Mechanobiologic signals regulate cellular responses under physiologic and pathologic conditions. Using synthetic biology and tissue engineering, we developed a mechanically responsive bioartificial tissue that responds to mechanical loading to produce a preprogrammed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. We engineered these cells into living tissues that respond to mechanical loading by producing the anti-inflammatory biologic drug interleukin-1 receptor antagonist. Chondrocyte TRPV4 is activated by osmotic loading and not by direct cellular deformation, suggesting that tissue loading is transduced into an osmotic signal that activates TRPV4. Either osmotic or mechanical loading of tissues transduced with TRPV4-responsive circuits protected constructs from inflammatory degradation by interleukin-1α. This synthetic mechanobiology approach was used to develop a mechanogenetic system to enable long-term, autonomously regulated drug delivery driven by physiologically relevant loading.

Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis but may cause significant adverse effects when delivered continuously at high doses. We used CRISPR-Cas9 genome editing of iPSCs to create a synthetic gene circuit that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic response. Cells were engineered into cartilaginous constructs that showed rapid activation and recovery in response to inflammation in vitro or in vivo. In the murine K/BxN model of inflammatory arthritis, bioengineered implants significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. Therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not achievable by current clinically available disease-modifying drugs. Combination tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

Transient, resolving inflammation plays a critical role in tissue repair and regeneration. In the context of joint disease, however, chronic inflammation following injury or with osteoarthritis can lead to irreversible articular cartilage degradation and joint pain. Developing tissue engineering strategies for the regeneration of articular cartilage remains challenging due to the harsh inflammatory environment of an injured or arthritic joint, which can promote degradation of engineered tissues as well as native articular cartilage. Here, we developed an artificial gene circuit for controlled, cell-based delivery of biologic drugs, based on a nuclear factor kappa-lightchain-enhancer of activated B cells (NF-kB)-responsive synthetic promoter. Using lentivirus-based gene therapy, we engineered murine induced pluripotent stem cells (iPSCs) capable of attenuating inflammation through controlled release of an anti-inflammatory drug, interleukin-1 receptor antagonist (IL-1Ra), subsequently inhibiting gene circuit activation in a self-regulating manner. Murine iPSCs were transduced with the synthetic gene circuit either in monolayer or through biomaterial-mediated transduction. Cells were maintained in monolayer or differentiated into cartilage constructs and stimulated with different doses of interleukin 1 alpha (IL-1a) to determine the ability of this synthetic NF-kB responsive system to inhibit inflammation and protect tissue-engineered constructs. In response to IL-1a, cells produced high levels of IL-1Ra, which inhibited inflammatory signaling and protected tissue-engineered cartilage from proteoglycan degradation. Our results show that the combination of gene therapy and tissue engineering can be used to successfully create iPSCs capable of producing biologic drugs in a controlled manner. This self-regulating system provides a tool for cellbased drug delivery as the basis for a novel therapeutic approach for a variety of diseases.

Mechanobiologic signals play critical roles in regulating cellular responses under both physiologic and pathologic conditions. Using a combination of synthetic biology and tissue engineering, we developed a mechanically-responsive bioartificial tissue that responds to mechanical loading to produce a pre-programmed therapeutic biologic drug. By deconstructing the signaling networks induced by activation of the mechanically-sensitive ion channel transient receptor potential vanilloid 4 (TRPV4), we created synthetic TRPV4-responsive genetic circuits in chondrocytes. These cells were then engineered into living tissues constructs that respond to mechanical compression to drive the production of the antiinflammatory biologic interleukin-1 receptor antagonist. Mechanical loading of these tissues constructs in the presence of the cytokine interleukin-1a protected constructs from inflammatory degradation. This "mechanogenetic" approach enables long-term autonomous delivery of therapeutic compounds that are driven by physiologically-relevant mechanical loading with cell-scale mechanical force resolution. The development of synthetic mechanogenetic gene circuits provides a novel approach for the autonomous regulation of cell-based drug delivery systems.