In utero and neonatal lipid nanoparticle delivery achieves robust editing in hematopoietic stem cells.

Abstract

Efficient delivery of genome editing reagents to hematopoietic stem cells (HSCs) has limited the development of in vivo gene editing therapies for hematologic disease. Here, we exploit developmental hematopoiesis to enable HSC targeting using clinically scalable lipid nanoparticles (LNPs). During fetal development, HSCs reside in the liver, a tissue that is efficiently accessed by LNPs. We show that in utero delivery of LNPs carrying Cre recombinase or CRISPR-Cas9 components results in transfection and genome editing of bona fide long-term repopulating HSCs. Edited HSCs maintain multilineage reconstitution capacity following transplantation, demonstrating preserved stem cell function. Comparative studies reveal that both fetal and early neonatal delivery permit HSC editing, with greater efficiency during fetal liver hematopoiesis. We further identify an LNP formulation that enhance HSC targeting and enable robust neonatal HSC editing without antibody-mediated targeting. Finally, combined delivery of Cas9 via LNPs and a repair template via adeno-associated virus in neonatal mice enables in vivo homology-directed repair in multiple tissues. Together, these findings establish the perinatal period as a therapeutic window for in vivo HSC genome editing and provide a scalable strategy for treating severe early-onset hematologic diseases.