Chimeric antigen receptor (CAR) T cells have achieved remarkable success in treating certain hematological malignancies. However, the effective engineering of primary cells remains a significant challenge. Coacervates are condensed droplets containing proteins or nucleic acids formed via liquid-liquid phase separation (LLPS). Here, we have identified a mammalian endogenous protein capable of forming coacervates with various nucleic acids (PCT/CN2024/099339). Our analysis revealed that these coacervates can encapsulate RNA molecules at a 1000-fold higher capacity compared to lipid nanoparticles (LNPs). Remarkably, these coacervates can enter various cell types, including human primary cells, and efficiently release nucleic acids within the cytoplasm. Based on this discovery, we have developed a non-viral, electroporation- and antibody-conjugated LNP-free, coacervate-based gene delivery system named PhaseX. We demonstrated that PhaseX can deliver mRNA encoding GFP protein into human primary T lymphocytes with over 90% efficiency and 95% viability. At day 12 post-treatment, 64% of cells still exhibited sustained GFP expression with high fluorescence intensity, indicating prolonged expression. Furthermore, we generated mRNA-based anti-CD19 CAR T cells using PhaseX. These CAR-T cells effectively eliminated tumor growth in NSG mice xenografted with CD19+ Raji cells after a single dose (5 x 106 CAR-T cells). To enhance CAR-T function, we simultaneously introduced Cas9 mRNA with sgRNAs into these CAR-T cells. This resulted in the successful knockout of multiple genes including PDCD1, and showed enhanced anti-tumor activity in vivo. For potential clinical applications, we have successfully scaled up the system to deliver mRNA into over 109 primary human T cells with over 90% efficiency. Future studies will focus on integrating genetic elements into the genome using this system. In summary, we have developed the first coacervate-based delivery system for primary cell engineering, including human primary T lymphocytes. This methodology will greatly benefit CAR-T applications, and the reagent is currently available for free testing upon request.
No relevant conflicts of interest to declare.
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