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

Abstract

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.

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

“…Next, to exclude potential artifacts caused by transient transfection, we stably integrated Eu eRF1 into human cells and measured frameshifting rates using the RLuc/Fluc reporter ( Figure 1 D). Additionally, to rule out an effect of the reporter gene sequences itself, we exchanged the RLuc/FLuc pair for NanoLuciferase (NLuc) and its temperature stable variant ThermoLuciferase 28 (TLuc) ( Figure 1 E) or the FLuc/NLuc dual-luciferase pair ( Figure 1 G) and observed comparable increase in frameshifting in all reporter constructs. ( Figures 1 F and 1H) Taken together, these results support the notion that Eu eRF1 elevates frameshifting rates in human cells at the consensus stop-frameshift heptamer sequence from Euplotes without the necessity of a secondary structure.…”

“…Rennila luciferase (RLuc) and Firefly luciferase (FLuc) were quantified using the Dual-Glo Luciferase Assay System (Promega AG, Switzerland) following the manufacturers instruction. Nanoluciferase (NLuc) and Termoluciferase (TLuc) were quantified as described before 28 using the Nano-Glo Luciferase Assay System (Promega AG, Switzerland) and 30 min 75°C for inactivation of NLuc before TLuc readout. Dual luciferase luminescence from FLuc and NLuc was quantified using the Nano-Glo Dual-Luciferase Reporter Assay System (Promega AG, Switzerland) according to the manufacturer’s instructions.…”

Small‐Molecule Regulators for Gene Switches to Program Mammalian Cell Behaviour

“…Besides small molecules, physical inputs can be used to trigger gene circuits [9] . However, although these physical strategies, including light, [8] temperature, [9] mechanical forces, [10] magnetic and electric fields, [11] offer non‐invasive, traceless, precise, and efficient means to activate a biological sensor domain, they may require expensive equipment, are often limited to specific applications, and can have other disadvantages. For example, the limited tissue penetration of light restricts the therapeutic applications of optogenetically engineered cell implants [8,12,13] .…”