Optimization of CRISPR/Cas9 Delivery to Human Hematopoietic Stem and Progenitor Cells for Therapeutic Genomic Rearrangements.

ABSTRACT: Editing the ?-globin locus in hematopoietic stem cells is an alternative therapeutic approach for gene therapy of ?-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 ?-globin locus associated with hereditary persistence of fetal hemoglobin (HPFH), a condition that mitigates the clinical phenotype of patients with ?-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 ?-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.

SUBMITTER: Lattanzi A 

PROVIDER: S-EPMC6318785 | biostudies-literature | 2019 Jan

REPOSITORIES: biostudies-literature