Biomembrane-inspired lipid nanoparticles enhance CRISPR-Cas9 editing for hemophilia A

Lipid nanoparticles (LNPs) have substantially advanced RNA-based therapies; however, their use for CRISPR-Cas9 remains limited by sub-optimal endosomal escape, innate immune activation, transient nuclease expression, and restricted tissue specificity. Here, we engineered biomembrane-inspired LNPs containing sphingomyelin and C18-galactosyl ceramide (C18-GalCer) to improve liver-targeted CRISPR delivery. The optimized formulation increased in vivo editing efficiency 2.3-fold relative to a benchmark ALC-0315 LNP. Compared with adeno-associated virus (AAV) vectors, transient mRNA delivery reduced the theoretical risk of insertional mutagenesis, lowered innate immune readouts, and allowed dose titration. By pairing AAV-mediated delivery of a therapeutic Factor VIII donor with LNP-mediated CRISPR-Cas9, we achieved efficient and durable gene correction in a hemophilia A mouse model. A single LNP dose restored plasma Factor VIII activity to >50 % of wild-type levels and maintained this correction for >12 weeks, with low systemic cytokine induction, undetectable off-target insertions, and no overt toxicity. The LNPs retained physicochemical properties and editing potency after prolonged storage and multiple freeze-thaw cycles, supporting their translational potential. Collectively, our findings position biomembrane-inspired LNPs as a safe and efficient non-viral CRISPR platform with potential applicability to other gene-editing therapies.