A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition

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Abstract

Disorders resulting from mutations in the hemoglobin subunit beta gene (HBB; which encodes β-globin), mainly sickle cell disease (SCD) and β-thalassemia, become symptomatic postnatally as fetal γ-globin expression from two paralogous genes, hemoglobin subunit gamma 1 (HBG1) and HBG2, decreases and adult β-globin expression increases, thereby shifting red blood cell (RBC) hemoglobin from the fetal (referred to as HbF or α2γ2) to adult (referred to as HbA or α2β2) form. These disorders are alleviated when postnatal expression of fetal γ-globin is maintained. For example, in hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition, mutations attenuate γ-globin-to-β-globin switching, causing high-level HbF expression throughout life. Co-inheritance of HPFH with β-thalassemia- or SCD-associated gene mutations alleviates their clinical manifestations. Here we performed CRISPR–Cas9-mediated genome editing of human blood progenitors to mutate a 13-nt sequence that is present in the promoters of the HBG1 and HBG2 genes, thereby recapitulating a naturally occurring HPFH-associated mutation. Edited progenitors produced RBCs with increased HbF levels that were sufficient to inhibit the pathological hypoxia-induced RBC morphology found in SCD. Our findings identify a potential DNA target for genome-editing-mediated therapy of β-hemoglobinopathies.

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Figure 1: Genome editing of the HBG1 and HBG2 promoters increases erythroid fetal hemoglobin (HbF) levels.
Figure 2: Spectrum of γ-globin-expression-inducing mutations caused by Cas9 and gRNA-1 expression.

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Acknowledgements

We are grateful to P. Mead, G. Neale, S. Olsen, C. Sherr and Y. Yasui for valuable discussions and technical expertise. DNA sequencing studies were performed at the Hartwell Center, St. Jude Children's Research Hospital (SJCRH). FISH studies were performed by the SJCRH Cytogenetics Resource. Lentivirus was produced by the SJCRH Hematology Department Vector Core Facility. Allophycocyanin (APC)-conjugated anti-Band3 was a gift from X. An (New York Blood Center). The all-in-one expression plasmid spCas9(BB)-2A-GFP (PX458, Addgene plasmid #48138) and lentiCRISPRv2 (Addgene plasmid #52961) were gifts from F. Zhang (Broad Institute). We thank D. Phillips for his support and encouragement. Support was provided by the US National Institutes of Health (grant no. 4R01HL119479 (G.A.B.), 2R56DK0658 (R.C.H.) and P30CA021765-37 (C.L.), and fellowship no. F30DK102291 (E.A.T.)), the American Lebanese Syrian Associated Charities (M.J.W.), the Assisi Foundation of Memphis (M.J.W.) and a Wellcome Strategic Award (J.R.H.). We thank our patients with SCD, who contributed samples for this study.

Author information

E.A.T., Y.Y. and M.J.W. designed experiments, analyzed data and wrote the manuscript; Y.-D.W. performed mutational analyses; K.J.W. performed off-target-site analysis; R.K. and Y.N. provided HUDEP-2 cell lines; J.R.H., R.C.H., G.A.B. and C.L. provided conceptual advice and technical expertise; M.J.W. supervised the study; and all of the authors discussed the results and assisted in the preparation of the manuscript.

Correspondence to Mitchell J Weiss.

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Supplementary Figures 1–7 and Supplementary Table 1 (PDF 2775 kb)

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Traxler, E., Yao, Y., Wang, Y. et al. A genome-editing strategy to treat β-hemoglobinopathies that recapitulates a mutation associated with a benign genetic condition. Nat Med 22, 987–990 (2016) doi:10.1038/nm.4170

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