504. Targeted Genome Engineering of Induced Pluripotent Stem Cells To Produce Auto-Regulated Inflammation Resistance for Musculoskeletal Regenerative Medicine

Abstract

Chronic inflammatory diseases such as arthritis are characterized by aberrant and dysregulated responses to cytokines such as tumor necrosis factor α (TNFα). While exogenous anti-cytokine therapies have been shown to diminish this inflammatory response, they are often used at high, unregulated doses that can induce significant side effects. Here, we propose a novel regenerative medicine approach to treating such chronic inflammatory diseases by generating custom-designed cells that can execute real-time, user-defined responses to environmental cues, including pro-inflammatory cytokines. We designed and deployed gene editing nucleases based on the CRISPR/Cas9 system to create stem cells with the ability to antagonize TNFα-mediated inflammation in an auto-regulated, feedback-controlled manner for musculoskeletal regenerative medicine applications. Specifically, the cytokine-responsive Ccl2 gene was reprogrammed by nuclease-mediated integration of the gene encoding soluble TNFα receptor I (sTNFRI), a TNFα antagonist, immediately downstream of the Ccl2 promoter. Using a combination of qRT-PCR and site-specific integration of luciferase reporters, we observed increased transgene transcription in response to endogenous Ccl2 activation by treatment with TNFα. Transgene expression from engineered cells was feedback-controlled with rapid on/off dynamics (Fig 1 A). Importantly, engineered cells showed the ability to mitigate the inflammatory effects of TNFα in an auto-regulated manner within 48 hr of induction (Fig 1 B). Finally, while cartilage produced from control stem cells showed significant tissue degradation in response to TNFα, cartilage derived from engineered stem cells was protected from a 3-day treatment with 20 ng/ml TNFα, as evidenced by qRT-PCR, biochemical, and histological analyses (Fig 1C-G).This work demonstrates the utility of programmable nucleases for the development of “designer” stem cells with properties customized for regenerative medicine. Using CRISPR/Cas9, we engineered pluripotent stem cells with the user-specified trait of pro-inflammatory cytokine resistance. Our results show that genome engineering facilitated the rewiring of endogenous cell circuits in order to define novel input/output relationships between inflammatory mediators and their antagonists, achieving therapeutic benefit coupled to a rapidly responding, auto-regulated system. The customization of intrinsic cellular signaling pathways in therapeutic stem cell populations, as demonstrated in this work, can transform the landscape of regenerative medicine and open innovative possibilities for safer and more effective treatments applicable to a wide variety of diseases. View Large Image | Download PowerPoint Slide