A mammalian protein-based coacervate platform enables scalable engineering for CAR-T therapy

Abstract The advancement of CAR-T cell therapies has reshaped treatment strategies for hematologic malignancies, yet the production of genetically engineered primary T cells remains dependent on complex, costly, and often cytotoxic delivery technologies. In response to these limitations, we have developed a novel gene transfer platform using mammalian protein-based coacervates, which form spontaneously via liquid–liquid phase separation with nucleic acids. These coacervate structures demonstrate extraordinary nucleic acid encapsulation capacity, rapid cellular entry, efficient cytoplasmic release, and broad compatibility across immune cell types including T cells, NK cells, and hematopoietic stem cells. Leveraging these properties, we developed the first coacervate-based system for gene delivery (EASY) system engineered from mammalian proteins (PCT/CN2024/124967). EASY supports a wide range of genetic cargoes for gene overexpression, knockout, and targeted integration. In primary human T lymphocytes, EASY mediated 90% GFP mRNA transfection efficiency with 90% viability. Expression remained detectable in more than 60% of cells for nearly two weeks, illustrating both the stability and potency of the system. We used EASY to generate mRNA-encoded anti-CD19 CAR-T cells that eliminated CD19⁺ Raji tumors in NSG mice following a single 5×10⁶-cell dose. In functional assays, CAR-T cells produced using EASY successfully cleared CD19⁺ Raji tumors in vivo after a single adoptive transfer, matching the performance of virally modified counterparts. Beyond transient expression, EASY also supports efficient gene editing applications. By co-delivering Cas9 mRNA or protein with sgRNAs, we demonstrated multiplexed knockout of TRAC, B2M, and PDCD1 loci with 80% editing efficiency in primary T cells. For targeted integration, we introduced a CAR transgene into the PD-1 locus using Cas9, sgRNA, and a single-stranded DNA template, yielding PD-1-deficient CAR-T cells with durable CAR expression.To support clinical translation, the EASY system has been successfully scaled to modify over 10⁹ primary human T cells per run while maintaining greater than 90% delivery efficiency. This scalability, combined with its non-viral, LNP-free, and electroporation-free nature, underscores its potential as a next-generation platform for CAR-T development. Ongoing studies are focused on expanding its capabilities for precise genome integration and multiplexed engineering in therapeutic applications.