Eukaryotic release factor 1 from Euplotes promotes frameshifting at premature stop codons in human cells.

Human physiology is highly susceptible to frameshift mutations within coding regions, and many hereditary diseases and cancers are caused by such indels. Presently, therapeutic options to counteract them are limited and, in the case of direct genome editing, risky. Here, we show that release factor 1 (eRF1) from Euplotes, an aquatic protist known for frequent +1 frameshifts in its coding regions, can enhance +1 ribosomal frameshifting at slippery heptameric sequences in human cells without an apparent requirement for an mRNA secondary structure. We further show an increase in frameshifting rate at the premature termination sequence found in the HEXA gene of Tay-Sachs disease patients, or a breast cancer cell line that harbors a tumor-driving frameshift mutation in GATA3. Although the overall increase in frameshifting would need further improvement for clinical applications, our results underscore the potential of exogenous factors, such as Eu eRF1, to increase frameshifting in human cells.

Controlling therapeutic protein expression via inhalation of a butter flavor molecule.

Precise control of the delivery of therapeutic proteins is critical for gene- and cell-based therapies, and expression should only be switched on in the presence of a specific trigger signal of appropriate magnitude. Focusing on the advantages of delivering the trigger by inhalation, we have developed a mammalian synthetic gene switch that enables regulation of transgene expression by exposure to the semi-volatile small molecule acetoin, a widely used, FDA-approved food flavor additive. The gene switch capitalizes on the bacterial regulatory protein AcoR fused to a mammalian transactivation domain, which binds to promoter regions with specific DNA sequences in the presence of acetoin and dose-dependently activates expression of downstream transgenes. Wild-type mice implanted with alginate-encapsulated cells transgenic for the acetoin gene switch showed a dose-dependent increase in blood levels of reporter protein in response to either administration of acetoin solution via oral gavage or longer exposure to acetoin aerosol generated by a commercial portable inhaler. Intake of typical acetoin-containing foods, such as butter, lychees and cheese, did not activate transgene expression. As a proof of concept, we show that blood glucose levels were normalized by acetoin aerosol inhalation in type-I diabetic mice implanted with acetoin-responsive insulin-producing cells. Delivery of trigger molecules using portable inhalers may facilitate regular administration of therapeutic proteins via next-generation cell-based therapies to treat chronic diseases for which frequent dosing is required.

Electrochemical Properties of PEDOT:PSS/Graphene Conductive Layers in Artificial Sweat.

Electrodes based on PEDOT:PSS are gaining increasing importance as conductive electrodes and functional layers in various sensors and biosensors due to their easy processing and biocompatibility. This study investigates PEDOT:PSS/graphene layers deposited via spray coating on flexible PET substrates. The layers are characterized in terms of their morphology, roughness (via AFM and SEM), and electrochemical properties in artificial sweat using electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The layers exhibit dominant capacitive behavior at low frequencies, with cut-off frequencies determined for thicker layers at 1 kHz. The equivalent circuit used to fit the EIS data reveals a resistance of about three orders of magnitude higher inside the layer compared to the charge transfer resistance at the solid/liquid interface. The capacitance values determined from the CV curves range from 54.3 to 122.0 mF m-2. After 500 CV cycles in a potential window of 1 V (from -0.3 to 0.7 V), capacitance retention for most layers is around 94%, with minimal surface changes being observed in the layers. The results suggest practical applications for PEDOT:PSS/graphene layers, both for high-frequency impedance measurements related to the functioning of individual organs and systems, such as impedance electrocardiography, impedance plethysmography, and respiratory monitoring, and as capacitive electrodes in the low-frequency range, realized as layered PEDOT:PSS/graphene conductive structures for biosignal recording.

Biomarker-driven feedback control of synthetic biology systems for next-generation personalized medicine.

Many current clinical therapies for chronic diseases involve administration of drugs using dosage and bioavailability parameters estimated for a generalized population. This standard approach carries the risk of under dosing, which may result in ineffective treatment, or overdosing, which may cause undesirable side effects. Consequently, maintaining a drug concentration in the therapeutic window often requires frequent monitoring, adversely affecting the patient's quality of life. In contrast, endogenous biosystems have evolved finely tuned feedback control loops that govern the physiological functions of the body based on multiple input parameters. To provide personalized treatment for chronic diseases, therefore, we require synthetic systems that can similarly generate a calibrated therapeutic response. Such engineered autonomous closed-loop devices should incorporate a sensor that actively tracks and evaluates the disease severity based on one or more biomarkers, as well as components that utilize these molecular inputs to bio compute and deliver the appropriate level of therapeutic output. Here, we review recent advances in applications of the closed-loop design principle in biomedical implants for treating severe and chronic diseases, highlighting translational studies of cellular therapies. We describe the engineering principles and components of closed-loop therapeutic devices, and discuss their potential to become a key pillar of personalized medicine.

Sunlight-Controllable Biopharmaceutical Production for Remote Emergency Supply of Directly Injectable Therapeutic Proteins.

Biopharmaceutical manufacturing requires specialized facilities and a long-range cold supply chain for the delivery of the therapeutics to patients. In order to produce biopharmaceuticals in locations lacking such infrastructure, a production process is designed that utilizes the trigger-inducible release of large quantities of a stored therapeutic protein from engineered endocrine cells within minutes to generate a directly injectable saline solution of the protein. To illustrate the versatility of this approach, it is shown that not only insulin, but also glucagon-like peptide 1 (GLP-1), nanoluciferase (NLuc), and the model biopharmaceutical erythropoietin (EPO) can be trigger-inducibly released, even when using biologically inactive insulin as a carrier. The facilitating beta cells are engineered with a controllable TRPV1-mediated Ca2+ influx that induces the fusion of cytoplasmic storage vesicles with the membrane, leading to the release of the stored protein. When required, the growth medium is exchanged for saline solution, and the system is stimulated with the small molecule capsaicin, with a hand-warming pack, or simply by using sunlight. Injection of insulin saline solution obtained in this way into a type-1 diabetes mouse model results in the regulation of blood glucose levels. It is believed that this system will be readily adaptable to deliver various biopharmaceutical proteins at remote locations.

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.

Substrate Plasticity of a Fungal Peptide α-N-Methyltransferase.

The methylation of amide nitrogen atoms can improve the stability, oral availability, and cell permeability of peptide therapeutics. Chemical N-methylation of peptides is challenging. Omphalotin A is a ribosomally synthesized, macrocylic dodecapeptide with nine backbone N-methylations. The fungal natural product is derived from the precursor protein, OphMA, harboring both the core peptide and a SAM-dependent peptide α-N-methyltransferase domain. OphMA forms a homodimer and its α-N-methyltransferase domain installs the methyl groups in trans on the hydrophobic core dodecapeptide and some additional C-terminal residues of the protomers. These post-translational backbone N-methylations occur in a processive manner from the N- to the C-terminus of the peptide substrate. We demonstrate that OphMA can methylate polar, aromatic, and charged residues when these are introduced into the core peptide. Some of these amino acids alter the efficiency and pattern of methylation. Proline, depending on its sequence context, can act as a tunable stop signal. Crystal structures of OphMA variants have allowed rationalization of these observations. Our results hint at the potential to control this fungal α-N-methyltransferase for biotechnological applications.