Hydrogel Encapsulation of Genome-Engineered Stem Cells for Long-Term Self-Regulating Anti-Cytokine Therapy

Abstract

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.

Keywords: autoimmune; designer cells; drug delivery; implant; induced pluripotent stem cells; rheumatoid arthritis.

Figure 1

(A) Schematic of agarose constructs of different thicknesses (2.4 mm [surface area = 36.8 mm²], 1.6 mm [29.2 mm²], and 0.75 mm [21.2 mm²]) loaded with Ccl2-IL1Ra iPSCs at the same cell densities and stimulated for chondrogenesis for 3 weeks. (B) After 24 h and 72 h of 1 ng/mL IL-1α treatment, the 2.4 and 1.6 mm constructs loaded with Ccl2-IL1Ra iPSCs produce similar levels of IL-1Ra, significantly exceeding the IL-1Ra production levels in the 1.6 mm construct loaded with Ccl2-Luc cells shown by dashed lines. (C) Fluorescent live/dead labeling (green = live; red = dead) show uniformly distributed live cells throughout the thickness of the 2.4 and 1.6 mm constructs, as well as relatively uniform proteoglycan staining by Safranin-O, scale bars: 500 µm. One-way ANOVA with Tukey’s post-hoc test, n = 4–5/group/timepoint. Different letters (a,b) indicate groups are significantly different from one another (p < 0.05).

Figure 2

(A) Custom-designed molds (left) were 3D-printed to generate cell-laden agarose rod constructs, which were aspirated into a wide-bore needle (middle) for minimally-invasive implantation subcutaneously through a small dorsal incision (right). (B) Ccl2-luc agarose rod constructs were challenged with 1 ng/mL IL-1α imaged via IVIS to observe activation over time, shown here is a time course of the same two constructs at 1 h, 4 h, and 12 h post IL-1α challenge. (C) Constructs showed robust activation with repeated washout (designated “W”) and reactivation with 1 ng/mL IL-1α. n = 2–6 samples/group/timepoint.

Figure 3

(A) Luminescence activity by IVIS 10 weeks after Ccl2-Luc agarose construct implantation indicates that cell activation peaked over the first 3 days after K/BxN serum challenge (B). (C) Luminescence activity by IVIS from Ccl2-Luc agarose constructs 28 weeks after implantation indicates cell activation after repeated K/BxN challenge. (D) Ccl2-Luc constructs demonstrated predominantly live cells upon explantation at 28 weeks, scale = 1 mm. (E,F) Ccl2-Luc constructs explanted after 1 and 28 weeks of implantation and challenged with 1 ng/mL IL-1α exhibited robust luminescence intensities ex vivo. n = 5–10 animals/group compared by 2-way repeated measures ANOVA and Dunnet’s or Tukey’s post-hoc test, n = 2–8/group/timepoint.

Figure 4

(A) Timeline showing in vivo K/BxN serum transfer arthritis (STA) studies at 1 week, 10 weeks, or 28 weeks post-subcutaneous implantation of Ccl2-IL1Ra agarose rods. Clinical scores of K/BxN STA animals 1 week after implantation of Ccl2-luc or Ccl2-IL1Ra agarose rods over 6 days of observation for (B) overall clinical scores, (C) hindpaws and (D) front paws. (E) IL-1Ra levels in serum at day 2 post K/BxN serum challenge, as measured by ELISA. IL1Ra constructs significantly mitigated mechanical allodynia by (F) Electronic von Frey and (G) pressure-pain hyperalgesia. STA overall clinical scores (H) 10 weeks and (I) 28 weeks after implantation of Ccl2-IL1Ra agarose rods. Note that the degree of disease mitigation observed after 28 weeks is similar to mitigation 1 week after implantation. Different letters indicate significant main effects of group, p < 0.05. N = 3–6/group, data were analyzed by 2-way repeated measures ANOVA with Dunnet’s or Tukey’s post-hoc test. Different letters indicate main effect of group p < 0.05.