Abstract

Homeostasis represents a critical balance between catabolic and anabolic states, which is especially evident in tissues like cartilage. When homeostasis is disrupted, such as in rheumatoid arthritis (RA), progressive joint destruction occurs. While inflammation is required in response to infection or tissue injury, aberrant immune activity can contribute to harmful outcomes in diseases like RA. The proinflammatory state in RA is perpetuated by cytokine signaling, notably including interleukin-1 (IL-1). Intracellular pathways activated by this signaling regulate networks such as the nuclear factor-κB (NF-κB) pathway, creating an environment of sustained inflammation. While no cure exists for RA, advances in disease modifying antirheumatic drugs (DMARDs), particularly in biologic drugs, have opened new opportunities for tailored treatment to target specific cytokines. For example, the targeted biologic IL-1 receptor antagonist (IL-1Ra; anakinra) is an FDA-approved DMARD. However, current treatment requires repeated, high doses to achieve clinical impact, resulting in significant side effects. Making disease management more challenging, patients with RA experience dynamic changes in inflammation across multiple timescales. On a short-term scale, inflammation peaks in the early morning each day, resulting in characteristic morning stiffness of joints. On a longer-term scale, disease varies between flares and remissions that can last weeks to months. Therefore, there is a need for drug delivery approaches that account for dynamic changes in disease state to optimize drug delivery. To address daily changes in disease, chronotherapy is an emerging concept that aligns therapeutics to the body’s circadian rhythm. While chronotherapy has shown success for synthetic DMARDs and anti-inflammatories in pre-clinical and clinical studies for RA, circadian medicine for biologic DMARDs has not been explored. Therefore, we sought to generate self-regulated drug delivery mechanisms for circadian IL-1Ra release. To accomplish this aim, we generated synthetic gene circuits driven by clock-controlled elements called E’-boxes, D-boxes, and RREs, which stimulate downstream gene expression according to the endogenous intercellular circadian feedback loops. We demonstrated that these chronogenetic gene circuits produced IL-1Ra at unique phases in a 24-hour cycle and protected an in vitro model of arthritis from circadian disruption and inflammatory degradation. Building on this concept, we engineered a dual-responsive synthetic gene circuit to address both short- and long-term dynamic inflammation. Utilizing a promoter with OR-gate logic driven by both inflammatory NF-κB-responsive elements and circadian E’-box elements, this design generated cyclical daily output to target circadian changes in inflammation plus enhanced therapeutic release on-demand in inflammatory environments, assessed in vitro. Finally, we translated the chronogenetic drug delivery mechanism in vivo, where we assessed hydrogel-encapsulated engineered cartilage constructs for self-regulated daily drug delivery in the K/BxN serum transfer arthritis (STA) model. We demonstrated that engineered cartilage cycled in vivo using bioluminescent reporters, and key clinical features of arthritis including joint swelling, pain, and bone erosion were improved with engineered implants. Together, this research adds to the toolkit for synthetic biology-based tailored drug delivery in arthritis. By utilizing disease-relevant input cues, the gene circuits described overcome limitations in current treatment approaches, demonstrated both in vitro and in vivo, and motivate new opportunities for personalized treatment.

Degree

Doctor of Philosophy (PhD)

Author's Department

Biomedical Engineering

Author's School

McKelvey School of Engineering

Document Type

Dissertation

Date of Award

6-26-2025

Language

English (en)

Download

Available for download on Friday, June 25, 2027

Share

COinS