T-cell-targeted fusogenic nanovesicles generate CAR-T cells in vivo for rheumatoid arthritis therapy.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic autoimmune disease in which pathogenic B cells drive persistent inflammation and joint damage. Although CD19-targeted CAR-T cell therapy enables effective B-cell depletion, conventional ex vivo manufacturing and viral vector-based genetic modification limit its scalability and clinical application. Strategies that allow efficient CAR-T cell generation directly in vivo may therefore provide a more practical approach to sustained immune modulation. METHODS: We developed FuNV(CAR), a T cell-targeted fusogenic nanovesicle system that delivers preformed αCD19 CAR proteins to T cells via membrane fusion. FuNV(CAR) was characterized for size, composition, and fusion specificity. CAR-T cell generation, persistence, and cytotoxic function were evaluated in vitro. In vivo CAR-T induction, B-cell depletion, therapeutic efficacy, and tissue-level outcomes were assessed in a collagen-induced arthritis (CIA) mouse model. RESULTS: FuNV(CAR) selectively fused with CD3⁺ T cells and enabled dose-dependent surface display of functional αCD19 CAR proteins without genetic modification. FuNV(CAR)-generated CAR-T cells exhibited antigen-specific cytotoxicity against CD19⁺ B cells in vitro. In vivo administration induced measurable CAR-T cell generation in blood and spleen, resulting in effective B-cell depletion, reduced autoantibody levels, attenuation of joint inflammation, and preservation of cartilage and bone architecture in CIA mice. FuNV(CAR) showed favorable tolerability with no overt organ toxicity. CONCLUSIONS: These findings demonstrate that FuNV(CAR) enables functional in vivo CAR-T cell generation and sustained disease modification in experimental arthritis. FuNV(CAR) represents a mechanistically distinct and scalable platform for CAR-T-based immunotherapy in RA and other B cell-driven autoimmune diseases.