A modular degron library for synthetic circuits in mammalian cells.

Tight control over protein degradation is a fundamental requirement for cells to respond rapidly to various stimuli and adapt to a fluctuating environment. Here we develop a versatile, easy-to-handle library of destabilizing tags (degrons) for the precise regulation of protein expression profiles in mammalian cells by modulating target protein half-lives in a predictable manner. Using the well-established tetracycline gene-regulation system as a model, we show that the dynamics of protein expression can be tuned by fusing appropriate degron tags to gene regulators. Next, we apply this degron library to tune a synthetic pulse-generating circuit in mammalian cells. With this toolbox we establish a set of pulse generators with tailored pulse lengths and magnitudes of protein expression. This methodology will prove useful in the functional roles of essential proteins, fine-tuning of gene-expression systems, and enabling a higher complexity in the design of synthetic biological systems in mammalian cells.

Sensing and responding to allergic response cytokines through a genetically encoded circuit.

While constantly rising, the prevalence of allergies is globally one of the highest among chronic diseases. Current treatments of allergic diseases include the application of anti-histamines, immunotherapy, steroids, and anti-immunoglobulin E (IgE) antibodies. Here we report mammalian cells engineered with a synthetic signaling cascade able to monitor extracellular pathophysiological levels of interleukin 4 and interleukin 13, two main cytokines orchestrating allergic inflammation. Upon activation of transgenic cells by these cytokines, designed ankyrin repeat protein (DARPin) E2_79, a non-immunogenic protein binding human IgE, is secreted in a precisely controlled and reversible manner. Using human whole blood cell culturing, we demonstrate that the mammalian dual T helper 2 cytokine sensor produces sufficient levels of DARPin E2_79 to dampen histamine release in allergic subjects exposed to allergens. Hence, therapeutic gene networks monitoring disease-associated cytokines coupled with in situ production, secretion and systemic delivery of immunomodulatory biologics may foster advances in the treatment of allergies.