A versatile coacervate based delivery system to overcome engineering barriers in immune cells

The clinical translation of immune cell engineering (e.g., CAR-T, TCR-T) and functional genomics research in hematologic malignancies are significantly hampered by the limitations of existing gene delivery systems. Viral vectors raise safety and cost concerns, while conventional non-viral methods often suffer from low efficiency and high cytotoxicity, particularly in primary immune cells and refractory hematopoietic cell lines. To overcome these barriers, we developed ProteanFect, the first endogenous protein-based coacervate delivery system. This platform is designed for high-efficiency delivery of diverse genetic payloads, including mRNA, DNA, siRNA, and CRISPR/Cas9 components (mRNA or RNP), enabling robust gene expression and precise gene editing. In primary human immune cells, ProteanFect achieved >90% mRNA transfection efficiency (e.g., GFP mRNA in T cells) with sustained transgene expression and maintained over 90% cell viability. It enabled the generation of functional anti-CD19 CAR-T cells that effectively suppressed tumor growth in vivo. Furthermore, ProteanFect demonstrated high-precision gene editing, achieving 88% knockout efficiency at the TRAC locus in primary T cells via Cas9 mRNA/sgRNA delivery. The platform also showed broad applicability, attaining >70% and >65% transfection efficiencies in natural killer (NK) cells and B cells, respectively, and robust performance in mouse primary immune cells. Simultaneously, ProteanFect effectively addressed transfection challenges in hard-to-transfect hematopoietic cell lines (e.g., Jurkat, K562, THP1, HL-60, Raji, Kasumi-1). It achieved 70-90% RNA and 40-70% DNA transfection efficiency, supported large plasmid (~15.7 kb) delivery with >50% efficiency, and enabled ~70% siRNA-mediated knockdown and ~80% CRISPR/Cas9-mediated knockout in Jurkat cells. Even in more refractory lines, RNA transfection efficiency reached ~80%. The system also facilitated co-transfection and multiplexed gene editing, highlighting its versatility. In summary, ProteanFect represents a transformative non-viral platform that uniquely synergizes high-efficiency nucleic acid delivery with precise gene editing capabilities, all while maintaining minimal cytotoxicity and demonstrating broad applicability across primary human and mouse immune cells as well as challenging hematologic cell lines. Its versatile support for diverse payloads-from mRNA and large plasmids to CRISPR RNP complexes-positions this technology as a pivotal tool that not only accelerates the production of engineered cell therapies but also expands the frontiers of basic research, functional genomics, and therapeutic development in immunology and hematology.