Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.
Editing the β-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of β-thalassemia and sickle cell disease. Using the CRISPR/Cas9 system, we genetically modified human hematopoietic stem and progenitor cells (HSPCs) to mimic the large rearrangements in the β-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with β-hemoglobinopathies. We optimized and compared the efficiency of plasmid-, lentiviral vector (LV)-, RNA-, and ribonucleoprotein complex (RNP)-based methods to deliver the CRISPR/Cas9 system into HSPCs. Plasmid delivery of Cas9 and gRNA pairs targeting two HPFH-like regions led to high frequency of genomic rearrangements and HbF reactivation in erythroblasts derived from sorted, Cas9+ HSPCs but was associated with significant cell toxicity. RNA-mediated delivery of CRISPR/Cas9 was similarly toxic but much less efficient in editing the β-globin locus. Transduction of HSPCs by LVs expressing Cas9 and gRNA pairs was robust and minimally toxic but resulted in poor genome-editing efficiency. Ribonucleoprotein (RNP)-based delivery of CRISPR/Cas9 exhibited a good balance between cytotoxicity and efficiency of genomic rearrangements as compared to the other delivery systems and resulted in HbF upregulation in erythroblasts derived from unselected edited HSPCs.
['Lattanzi|Annalisa|A|', 'Meneghini|Vasco|V|', 'Pavani|Giulia|G|', 'Amor|Fatima|F|', 'Ramadier|Sophie|S|', 'Felix|Tristan|T|', 'Antoniani|Chiara|C|', 'Masson|Cecile|C|', 'Alibeu|Olivier|O|', 'Lee|Ciaran|C|', 'Porteus|Matthew H|MH|', 'Bao|Gang|G|', 'Amendola|Mario|M|', 'Mavilio|Fulvio|F|', 'Miccio|Annarita|A|']
D000755:Anemia, Sickle Cell D000076987:CRISPR-Associated Protein 9 D064113:CRISPR-Cas Systems D000072669:Gene Editing D015316:Genetic Therapy D006412:Hematopoietic Stem Cells D006453:Hemoglobinopathies D034441:Heterogeneous-Nuclear Ribonucleoproteins D006801:Humans D010957:Plasmids D017394:RNA, Guide D017086:beta-Thalassemia
CRISPR/Cas9 delivery β-hemoglobinopathies genome editing