(B-51) PEG Hydrogels as a Platform for Self-Regulating Anti-Cytokine Therapy in a Model of Rheumatoid Arthritis

Introduction:: Rheumatoid arthritis (RA) is a debilitating, progressive, and chronic inflammatory disease that leads to joint swelling and destruction. Current treatment strategies involve the administration of biologic anti-cytokine drugs. While effective, biologics are administered at high doses continuously despite the rapidly varying levels of inflammatory cytokine associated with an RA flare. The goal of this study was to develop an implantable cell-based system that can provide on-demand anti-cytokine therapy proportional to the levels of inflammatory cytokines released during flares. Our laboratory has established a murine induced pluripotent stem cell (iPSC) line using CRISPR-Cas9 genome editing that responds to elevated TNF-α by synthesizing its inhibitor, soluble TNF receptor 1 (sTNFR1)¹. We designed a poly(ethylene glycol) (PEG) hydrogel niche for these therapeutic cells using thiol-ene click chemistry, which allows for precise tunability over mechanical properties as well as incorporation of functional biochemical cues. TNF-α is a potent cytokine implicated in RA progression, and anti-TNF therapies are among the most common and successful biologic treatments for RA, making TNF-α an ideal target with which to test our hydrogel system. Therapeutic hydrogels were tested in vitro for viability, extracellular matrix (ECM) deposition, and secretion of sTNFR1. Using a subcutaneous implant model, therapeutic hydrogels were then tested in vivo in a model of TNF-α-mediated inflammatory arthritis. Our preliminary results highlight the promise of PEG hydrogels as a drug delivery platform that can stably provide continuous, long-term delivery of proportional anti-cytokine therapy to mitigate disease.


Materials and Methods:: For cell-laden hydrogels, 9% (w/w) 8-arm PEG-norbornene was crosslinked with PEG-dithiol at a 1:1 molar thiol:ene ratio with 0.1 mM cell adhesion peptide (GCRGDS) and 1% thiolated chondroitin sulfate². Murine iPSCs edited at the Ccl2 locus to express sTNFR1 (Ccl2_sTNFR1) or luciferase reporter (Ccl2_luc) were incorporated with the polymer precursor solution at 100E6 cells/mL and polymerized under 6mW/cm² UV light with the photoinitiator Irgacure 2959 (Fig 1a). Cell-laden hydrogel disks were cultured for 21 days in chondrogenic medium and evaluated for viability by live/dead staining. Hydrogels were then challenged with 20 ng/mL TNF-α and evaluated for sTNFR1 secretion (ELISA), ECM deposition (safranin-O staining) and glycosaminoglycan (GAG) content. Hydrogel rods were prepared for subcutaneous injection via a wide bore needle in vivo. We utilized bone marrow chimeras wherein seven-week-old C57BL/6 CD45.2 mice were lethally irradiated and received donor bone marrow (BM) from either wildtype (WT) or TNF^ΔARE/+ CD45.1 mice. Engraftment was confirmed >95% by flow cytometry. Mice were euthanized after 16 weeks and their hind paws were evaluated for inflammation histologically and for bone erosion by micro computed tomography (μCT, Bruker SkyScan 1176). All quantitative data were analyzed using two-tailed student t-tests or ANOVA with Tukey post-hoc comparisons (α = 0.05) where appropriate. All animal protocols were approved by the IACUC and follow the NIH guidelines for animal use and care.


Results, Conclusions, and Discussions:: Our results show that PEG hydrogels provide a drug delivery platform which maintains cell viability and responsiveness. In vitro testing of therapeutic constructs revealed nearly 100% live encapsulated cells by live/dead staining after 21 days of culture (Fig 1b). PEG hydrogels supported differentiation and viability of the encapsulated iPSCs, with increased GAG/DNA content and safranin-O staining over the culture period (Fig 1c). Upon inflammatory challenge, the hydrogels supported diffusion of sTNFR1 to protect the tissue engineered construct from TNF-α-induced damage and GAG loss compared to luciferase controls (Fig 1d-f).

To assess the efficacy of therapeutic cell-laden PEG hydrogel constructs in vivo, we utilized TNF^ΔARE/+ mice, which exhibit systemic overexpression of translated TNF-α resulting in a spontaneous inflammatory phenotype. We have shown that an RA phenotype with elevated plasma TNF-α level is inducible in WT mice through BM transplantation from TNF^ΔARE/+ mice, allowing for controlled onset of disease. TNF^ΔARE/+ BM chimeras weighed less than WT controls and had significant bone erosion in their hind paws exemplified by lower bone mineral density (BMD), higher bone surface to bone volume ratio (BS/BV), and lower bone volume to total volume ratio (BV/TV) (Fig 2a). Histological assessment revealed hallmarks of RA, including cartilage degradation and synovial inflammation. This phenotype was prevented by treatment with anti-TNF-α antibody (not shown). We then injected Ccl2_sTNFR1-laden PEG hydrogel rods subcutaneously in TNF^ΔARE/+ BM chimeras. Preliminary data shows that weight loss was mitigated in mice that received an sTNFR1-producing implant, and that TNF^ΔARE/+ BM chimeras that received implants had better bone outcomes by μCT than untreated controls, though these differences were not significant (Fig 2b,c). Ongoing work aims to optimize dose and implant geometry to improve outcomes.

There remains a need for sensitive and proportional anti-cytokine therapy to treat RA flares in a controlled manner. Here, we demonstrate in a TNF-mediated model that PEG hydrogels are a promising platform for the delivery of gene-edited cells for anti-cytokine therapy. This platform can be expanded to create dynamic, responsive stem cells to a broad range of inflammatory mediators involved in RA, offering a highly tunable and self-regulating system for drug delivery.


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References (Optional): : [1] Brunger+, Stem Cell Reports, 2017. [2] Aisenbrey+, J Mater Chem B, 2016.