A Synthetic Multifunctional Mammalian pH Sensor and CO2 Transgene-Control Device

Abstract

All metabolic activities operate within a narrow pH range that is controlled by the CO2-bicarbonate buffering system. We hypothesized that pH could serve as surrogate signal to monitor and respond to the physiological state. By functionally rewiring the human proton-activated cell-surface receptor TDAG8 to chimeric promoters, we created a synthetic signaling cascade that precisely monitors extracellular pH within the physiological range. The synthetic pH sensor could be adjusted by organic acids as well as gaseous CO2 that shifts the CO2-bicarbonate balance toward hydrogen ions. This enabled the design of gas-programmable logic gates, provided remote control of cellular behavior inside microfluidic devices, and allowed for CO2-triggered production of biopharmaceuticals in standard bioreactors. When implanting cells containing the synthetic pH sensor linked to production of insulin into type 1 diabetic mice developing diabetic ketoacidosis, the prosthetic network automatically scored acidic pH and coordinated an insulin expression response that corrected ketoacidosis.

Engineering Gene Circuits for Mammalian Cell–Based Applications

“…The latter harbors a synthetic promoter sequence with cognate DNA response elements (CRE, SRE, NFAT-RE) that recruit signaling pathway-specific transcription factors. Thus far, synthetic GPCRbased gene control systems have been engineered that respond to metabolites, hormones, and drug ligands, including luteinizing hormone (Kemmer et al 2011), histamine (Ausländer et al 2014b), dopamine (Rössger et al 2013a), and guanabenz (Ye et al 2013), in addition to other signals, such as pH changes (Ausländer et al 2014a), heat (Stanley et al 2012), and blue light (Ye et al 2011). The orthogonal usage of multiple GPCRs in individual cells is impeded if they use the same signaling pathway.…”

“…Circuit topology can also be applied in biology to show synthetic biological networks and to describe input -output functions. Gene circuits with different topologies, such as oscillators (Tigges et al 2009), logic gates (Kramer et al 2004;Rinaudo et al 2007;Smolke 2007, 2008;Ausländer et al 2012aAusländer et al , 2014a, hysteresis (Kramer and Fussenegger 2005), band-pass (Greber and Fussenegger 2010), time delay Lapique and Benenson 2014), bow-tie (Prochazka et al 2014), and memory devices (Burrill et al 2012) have already been constructed in living mammalian cells. Because the above-described synthetic gene switches are able to process input information and to produce a specific output, they are well-suited building blocks for the design of programmable gene circuits reminiscent of electronic circuits.…”

“…In a recent publication, sets of different building blocks were used to resemble logic gates in mammalian cells (Fig. 3B) (Ausländer et al 2014a). In particular, a GPCR-based CO 2 / H þ -inducible gene-regulation system was combined with bacterial repressor-and protein dimerization -based systems to build gas-inducible gene circuits.…”

“…4B) (Rössger et al 2013b); (3) diabetic ketoacidosis, sensing reduced blood pH levels and producing the hormone insulin, thereby restoring blood glucose and pH homeostasis (Fig. 4C) (Ausländer et al 2014a); (4) Graves disease, sensing thyroid hormone levels and producing an engineered variant of the human thyroid-stimulating hormone (TSH), thereby restoring the thyrotrophic feedback control of the hypothalamus -pituitarythyroid axis (Fig. 4D) (Saxena et al 2016); and (5) psoriasis, sensing proinflammatory cytokines TNF as well as interleukin 22 (IL22) and producing anti-inflammatory cytokines IL4 and IL10 only in the presence of both input signals (AND-gate-expression logic), thereby preventing or attenuating psoriatic flares ( Fig.…”

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation

“…Of particular interest are sensor-effector designs that can autonomously detect and treat various pathological conditions, such as a malfunction in urate homeostasis, 12 diet-induced obesity, 13 and diabetic ketoacidosis. 14 Recently, an innovative designer cell device to treat experimental psoriasis by converting the disease-relevant inputs into the therapeutic output was presented. 15 The therapeutic challenge in therapy of inflammatory diseases is to detect and suppress inflammatory flare-ups as soon as possible before they are amplified through positive-inflammatory-feedback loops and cause damage.…”