Synthetic gene circuits as tools for drug discovery

Abstract

Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS). By tracking events and cellular states across multiple length and time scales, circuits will transform how we decipher the causal link between molecular events and phenotypes to improve the selectivity and sensitivity of cell-based assays. Enhancing phenotypic drug discovery with synthetic biologyDevelopment of new drugs requires identification and optimization of functional molecules that often proceeds sequentially through high-throughput discovery screening, hit validation, hit expansion and optimization, and preclinical disease modeling (see Glossary) (Figure 1, Key figure)[1]. Target-based drug discovery (TDD) programs aim to identify drug candidates that modulate a defined biological target that is hypothesized to play a causal role in initiating or sustaining a disease. TDD programs offer precision by directly screening for drug candidate efficacy using purified molecular targets [2]. By contrast, phenotypic drug discovery (PDD) programs aim to identify drug candidates that modulate a physiologically-relevant biological system or cellular signaling pathway and screen for drug efficacy using cell-based assays [2]. Cell-based assays are target-agnostic and provide the opportunity to identify therapeutics that resolve diseaseassociated phenotypic deficits even when the disease etiology remains obscure and targets are undefined. Thus, PDD has recently received renewed interest for its potential to identify first-in-class drugs that act via a novel mechanism of action (MoA) as well as identify drugs for disease with complex or unknown etiology [1][2][3][4]. In order to identify drug candidates that translate to effective therapeutics in the clinic, PDD programs require cell-based assays that faithfully recapitulate and report on disease-relevant processes. To efficiently and effectively screen in phenotypic models, assay development for initial discovery screens must balance throughput with the ability to capture these disease processes [1]. As PDD probes a larger biological space than TDD to identify potential therapeutics, the number of hits from an initial discovery screen in a PDD program may be very large [2]. Following initial discovery screens, hit validation and triage are required to produce a small number of high-confidence candidates to proceed to the resource-intensive stages of hit expansion, compound optimization, and hig...

Supercoiling-mediated feedback rapidly couples and tunes transcription

“…Cells coordinate complex behaviors through precise spatiotemporal control of gene expression. To rapidly advance gene- and cell-based therapies, synthetic biology aims to harness the power of native biology by constructing synthetic gene regulatory networks capable of dynamically prescribing cellular processes, states, and identities ( Chen et al, 2012 ; Beitz et al, 2022 ; Purnick and Weiss, 2009 ; Elowitz and Lim, 2010 ). Synthetic networks process diverse inputs into complex logical and temporal responses ( Weinberg et al, 2017 ; Xie et al, 2011 ; Tabor et al, 2009 ).…”

Precise programming of multigene expression stoichiometry in mammalian cells by a modular and programmable transcriptional system

“…One possible challenge for the host-independent system is that it may take too much translational resources and induce unexpected cellular burden or toxicity for mammalian cells. Other challenges would be the resource-limiting factors coming from translational process and other regulatory processes [31][32][33] . For example, ribosomes and other translational factors were found to be the limiting resources for expressing multiple genes 31 .…”

“…For example, ribosomes and other translational factors were found to be the limiting resources for expressing multiple genes 31 . Dicer proteins as a common cofactor of miRNA regulations would be a limiting factor for the regulatory functions of multiple miRNAs, which could result in retroactivity for genetic circuits composed of multiple miRNAs 32 .…”

Supercoiling-mediated feedback rapidly couples and tunes transcription

“…Cells coordinate complex behaviors through precise spatiotemporal control of gene expression. To rapidly advance gene and cell-based therapies, synthetic biology aims to harness the power of native biology by constructing synthetic gene regulatory networks capable of dynamically prescribing cellular processes, states, and identities [1][2][3][4]. Synthetic networks process diverse inputs into complex logical and temporal responses [5][6][7].…”