LNP-mediated BCL11A Editing Corrects Sickling Phenotypes and Preserves HSPC Fitness Compared to Electroporation

Hemoglobinopathies, including sickle cell disease (SCD) and thalassemia syndromes, affect millions of individuals worldwide who have limited access to curative therapies. Autologous hematopoietic stem cell transplant following ex vivo CRISPR editing of the BCL11A erythroid enhancer reactivates fetal hemoglobin (HbF) and achieves an effective cure, but the resource constraints of clinically approved procedures for editing by electroporation (EP) severely limit widespread implementation. We directly compared the functional outcomes of EP delivery of Cas9 ribonucleoprotein with lipid nanoparticle (LNP) delivery of Cas9 mRNA in primary human HSPCs obtained from healthy HbAA donors and from patients with SCD. While higher editing rates are achieved with EP, LNP-treated HSPCs exhibited greater viability and cell yields that persisted throughout a multi-stage in vitro erythroid differentiation protocol. By day 20, the yield of mature red blood cells (CD71 low CD235a high ) was lowest in the EP cohorts. Across treatment groups, we observed HbF induction proportional to indel frequency. LNP editing of SCD patientderived HSPCs as low as 25% modified alleles still caused HbF production and reduced the propensity for sickling of in vitro differentiated RBCs. These findings highlight the critical trade-offs among manufacturing ease, delivery-associated toxicity, and functional performance across two modalities of therapeutic genome editing for hemoglobinopathies.

Although RNP electroporation achieves higher CRISPR editing rates, it reduces HSPC recovery and RBC differentiation; conversely, mRNA-LNP delivery achieves modest rates while preserving HSPC fitness, progenitor expansion, and RBC enucleation efficiencies.Despite lower editing rates, mRNA-LNP delivery achieves a sufficient therapeutic threshold to reduce hypoxia-induced sickling without altering bulk oxygen affinity.

Abstract from PubMed Central (PMID 42922501, PMC13684507). This entry was added automatically by our daily literature monitor because it matches the topics we follow; read the full paper at the original source.