A mammalian coacervate-based platform for high-efficiency gene editing in hematopoietic stem cells Free

Gene editing in hematopoietic stem cells (HSCs) holds tremendous promise for treating inherited blood disorders such as sickle cell disease and β-thalassemia. However, commonly used delivery systems like viral vectors, electroporation, and lipid nanoparticles often suffer from high toxicity, immunogenicity, and suboptimal efficiency, especially when delivering large or complex cargo. Here, we report the identification of a mammalian endogenous protein capable of forming coacervates with various nucleic acids. These coacervates exhibit an mRNA encapsulation capacity 1000-fold greater than LNPs, demonstrating efficient cellular entry, robust cytoplasmic release of nucleic acids, and broad compatibility with diverse cell types. Building on this, we developed the first mammalian coacervate-based gene delivery system, named EASY, utilizing mammalian endogenous proteins (PCT/CN2024/124967). EASY supports various gene delivery applications, including mRNA transfection, gene knockout via CRISPR, and antisense-mediated knockdown.

In primary human CD34⁺ HSCs, we achieved over 90% transfection efficiency for mRNA encoding green fluorescent protein (GFP), while maintaining 90% cell viability. GFP expression persisted in 64% of cells up to 12 days post-transfection, highlighting its high payload capacity. Notably, EASY facilitated efficient gene knockout by co-delivering Cas9 mRNA and sgRNA. We demonstrated that knocking out BCL11A in HSCs, a transcription factor that represses γ-globin and fetal hemoglobin expression in erythroid cells, significantly increases fetal hemoglobin expression without affecting cell proliferation and differentiation, offering a potential therapeutic strategy for hemoglobinopathies. Our results showed that approximately 80% of alleles at this locus were successfully modified, with no evidence of off-target editing in HSCs using EASY, highlighting its potential for advancing cell therapy.

In summary, EASY is a versatile, non-viral, LNP- and electroporation-free gene delivery platform with high efficiency, low toxicity, and ease of use. By avoiding insertional mutagenesis and specialized equipment, EASY offers a safer, scalable solution for gene editing and HSC therapy, advancing cell therapy manufacturing.