Date of Award
Doctor of Philosophy (PhD)
The recent advances in the fields of synthetic biology and genome engineering open up new possibilities for creating cell-based therapies. We combined these tools to target repair of articular cartilage, a tissue that lacks a natural ability to regenerate, in the presence of arthritic diseases. To this end, we developed cell-based therapies that harness disease pathways and the unique properties of articular cartilage for prescribed, localized, and controlled delivery of biologics, creating the next generation of cell therapies and new classes of synthetic circuits. We created tissue engineered cartilage from murine induced pluripotent stem cells that had the ability to sense inflammatory stimuli to produce an anti-cytokine biologic to self-regulate and inhibit inflammation. To create this gene circuit, we developed a synthetic promoter activated by NF-κB signaling, a key inflammatory pathway activated within chondrocytes in arthritis. This lentiviral system was capable of producing an anti-cytokine therapeutic, IL-1Ra, and protecting tissue engineered cartilage from inflammation-mediated degradation. Chondrocytes within articular cartilage respond to mechanobiologic signals through ion channels, such as the TRPV4 ion channel, involved in mechanotransduction. We developed synthetic cell-based therapies that could sense mechanical stimuli, such as activation of TRPV4, and produce prescribed biologic drugs in response to mechanical stimuli. With this approach, we created two novel mechanogenetic circuits activated by TRPV4 that produced our therapeutic transgene with different drug release kinetics. The cartilage circadian clock plays a key role in maintaining cartilage homeostasis and integrity. When the circadian clock is desynchronized, such as in the presence of inflammation, articular cartilage begins to degrade. Therefore, we created clock-preserving synthetic circuits that are capable of preserving circadian rhythms even in the presence of inflammation. In addition to creating these circuits, we also characterized the circadian clock throughout chondrogenic differentiation and uncovered interesting characteristics between circadian disruption and extracellular matrix (ECM) degradation that can be further examined to better understand the relationship between inflammation and circadian rhythm disruption. Finally, we developed the newest generation of cell-based therapies by creating chronogenetic therapies. Expanding beyond preserving circadian rhythms, we developed synthetic chronogenetic circuits driven by the circadian clock for temporal delivery of biologic drugs at specific times of day. This approach was motivated by the field of chronotherapy and the increase in efficacy of drugs when administered at specific times of day. We developed the first cell-based chronotherapy capable of producing an anti-inflammatory biologic at a specific time to combat the peak of inflammatory flares exhibited by patients with chronic inflammation. Overall, the work in this dissertation builds upon existing synthetic biology and genome engineering tools to create smart cell therapies that are activated by a prescribed input and can produce a therapeutic transgene in a controlled manner. These synthetic circuits provide novel strategies to target inflammation in an arthritic joint and can be expanded for other applications to create better and more effective therapeutics to treat disease.
William Buchser, Erik Herzog, Nathaniel Huebsch, Jeffrey Millman,
Available for download on Sunday, May 15, 2022