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Mol Ther. 2019 Jan 2;27(1):137-150. doi: 10.1016/j.ymthe.2018.10.008. Epub 2018 Oct 17.

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

Author information

1
Genethon, INSERM UMR951, Evry 91000, France.
2
Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, INSERM UMR1163, Paris 75015, France; Paris Descartes, Sorbonne Paris Cité University, Imagine Institute, Paris 75015, France.
3
Paris-Descartes Bioinformatics Platform, Imagine Institute, Paris 75015, France.
4
Genomic Platform, Imagine Institute, Paris 75015, France.
5
Department of Bioengineering, Rice University, Houston, TX 77006, USA.
6
Department of Pediatrics, Stanford University, Stanford, CA 94305, USA.
7
Paris Descartes, Sorbonne Paris Cité University, Imagine Institute, Paris 75015, France; Department of Life Sciences, University of Modena and Reggio Emilia, 41125 Modena, Italy.
8
Genethon, INSERM UMR951, Evry 91000, France; Laboratory of Chromatin and Gene Regulation During Development, Imagine Institute, INSERM UMR1163, Paris 75015, France; Paris Descartes, Sorbonne Paris Cité University, Imagine Institute, Paris 75015, France. Electronic address: annarita.miccio@institutimagine.org.

Abstract

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.

KEYWORDS:

CRISPR/Cas9 delivery; genome editing; β-hemoglobinopathies

PMID:
30424953
PMCID:
PMC6318785
[Available on 2020-01-02]
DOI:
10.1016/j.ymthe.2018.10.008

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