Rewiring Cellular Input-Output to Engineer Cell-Based Therapies That Interface Robustly with Human Physiology

Cell-based therapies have proven to be useful diagnostic and therapeutic tools for treating a wide variety of diseases such as arthritis, infectious diseases, and cancer.  Treating cancer with genetically engineered T cells is particularly promising, and for patients with certain types of “liquid” cancers, this approach has already delivered astounding clinical benefits.  However, current clinical experience indicates that extending these benefits to other types of cancer, as well as to other diseases, will require new therapeutic capabilities. In particular, there yet exists a need for technologies enabling one to program cells to sense and respond to their environment in a defined fashion.  Toward this goal, our lab has developed a platform for engineering novel protein biosensors for extracellular cues, which we have termed modular extracellular sensor architecture (MESA).  MESA comprises a self-contained receptor-signal transduction system in which ligand binding induces receptor dimerization, which then releases a sequestered transcription factor to regulate expression of an “output” gene or genes.

Here we present two expansions of the basic MESA capabilities, which make key strides towards meeting the needs identified above: 1) integrating ligand-specific MESA receptors with intracellular gene circuits to enable the cell to “process” multiparametric environmental cues, and (2) rewiring cellular input-output by coupling the sensing of biologically relevant extracellular signals to modulation of endogenous gene expression.  To engineer biosensing of novel input ligands, we demonstrated that both camelid antibody analogs termed  “nanobodies” and scFv antibody fragments could be incorporated as novel MESA ligand-binding domains. To achieve MESA-regulated induction of endogenous target gene expression, we integrated dCas9-based transcription factors as receptor “outputs”.  Altogether, these key proof-of-principle studies identified readily generalizable strategies for rewiring cellular input-output behavior in a user-defined fashion. This work lays the foundation for leveraging MESA receptors for a range of applications in both fundamental research as well as advanced cell-based therapies