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.

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.

In vitro analysis of genome-engineered muscle-derived stem cells for autoregulated anti-inflammatory and antifibrotic activity.

Traumatic muscle injury leads to chronic and pathologic fibrosis in skeletal muscles, primarily driven through upregulation of transforming growth factor-ß1 (TGF-ß1). Cell-based therapies, such as injection of muscle-derived stem cells (MDSCs), have shown promise in muscle repair. However, injected MDSCs in injured skeletal muscle can differentiate into myofibroblasts under the influence of TGF-ß1, and contribute to the development of fibrosis, limiting their regenerative potential. In this study, we created a "smart" cell-based drug delivery system using CRISPR-Cas9 to genetically engineer MDSCs capable of sensing TGF-ß1 and producing an antifibrotic biologic, decorin. These gene-edited smart cells, capable of inhibiting fibrosis in a dose-dependent and autoregulating manner, show anti-inflammatory and antifibrotic properties in vitro, without changing the expression of myogenic and stem cell markers as well as their cell proliferation and myogenic differentiation. Additionally, differentiation down a fibrotic lineage is reduced or eliminated in response to TGF-ß1. Our results show that gene editing can be used to successfully create smart stem cells capable of producing biologic drugs with antifibrotic capabilities in a controlled and localized manner. This system provides a tool for cell-based drug delivery as the basis for a novel therapeutic approach for a variety of diseases.

Mechanogenetics: harnessing mechanobiology for cellular engineering.

'Mechanogenetics,' a new field at the convergence of mechanobiology and synthetic biology, presents an innovative strategy to treat, repair, or restore diseased cells and tissues by harnessing mechanical signal transduction pathways to control gene expression. While the role of mechanical forces in regulating development, homeostasis, and disease is well established, only recently have we identified the specific mechanosensors and downstream signaling pathways involved in these processes. Simultaneously, synthetic biological systems are developing increasingly sophisticated approaches of controlling mammalian cellular responses. Continued mechanistic refinement and identification of how cellular mechanosensors respond to homeostatic and pathological mechanical forces, combined with synthetic tools to integrate and respond to these inputs, promises to extend the development of new therapeutic approaches for treating disease.

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery.

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.

Immunoengineering the next generation of arthritis therapies.

Immunoengineering continues to revolutionize healthcare, generating new approaches for treating previously intractable diseases, particularly in regard to cancer immunotherapy. In joint diseases, such as osteoarthritis (OA) and rheumatoid arthritis (RA), biomaterials and anti-cytokine treatments have previously been at that forefront of therapeutic innovation. However, while many of the existing anti-cytokine treatments are successful for a subset of patients, these treatments can also pose severe risks, adverse events and off-target effects due to continuous delivery at high dosages or a lack of disease-specific targets. The inadequacy of these current treatments has motivated the development of new immunoengineering strategies that offer safer and more efficacious alternative therapies through the precise and controlled targeting of specific upstream immune responses, including direct and mechanistically-driven immunoengineering approaches. Advances in the understanding of the immunomodulatory pathways involved in musculoskeletal disease, in combination with the growing emphasis on personalized medicine, stress the need for carefully considering the delivery strategies and therapeutic targets when designing therapeutics to better treat RA and OA. Here, we focus on recent advances in biomaterial and cell-based immunomodulation, in combination with genetic engineering, for therapeutic applications in joint diseases. The application of immunoengineering principles to the study of joint disease will not only help to elucidate the mechanisms of disease pathogenesis but will also generate novel disease-specific therapeutics by harnessing cellular and biomaterial responses. STATEMENT OF SIGNIFICANCE: It is now apparent that joint diseases such as osteoarthritis and rheumatoid arthritis involve the immune system at both local (i.e., within the joint) and systemic levels. In this regard, targeting the immune system using both biomaterial-based or cellular approaches may generate new joint-specific treatment strategies that are well-controlled, safe, and efficacious. In this review, we focus on recent advances in immunoengineering that leverage biomaterials and/or genetically engineered cells for therapeutic applications in joint diseases. The application of such approaches, especially synergistic strategies that target multiple immunoregulatory pathways, has the potential to revolutionize our understanding, treatment, and prevention of joint diseases.

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 Synthetic Gene Circuit for Self-Regulating Delivery of Biologic Drugs in Engineered Tissues.

IMPACT STATEMENT: We engineered a synthetic transcription system based on nuclear factor kappa-light-chain-enhancer of activated B cells signaling that can attenuate the effects of the inflammatory cytokine interleukin (IL)-1α in a self-regulating manner. This system responds in a time- and dose-dependent manner to rapidly produce therapeutic levels of IL-1 receptor antagonist (IL-1Ra). The use of lentiviral gene therapy allows this system to be utilized through different transduction methods and in different cell types for a variety of applications. Broadly, this approach may be applicable in developing autoregulated biologic systems for tissue engineering and drug delivery in a range of disease applications.

Designer Stem Cells: Genome Engineering and the Next Generation of Cell-Based Therapies.

Stem cells provide tremendous promise for the development of new therapeutic approaches for musculoskeletal conditions. In addition to their multipotency, certain types of stem cells exhibit immunomodulatory effects that can mitigate inflammation and enhance tissue repair. However, the translation of stem cell therapies to clinical practice has proven difficult due to challenges in intradonor and interdonor variability, engraftment, variability in recipient microenvironment and patient indications, and limited therapeutic biological activity. In this regard, the success of stem cell-based therapies may benefit from cellular engineering approaches to enhance factors such as purification, homing and cell survival, trophic effects, or immunomodulatory signaling. By combining recent advances in gene editing, synthetic biology, and tissue engineering, the potential exists to create new classes of "designer" cells that have prescribed cell-surface molecules and receptors as well as synthetic gene circuits that provide for autoregulated drug delivery or enhanced tissue repair. Published by Wiley Periodicals, Inc. J Orthop Res 37:1287-1293, 2019.

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 Camelidae 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.