engineered components. We first developed
a set of hybrid promoters that exhibited AND gate activation by two
transcription factors. We then evaluated these promoters when paired
with two MESA receptors and various ligand combinations. Unexpectedly,
although the multiplexed system exhibited distinct responses to ligands
applied individually and in combination, the same synergy was not
observed as when promoters were characterized with soluble transcription
factors. Therefore, we developed a mechanistic computational model
leveraging these observations, to both improve our understanding of
how the receptors and promoters interface and to guide the design
and implementation of future systems. Notably, the model explicitly
accounts for the impact of intercellular variation on system characterization
and performance. Model analysis identified key factors that affect
the current receptors and promoters, and enabled an in silico exploration of potential modifications that inform the design of
improved logic gates and their robustness to intercellular variation.
Ultimately, this quantitative design-driven approach may guide the
use and multiplexing of synthetic receptors for diverse custom biological
functions beyond the case study considered here.