Engineering a Rapid Insulin Release System Controlled By Oral Drug Administration.

Rapid insulin release plays an essential role in maintaining blood-glucose homeostasis in mammalians. Patients diagnosed with type-I diabetes mellitus experience chronic and remarkably high blood-sugar levels, and require lifelong insulin injection therapy, so there is a need for more convenient and less invasive insulin delivery systems to increase patients' compliance and also to enhance their quality of life. Here, an endoplasmic-reticulum-localized split sec-tobacco etch virus protease (TEVp)-based rapamycin-actuated protein-induction device (RAPID) is engineered, which is composed of the rapamycin-inducible dimerization domains FK506 binding protein (FKBP) and FKBP-rapamycin binding protein fused with modified split sec-TEVp components. Insulin accumulation inside the endoplasmic reticulum (ER) is achieved through tagging its C-terminus with KDEL, an ER-retention signal, spaced by a TEVp cleavage site. In the presence of rapamycin, the split sec-TEVp-based RAPID components dimerize, regain their proteolytic activity, and remove the KDEL retention signal from insulin. This leads to rapid secretion of accumulated insulin from cells within few minutes. Using liver hydrodynamic transfection methodology, it is shown that RAPID quickly restores glucose homeostasis in type-1-diabetic (T1DM) mice treated with an oral dose of clinically licensed rapamycin. This rapid-release technology may become the foundation for other cell-based therapies requiring instantaneous biopharmaceutical availability.

Genetically Encoded Protein Thermometer Enables Precise Electrothermal Control of Transgene Expression.

Body temperature is maintained at around 37 °C in humans, but may rise to 40 °C or more during high-grade fever, which occurs in most adults who are seriously ill. However, endogenous temperature sensors, such as ion channels and heat-shock promoters, are fully activated only at noxious temperatures above this range, making them unsuitable for medical applications. Here, a genetically encoded protein thermometer (human enhanced gene activation thermometer; HEAT) is designed that can trigger transgene expression in the range of 37-40 °C by linking a mutant coiled-coil temperature-responsive protein sensor to a synthetic transcription factor. To validate the construct, a HEAT-transgenic monoclonal human cell line, FeverSense, is generated and it is confirmed that it works as a fever sensor that can temperature- and exposure-time-dependently trigger reporter gene expression in vitro and in vivo. For translational proof of concept, microencapsulated designer cells stably expressing a HEAT-controlled insulin production cassette in a mouse model of type-1 diabetes are subcutaneously implanted and topical heating patches are used to apply heat corresponding to a warm sensation in humans. Insulin release is induced, restoring normoglycemia. Thus, HEAT appears to be suitable for practical electrothermal control of cell-based therapy, and may also have potential for next-generation treatment of fever-associated medical conditions.

A fully human transgene switch to regulate therapeutic protein production by cooling sensation.

The ability to safely control transgene expression with simple synthetic gene switches is critical for effective gene- and cell-based therapies. In the present study, the signaling pathway controlled by human transient receptor potential (TRP) melastatin 8 (hTRPM8), a TRP channel family member1, is harnessed to control transgene expression. Human TRPM8 signaling is stimulated by menthol, an innocuous, natural, cooling compound, or by exposure to a cool environment (15-18 °C). By functionally linking hTRPM8-induced signaling to a synthetic promoter containing elements that bind nuclear factor of activated T cells, a synthetic gene circuit was designed that can be adjusted by exposure to either a cool environment or menthol. It was shown that this gene switch is functional in various cell types and human primary cells, as well as in mice implanted with engineered cells. In response to transdermal delivery of menthol, microencapsulated cell implants harboring this gene circuit, coupled to expression of either of two therapeutic proteins, insulin or a modified, activin type IIB, receptor ligand trap protein (mActRIIBECD-hFc), could alleviate hyperglycemia in alloxan-treated mice (a model of type 1 diabetes) or reverse muscle atrophy in dexamethasone-treated mice (a model of muscle wasting), respectively. This fully human-derived orthogonal transgene switch should be amenable to a wide range of clinical applications.

Caffeine-inducible gene switches controlling experimental diabetes.

Programming cellular behavior using trigger-inducible gene switches is integral to synthetic biology. Although significant progress has been achieved in trigger-induced transgene expression, side-effect-free remote control of transgenes continues to challenge cell-based therapies. Here, utilizing a caffeine-binding single-domain antibody we establish a caffeine-inducible protein dimerization system, enabling synthetic transcription factors and cell-surface receptors that enable transgene expression in response to physiologically relevant concentrations of caffeine generated by routine intake of beverages such as tea and coffee. Coffee containing different caffeine concentrations dose-dependently and reversibly controlled transgene expression by designer cells with this caffeine-stimulated advanced regulators (C-STAR) system. Type-2 diabetic mice implanted with microencapsulated, C-STAR-equipped cells for caffeine-sensitive expression of glucagon-like peptide 1 showed substantially improved glucose homeostasis after coffee consumption compared to untreated mice. Biopharmaceutical production control by caffeine, which is non-toxic, inexpensive and only present in specific beverages, is expected to improve patient compliance by integrating therapy with lifestyle.

Designer exosomes produced by implanted cells intracerebrally deliver therapeutic cargo for Parkinson's disease treatment.

Exosomes are cell-derived nanovesicles (50-150 nm), which mediate intercellular communication, and are candidate therapeutic agents. However, inefficiency of exosomal message transfer, such as mRNA, and lack of methods to create designer exosomes have hampered their development into therapeutic interventions. Here, we report a set of EXOsomal transfer into cells (EXOtic) devices that enable efficient, customizable production of designer exosomes in engineered mammalian cells. These genetically encoded devices in exosome producer cells enhance exosome production, specific mRNA packaging, and delivery of the mRNA into the cytosol of target cells, enabling efficient cell-to-cell communication without the need to concentrate exosomes. Further, engineered producer cells implanted in living mice could consistently deliver cargo mRNA to the brain. Therapeutic catalase mRNA delivery by designer exosomes attenuated neurotoxicity and neuroinflammation in in vitro and in vivo models of Parkinson's disease, indicating the potential usefulness of the EXOtic devices for RNA delivery-based therapeutic applications.

Antagonistic control of a dual-input mammalian gene switch by food additives.

Synthetic biology has significantly advanced the design of mammalian trigger-inducible transgene-control devices that are able to programme complex cellular behaviour. Fruit-based benzoate derivatives licensed as food additives, such as flavours (e.g. vanillate) and preservatives (e.g. benzoate), are a particularly attractive class of trigger compounds for orthogonal mammalian transgene control devices because of their innocuousness, physiological compatibility and simple oral administration. Capitalizing on the genetic componentry of the soil bacterium Comamonas testosteroni, which has evolved to catabolize a variety of aromatic compounds, we have designed different mammalian gene expression systems that could be induced and repressed by the food additives benzoate and vanillate. When implanting designer cells engineered for gene switch-driven expression of the human placental secreted alkaline phosphatase (SEAP) into mice, blood SEAP levels of treated animals directly correlated with a benzoate-enriched drinking programme. Additionally, the benzoate-/vanillate-responsive device was compatible with other transgene control systems and could be assembled into higher-order control networks providing expression dynamics reminiscent of a lap-timing stopwatch. Designer gene switches using licensed food additives as trigger compounds to achieve antagonistic dual-input expression profiles and provide novel control topologies and regulation dynamics may advance future gene- and cell-based therapies.