Christian Kemmer

Self-sufficient control of urate homeostasis in mice by a synthetic circuit

Synthetic biology has shown that the metabolic behavior of mammalian cells can be altered by genetic devices such as epigenetic and hysteretic switches, timers and oscillators, biocomputers, hormone systems and heterologous metabolic shunts. To explore the potential of such devices for therapeutic strategies, we designed a synthetic mammalian circuit to maintain uric acid homeostasis in the bloodstream, disturbance of which is associated with tumor lysis syndrome and gout. This synthetic device consists of a modified Deinococcus radiodurans-derived protein that senses uric acids levels and triggers dose-dependent derepression of a secretion-engineered Aspergillus flavus urate oxidase that eliminates uric acid. In urate oxidase–deficient mice, which develop acute hyperuricemia, the synthetic circuit decreased blood urate concentration to stable sub-pathologic levels in a dose-dependent manner and reduced uric acid crystal deposits in the kidney. Synthetic gene-network devices providing self-sufficient control of pathologic metabolites represent molecular prostheses, which may foster advances in future gene- and cell-based therapies.

Controlling transgene expression in subcutaneous implants using a skin lotion containing the apple metabolite phloretin

Adjustable control of therapeutic transgenes in engineered cell implants after transdermal and topical delivery of nontoxic trigger molecules would increase convenience, patient compliance, and elimination of hepatic first-pass effect in future therapies. Pseudomonas putida DOT-T1E has evolved the flavonoid-triggered TtgR operon, which controls expression of a multisubstrate-specific efflux pump (TtgABC) to resist plant-derived defense metabolites in its rhizosphere habitat. Taking advantage of the TtgR operon, we have engineered a hybrid P. putida–mammalian genetic unit responsive to phloretin. This flavonoid is contained in apples, and, as such, or as dietary supplement, regularly consumed by humans. The engineered mammalian phloretin-adjustable control element (PEACE) enabled adjustable and reversible transgene expression in different mammalian cell lines and primary cells. Due to the short half-life of phloretin in culture, PEACE could also be used to program expression of difficult-to-produce protein therapeutics during standard bioreactor operation. When formulated in skin lotions and applied to the skin of mice harboring transgenic cell implants, phloretin was able to fine-tune target genes and adjust heterologous protein levels in the bloodstream of treated mice. PEACE-controlled target gene expression could foster advances in biopharmaceutical manufacturing as well as gene- and cell-based therapies.

A designer network coordinating bovine artificial insemination by ovulation-triggered release of implanted sperms

Synthetic biology has been successfully used to program novel metabolic function in mammalian cells and to design the first-generation of prosthetic networks that have shown the potential for the treatment of obesity, hormone-related disorders and hyperuricemia in small-animal model systems. By functionally rewiring luteinizing hormone receptor signaling to CREB1 (cAMP-responsive element binding protein 1)-mediated transgene expression via the common cyclic adenosine monophosphate (cAMP) second messenger pool we have designed an artificial insemination device which enables lutropin-triggered in-utero release of sperms protected inside cellulose-based implants. Swiss dairy cows treated with such in-utero implants containing spermatozoa and mammalian cells transgenic for luteinizing hormone receptor and CREB1-inducible expression of an engineered cellulase showed ovulation-triggered implant degradation and sperm release leading to successful fertilization of the animals. Synthetic devices plugged into endogenous control circuitry enable the body to automatically control spatio-temporal metabolic activities that could improve the economics of cattle breeding and provide novel opportunities for future therapeutic interventions.

The food additive vanillic acid controls transgene expression in mammalian cells and mice

Trigger-inducible transcription-control devices that reversibly fine-tune transgene expression in response to molecular cues have significantly advanced the rational reprogramming of mammalian cells. When designed for use in future gene- and cell-based therapies the trigger molecules have to be carefully chosen in order to provide maximum specificity, minimal side-effects and optimal pharmacokinetics in a mammalian organism. Capitalizing on control components that enable Caulobacter crescentus to metabolize vanillic acid originating from lignin degradation that occurs in its oligotrophic freshwater habitat, we have designed synthetic devices that specifically adjust transgene expression in mammalian cells when exposed to vanillic acid. Even in mice transgene expression was robust, precise and tunable in response to vanillic acid. As a licensed food additive that is regularly consumed by humans via flavoured convenience food and specific fresh vegetable and fruits, vanillic acid can be considered as a safe trigger molecule that could be used for diet-controlled transgene expression in future gene- and cell-based therapies.

A novel system for trigger-controlled drug release from polymer capsules

Technologies currently available for the controlled release of protein therapeutics involve either continuous or tissue-specific discharge from implants or engineered extracellular matrix mimetics. For some therapeutic applications the trigger-controlled release of protein cargo from a synthetic implant could be highly desirable. We have designed the CellEase technology, a two-component system consisting of cellulose sulfate (CS) poly-diallyldimethyl ammonium chloride (pDADMAC) capsules harboring mammalian sensor cells transgenic for trigger-inducible expression of an engineered secreted mammalian cellulase (SecCell). SecCell is a Bacillus subtilis-derived (1-4)-beta-glucanase, which was modified by replacing the N-terminal part of the bacterial enzyme with a murine Igkappa-chain V-12-C region-derived secretion signal. SecCell was engineered for doxycycline- or erythromycin-inducible expression to enable trigger-controlled secretion by mammalian cells. Detailed characterization of SecCell showed that it was glycosylated and efficiently secreted by a variety of mammalian sensor cells such that it could internally rupture CS-pDADMAC capsules within which the cells had been encapsulated. When SecCell was inducibly expressed in sender cells, that were co-encapsulated with producer cell lines expressing therapeutic proteins, the removal of relevant inducer molecules enabled the time-dependent discharge of these therapeutic proteins, the kinetics of which could be modified by varying the concentration of inducer molecules or the amount of encapsulated sender cells. SecCells capacity to rupture CS-pDADMAC capsules exclusively internally also enabled the independent trigger-induced release of different proteins from two capsule populations harboring different inducible SecCell sensor cells. CellEase-based protein release was demonstrated in vivo using capsules implanted intraperitoneally into mice that enabled the doxycycline-controlled release of a model glycoprotein and accumulation in the bloodstream of treated animals. Trigger-induced breakdown of tissue-compatible implants which provide a timely controlled release of biologics may foster novel opportunities in human therapy.

A designer cell-based histamine-specific human allergy profiler

Allergic disorders are markedly increasing in industrialized countries. The identification of compounds that trigger the immunoglobulin E-dependent allergic reaction remains the key to limit patients’ exposure to critical allergens and improve their quality of life. Here we use synthetic biology principles to design a mammalian cell-based allergy profiler that scores the allergen-triggered release of histamine from whole-blood-derived human basophils. A synthetic signalling cascade engineered within the allergy profiler rewires histamine input to the production of reporter protein, thereby integrating histamine levels in whole-blood samples with remarkable sensitivity and a wide dynamic range, allowing for rapid results or long-term storage of output, respectively. This approach provides non-intrusive allergy profiles for the personalized medicine era.

Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420

Countering TB prodrug resistance The arsenal of antibiotics for treating tuberculosis (TB) contains many prodrugs, such as ethionamide, which need activation by normal metabolism to release their toxic effects. Ethionamide is potentiated by small molecules. Blondiaux et al. screened for more potent analogs and identified a lead compound called SMARt-420. This small molecule inactivates a TetR-like repressor, EthR2, and boosts ethionamide activation. SMARt-420 successfully promoted clearance of multidrug-resistant strains of Mycobacterium tuberculosis from the lungs of mice. Science, this issue p. 1206 Resistance to an antituberculosis drug can be reversed by small molecules that activate a cryptic enzymatic pathway. Antibiotic resistance is one of the biggest threats to human health globally. Alarmingly, multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis have now spread worldwide. Some key antituberculosis antibiotics are prodrugs, for which resistance mechanisms are mainly driven by mutations in the bacterial enzymatic pathway required for their bioactivation. We have developed drug-like molecules that activate a cryptic alternative bioactivation pathway of ethionamide in M. tuberculosis, circumventing the classic activation pathway in which resistance mutations have now been observed. The first-of-its-kind molecule, named SMARt-420 (Small Molecule Aborting Resistance), not only fully reverses ethionamide-acquired resistance and clears ethionamide-resistant infection in mice, it also increases the basal sensitivity of bacteria to ethionamide.

A novel genome editing platform for drug resistant Acinetobacter baumannii revealed an AdeR-unrelated tigecycline resistance mechanism.

ABSTRACT Infections with the Gram-negative coccobacillus Acinetobacter baumannii are a major threat in hospital settings. The progressing emergence of multidrug-resistant clinical strains significantly reduces the treatment options for clinicians to fight A. baumannii infections. The current lack of robust methods to genetically manipulate drug-resistant A. baumannii isolates impedes research on resistance and virulence mechanisms in clinically relevant strains. In this study, we developed a highly efficient and versatile genome-editing platform enabling the markerless modification of the genome of A. baumannii clinical and laboratory strains, regardless of their resistance profiles. We applied this method for the deletion of AdeR, a transcription factor that regulates the expression of the AdeABC efflux pump in tigecycline-resistant A. baumannii, to evaluate its function as a putative drug target. Loss of adeR reduced the MIC90 of tigecycline from 25 μg/ml in the parental strains to 3.1 μg/ml in the ΔadeR mutants, indicating its importance in the drug resistance phenotype. However, 60% of the clinical isolates remained nonsusceptible to tigecycline after adeR deletion. Evolution of artificial tigecycline resistance in two strains followed by whole-genome sequencing revealed loss-of-function mutations in trm, suggesting its role in an alternative AdeABC-independent tigecycline resistance mechanism. This finding was strengthened by the confirmation of trm disruption in the majority of the tigecycline-resistant clinical isolates. This study highlights the development and application of a powerful genome-editing platform for A. baumannii enabling future research on drug resistance and virulence pathways in clinically relevant strains.