Optimized Lipid Nanoparticle Formulation Enhances Targeted Delivery of CRISPR/Cas9 Ribonucleoproteins to Hematopoietic Stem Cells for Sickle Cell Disease Gene Editing

Gene editing via CRISPR/Cas9 ribonucleoprotein (RNP) complexes offers immense therapeutic potential for sickle cell disease (SCD) through the targeted disruption of the BCL11A erythroid enhancer, leading to the reactivation of fetal hemoglobin (HbF). However, safe and efficient non-viral delivery into primary CD34+ hematopoietic stem and progenitor cells (HSPCs) without compromising cell viability or stemness remains a central challenge in translation. In this study, we engineered an optimized ionizable lipid nanoparticle (LNP) formulation functionalized with anti-CD117 (c-Kit) single-chain variable fragments (scFv) to achieve targeted cytosolic delivery of Cas9 RNPs targeting the BCL11A enhancer. Physical characterization confirmed uniform LNP morphology, high encapsulation efficiency (>88%), and robust pH-responsive endosomal escape properties. In primary human CD34+ HSPCs, targeted LNP delivery yielded an average BCL11A gene editing efficiency of 84.3%, matching traditional electroporation while reducing treatment-associated apoptosis by over 60%. Edited HSPCs retained their primitive CD34+CD38- subpopulation phenotype and multilineage differentiation potential in colony-forming assays. Furthermore, erythroid progeny derived from edited cells exhibited a marked increase in HbF expression (>45% F-cells). Xenotransplantation into immunodeficient mice confirmed long-term repopulation capacity with sustained target gene editing in bone marrow engrafted cells at 16 weeks post-transplantation. This targeted LNP platform provides a scalable, low-toxicity non-viral strategy for ex vivo and potential in vivo HSC gene therapy in sickle cell disease.