Fluoropolymer-mediated delivery of a dual TSHR/IGF1R-targeting CRISPR-Cas9 system for localized gene therapy in thyroid-associated ophthalmopathy
Thyroid-associated ophthalmopathy (TAO), a vision-threatening and disfiguring autoimmune orbital disorder, remains a therapeutic challenge due to the lack of therapies with orbital specificity, sustained efficacy, and minor side effects. Herein, we present G4F7-CRISPR, a fluoropolymer-based CRISPR-Cas9 delivery platform designed for localized and efficient gene disruption of thyroid-stimulating hormone receptor (TSHR) and insulin-like growth factor 1 receptor (IGF1R), two key mediators of TAO pathogenesis. G4F7-CRISPR achieved high insertion/deletion frequencies in primary orbital fibroblasts (Tshr: 37.2%; Igf1r: 42.8%) and mature adipocytes (Tshr: 22.4%; Igf1r: 24.3%), and maintained robust editing efficiency in orbital adipose tissue of TAO mouse models (Tshr: 30.7%; Igf1r: 32.4%), with no detectable off-target effects. In vivo, dual-gene editing of Tshr and Igf1r significantly reduced orbital adipogenesis, inflammation, and fibrosis, demonstrating superior therapeutic efficacy over either single-gene approaches. Notably, G4F7-CRISPR exhibited excellent short- and long-term ocular and systemic safety in TAO mouse models. These therapeutic benefits were further validated in 3D human orbital organoids, highlighting the translational potential of G4F7-CRISPR as a safe, precise, and clinically viable gene therapy strategy for TAO.