Genome Engineering of Stem Cells for Autonomously Regulated, Closed-Loop Delivery of Biologic Drugs.

Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response but have significant side effects and are used at high, constant doses that do not reflect the dynamic nature of disease activity. Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells, as demonstrated here, opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases.

CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.

OBJECTIVE: Proinflammatory cytokines such as interleukin-1 (IL-1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation-resistant murine induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues. METHODS: Targeted deletion of the IL-1 receptor type I (IL-1RI) gene in murine iPSCs was achieved using the RNA-guided, site-specific clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histologic, and biomechanical assays after a 3-day treatment with 1 ng/ml of IL-1α. RESULTS: Three of 41 clones isolated possessed the IL-1RI+/- genotype. Four clones possessed the IL-1RI-/- genotype, and flow cytometry confirmed loss of IL-1RI on the surface of these cells, which led to an absence of NF-κB transcription activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. In contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade. CONCLUSION: This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to proinflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering.