Authors: Alexa N Gormick, Adam M Zahm, Justin G English
Mentors: Justin G English
Insitution: University of Utah
Recent advancements in gene therapy have pushed towards the prevention and treatment of a diverse spectrum of disorders and diseases that are caused by misregulation of gene expression programs and their transcriptional regulators. However, the profoundness of the field means that much of the mechanisms and effects of regulation are unknown and understudied.
Here, we explore the limits of flexible exogenous gene expression and its potential use in optimizing efficacy and specificity in gene therapy interventions while minimizing the possible associated risks. This is made possible by exploiting the Tet-On system of inducible transcriptional regulation, which allows the expression of any target gene to be reversibly, specifically, and differentially controlled. In this system, the tetracycline repressor (TetR) binds the tetracycline operator (TetO), impeding transcription of any downstream gene embedded by the researcher; tetracycline dosing causes TetR to adopt a new conformation that removes it from TetO, inducing gene expression on command (Das et al., 2016). Because of the diverse utility of this system, we are in pursuit of developing novel TetR-TetO orthologous pairs that do not interfere with this wild-type circuit and can be used to regulate gene expression in parallel. As a first step to generating TetR-TetO orthologs, we mapped the usage of TetO by TetR in a massively parallel reporter assay (MPRA) by engineering an extensive library of mutant TetOs and quantified the resulting range of TetR regulation through reporter gene expression. From this screen, we identified candidate TetO mutants to direct the evolution of the wild-type TetR towards complementary states to those TetO mutant sequences.
Our preliminary findings indicate that the engineering of distinct synthetic expression cassettes based on the TetR-TetO operon is feasible. These novel tools may ultimately allow us to build a synthetic genetic circuit to model regulatory feedback loops that can help discover malfunctions in cell growth, reproduction, and cycling that can arise from genetic disorders and can lead to disease.
1. Das, A. T., Tenenbaum, L., & Berkhout, B. (2016). Tet-On Systems For
Doxycycline-inducible Gene Expression. Current Gene Therapy, 16(3), 156–167. https://doi.org/10.2174/1566523216666160524144041