Synthetic biology in the clinic: engineering vaccines, diagnostics, and therapeutics.

Synthetic biology is a design-driven discipline centered on engineering novel biological functions through the discovery, characterization, and repurposing of molecular parts. Several synthetic biological solutions to critical biomedical problems are on the verge of widespread adoption and demonstrate the burgeoning maturation of the field. Here, we highlight applications of synthetic biology in vaccine development, molecular diagnostics, and cell-based therapeutics, emphasizing technologies approved for clinical use or in active clinical trials. We conclude by drawing attention to recent innovations in synthetic biology that are likely to have a significant impact on future applications in biomedicine.

Engineering digitizer circuits for chemical and genetic screens in human cells.

Cell-based transcriptional reporters are invaluable in high-throughput compound and CRISPR screens for identifying compounds or genes that can impact a pathway of interest. However, many transcriptional reporters have weak activities and transient responses. This can result in overlooking therapeutic targets and compounds that are difficult to detect, necessitating the resource-consuming process of running multiple screens at various timepoints. Here, we present RADAR, a digitizer circuit for amplifying reporter activity and retaining memory of pathway activation. Reporting on the AP-1 pathway, our circuit identifies compounds with known activity against PKC-related pathways and shows an enhanced dynamic range with improved sensitivity compared to a classical reporter in compound screens. In the first genome-wide pooled CRISPR screen for the AP-1 pathway, RADAR identifies canonical genes from the MAPK and PKC pathways, as well as non-canonical regulators. Thus, our scalable system highlights the benefit and versatility of using genetic circuits in large-scale cell-based screening.

Quantitative characterization of recombinase-based digitizer circuits enables predictable amplification of biological signals.

Many synthetic gene circuits are restricted to single-use applications or require iterative refinement for incorporation into complex systems. One example is the recombinase-based digitizer circuit, which has been used to improve weak or leaky biological signals. Here we present a workflow to quantitatively define digitizer performance and predict responses to different input signals. Using a combination of signal-to-noise ratio (SNR), area under a receiver operating characteristic curve (AUC), and fold change (FC), we evaluate three small-molecule inducible digitizer designs demonstrating FC up to 508x and SNR up to 3.77 dB. To study their behavior further and improve modularity, we develop a mixed phenotypic/mechanistic model capable of predicting digitizer configurations that amplify a synNotch cell-to-cell communication signal (Δ SNR up to 2.8 dB). We hope the metrics and modeling approaches here will facilitate incorporation of these digitizers into other systems while providing an improved workflow for gene circuit characterization.

Mechanical confinement via a PEG/Collagen interpenetrating network inhibits behavior characteristic of malignant cells in the triple negative breast cancer cell line MDA.MB.231.

To decouple the effects of collagen fiber density and network mechanics on cancer cell behavior, we describe a highly tunable in vitro 3D interpenetrating network (IPN) consisting of a primary fibrillar collagen network reinforced by a secondary visible light-mediated thiol-ene poly(ethylene glycol) (PEG) network. This PEG/Collagen IPN platform is cytocompatible, inherently bioactive via native cellular adhesion sites, and mechanically tunable over several orders of magnitude-mimicking both healthy and cancerous breast tissue. Furthermore, we use the PEG/Collagen IPN platform to investigate the effect of mechanical confinement on cancer cell behavior as it is hypothesized that cells within tumors that have yet to invade into the surrounding tissue experience mechanical confinement. We find that mechanical confinement via the IPN impairs behavior characteristic of malignant cells (i.e., viability, proliferation, and cellular motility) in the triple negative breast cancer cell line MDA.MB.231, and is more effective than removal of soluble growth signals. The PEG/Collagen IPN platform is a useful tool for studying mechanotransductive signaling pathways and motivates further investigation into the role of mechanical confinement in cancer progression. STATEMENT OF SIGNIFICANCE: In this study, we have developed, optimized, and applied a novel 3D in vitro cell culture platform composed of an interpenetrating network (IPN) that is both mechanically tunable and inherently bioactive. The IPN consists of a primary fibrillar collagen type-1 network reinforced by a secondary thiol-ene poly(ethylene glycol) (PEG) network. The IPNs are formed via a novel strategy in which cell-laden collagen gels are formed first, and soluble PEG monomers are added later and crosslinked via visible light. This approach ensures that the collagen gels contain a fibrillar architecture similar to the collagen architecture present in vivo. We applied our IPN platform to study the effect of mechanical confinement on cancer cell behavior and found that it inhibits malignant-like behavior.