Synthetic Gene Circuits for Self-Regulating and Temporal Delivery of Anti-Inflammatory Biologic Drugs in Engineered Tissues

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

Hydrogel encapsulation of genome-engineered stem cells for long-term self-regulating anti-cytokine therapy

Biologic therapies have revolutionized treatment options for rheumatoid arthritis (RA) but their continuous administration at high doses may lead to adverse events. Thus, the development of improved drug delivery systems that can sense and respond commensurately to disease flares represents an unmet medical need. Toward this end, we generated induced pluripotent stem cells (iPSCs) that express interleukin-1 receptor antagonist (IL-1Ra, an inhibitor of IL-1) in a feedback-controlled manner driven by the macrophage chemoattractant protein-1 (Ccl2) promoter. Cells were seeded in agarose hydrogel constructs made from 3D printed molds that can be injected subcutaneously via a blunt needle, thus simplifying implantation of the constructs, and the translational potential. We demonstrated that the subcutaneously injected agarose hydrogels containing genome-edited Ccl2-IL1Ra iPSCs showed significant therapeutic efficacy in the K/BxN model of inflammatory arthritis, with nearly complete abolishment of disease severity in the front paws. These implants also exhibited improved implant longevity as compared to the previous studies using 3D woven scaffolds, which require surgical implantation. This minimally invasive cell-based drug delivery strategy may be adapted for the treatment of other autoimmune or chronic diseases, potentially accelerating translation to the clinic

A synthetic mechanogenetic gene circuit for autonomous drug delivery in engineered tissues

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

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery

[Figure: see text]

Quantitatively Predictable Control of Cellular Protein Levels through Proteasomal Degradation

Protein function is typically studied and engineered by modulating protein levels within the complex cellular environment. To achieve fast, targeted, and predictable control of cellular protein levels without genetic manipulation of the target, we developed a technology for post-translational depletion based on a bifunctional molecule (NanoDeg) consisting of the antigen-binding fragment from the <i>Camelidae</i> species heavy-chain antibody (nanobody) fused to a degron signal that mediates degradation through the proteasome. We provide proof-of-principle demonstration of targeted degradation using a nanobody against the green fluorescent protein (GFP). Guided by predictive modeling, we show that customizing the NanoDeg rate of synthesis, rate of degradation, and mode of degradation enables quantitative and predictable control over the target’s levels. Integrating the GFP-specific NanoDeg within a genetic circuit based on stimulus-dependent GFP output results in enhanced dynamic range and resolution of the output signal. By providing predictable control over cellular proteins’ levels, the NanoDeg system could be readily used for a variety of systems-level analyses of cellular protein function

Synthetic gene circuits for preventing disruption of the circadian clock due to interleukin-1-induced inflammation

The circadian clock regulates tissue homeostasis through temporal control of tissue-specific clock-controlled genes. In articular cartilage, disruptions in the circadian clock are linked to a procatabolic state. In the presence of inflammation, the cartilage circadian clock is disrupted, which further contributes to the pathogenesis of diseases such as osteoarthritis. Using synthetic biology and tissue engineering, we developed and tested genetically engineered cartilage from murine induced pluripotent stem cells (miPSCs) capable of preserving the circadian clock in the presence of inflammation. We found that circadian rhythms arise following chondrogenic differentiation of miPSCs. Exposure of tissue-engineered cartilage to the inflammatory cytokine interleukin-1 (IL-1) disrupted circadian rhythms and degraded the cartilage matrix. All three inflammation-resistant approaches showed protection against IL-1-induced degradation and loss of circadian rhythms. These synthetic gene circuits reveal a unique approach to support daily rhythms in cartilage and provide a strategy for creating cell-based therapies to preserve the circadian clock

Synthetic gene circuits for preventing disruption of the circadian clock due to interleukin-1–induced inflammation

The circadian clock regulates tissue homeostasis through temporal control of tissue-specific clock-controlled genes. In articular cartilage, disruptions in the circadian clock are linked to a procatabolic state. In the presence of inflammation, the cartilage circadian clock is disrupted, which further contributes to the pathogenesis of diseases such as osteoarthritis. Using synthetic biology and tissue engineering, we developed and tested genetically engineered cartilage from murine induced pluripotent stem cells (miPSCs) capable of preserving the circadian clock in the presence of inflammation. We found that circadian rhythms arise following chondrogenic differentiation of miPSCs. Exposure of tissue-engineered cartilage to the inflammatory cytokine interleukin-1 (IL-1) disrupted circadian rhythms and degraded the cartilage matrix. All three inflammation-resistant approaches showed protection against IL-1–induced degradation and loss of circadian rhythms. These synthetic gene circuits reveal a unique approach to support daily rhythms in cartilage and provide a strategy for creating cell-based therapies to preserve the circadian clock