In vivo engineering of hematopoietic stem cells by targeted lipid nanoparticles within humanized mice

In vivo genetic engineering of hematopoietic stem and progenitor cells (HSPCs) holds the potential to revolutionize the treatment landscape for numerous diseases with unmet clinical needs. An optimal delivery system for genetic medicines must target quiescent human HSCs to ensure long-term therapeutic efficacy. Here, we screened 15 lipid nanoparticles (LNPs) with different PEG-lipid structures and reported an LNP named LNPDP that demonstrated efficient delivery of reporter mRNA to human HSPCs both in ex vivo and in vivo settings, when conjugated with the anti-CD34 antibody (CD34/LNPDP). Using CRISPR/Cas editing cargos, CD34/LNPDP achieved high editing efficiency in human HSPCs ex vivo. Subtle transcriptomic alterations were detected following LNP treatment, primarily attributed to the cellular uptake of exogenous cholesterol. Intrafemoral administration of CD34/LNPDP in humanized mice resulted in efficient editing of the erythroid-specific BCL11A enhancer within human HSPCs, which enables the sustained long-term reactivation of fetal hemoglobin (HbF) expression in erythroid cells. Furthermore, utilizing an additional humanized neutropenia model harboring an ELANE mutation, we demonstrated that intrafemoral administration of CD34/LNPDP can achieve robust editing targeting exon 2 of ELANE in human HSPCs, thereby attenuating the expression of mutant ELANE and partially restoring neutrophil development impairment under long-term observation. Notably, the genetically modified HSPCs exhibited unbiased repopulation of the entire hematopoietic hierarchy following LNP administration. Collectively, targeting CD34 on human HSPCs provides a novel platform to modify HSPCs for in vivo gene therapy without perturbing normal hematopoietic output, making it a promising candidate for clinical translation.