Synthetic Circuit-Driven Expression of Heterologous Enzymes for Disease Detection

Abstract

The integration of nanotechnology and synthetic biology could lay the framework for new classes of engineered biosensors that produce amplified readouts of disease states. As a proof-of-concept demonstration of this vision, here we present an engineered gene circuit that, in response to cancer-associated transcriptional deregulation, expresses heterologous enzyme biomarkers whose activity can be measured by nanoparticle sensors that generate amplified detection readouts. Specifically, we designed an AND-gate gene circuit that integrates the activity of two ovarian cancer-specific synthetic promoters to drive the expression of a heterologous protein output, secreted Tobacco Etch Virus (TEV) protease, exclusively from within tumor cells. Nanoparticle probes were engineered to carry a TEV-specific peptide substrate in order to measure the activity of the circuit-generated enzyme to yield amplified detection signals measurable in the urine or blood. We applied our integrated sense-and-respond system in a mouse model of disseminated ovarian cancer, where we demonstrated measurement of circuit-specific TEV protease activity both in vivo using exogenously administered nanoparticle sensors and ex vivo using quenched fluorescent probes. We envision that this work will lay the foundation for how synthetic biology and nanotechnology can be meaningfully integrated to achieve next-generation engineered biosensors.

Protease Activity Analysis: A Toolkit for Analyzing Enzyme Activity Data

“… 11 , 37 To this end, our group has developed activity-based nanosensors, probes that detect the activity of aberrant proteases within the body and generate exogenous urinary reporters that reflect the degree of proteolytic cleavage encountered in vivo . 12 − 14 , 16 , 918 , 38 − 42 These nanosensors consist of an inert scaffold whose surface is decorated with peptide substrates, designed to be cleaved by proteases dysregulated in the disease state of interest. Each substrate is marked with a mass-encoded peptide barcode, which is released upon interaction of the nanosensor with target proteases and then concentrated in the urine.…”

“…Because proteases play critical functional roles in a variety of disease processes, recent years have seen the emergence of new classes of activity-based diagnostics that are engineered to measure the activity of endogenous enzymes at the site of disease and to generate an output signal that can be read out externally. , To this end, our group has developed activity-based nanosensors, probes that detect the activity of aberrant proteases within the body and generate exogenous urinary reporters that reflect the degree of proteolytic cleavage encountered in vivo . − ,,,− These nanosensors consist of an inert scaffold whose surface is decorated with peptide substrates, designed to be cleaved by proteases dysregulated in the disease state of interest. Each substrate is marked with a mass-encoded peptide barcode, which is released upon interaction of the nanosensor with target proteases and then concentrated in the urine.…”

Protease Activity Analysis: A Toolkit for Analyzing Enzyme Activity Data

“…11,34 To this end, our group has developed activity-based nanosensors (ABNs), which are probes that detect the activity of aberrant proteases within the body, thereby generating exogenous urinary reporters that reflect the degree of proteolytic cleavage encountered in vivo. [12][13][14]16,[35][36][37][38][39] These nanosensors consist of an inert scaffold whose surface is decorated with peptide substrates, designed to be cleaved by proteases dysregulated in the disease state of interest. Each substrate is marked with a mass-encoded peptide barcode, which is released upon interaction of the nanosensor with target proteases and then concentrated in the urine.…”

The Proteolytic Landscape of Ovarian Cancer: Applications in Nanomedicine

“…Subsequently, a 40 kDa eight-arm PEG conjugated to TEV substrate (Biotin-eGvndneeGffsar-K(FAM)-dGGENLYFQGGGC) with reporter molecule (glutamate fibrinopeptide B) or fluorescent marker was systemically administered to tumor-bearing mice and readout recorded by urine immunoassay or blood fluorescence, respectively. Although an interesting concept, reporters for TEV protease, indicating the OvCa, were detected at only a 1.6 fold change ( p = 0.0078) and 1.2 fold change ( p = 0.0027) in the urine and blood samples of OvCa mice versus healthy mice, respectively, representing a change that may be challenging to detect in clinical settings [ 240 ]. Nonetheless, synthetic biology may have a unique role in amplifying protease diagnostics for detection of OvCa.…”