Format

Send to

Choose Destination
Blood Adv. 2019 Nov 12;3(21):3379-3392. doi: 10.1182/bloodadvances.2019000820.

Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.

Author information

1
Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.
2
Division of Hematology/Oncology, Boston Children's Hospital, Boston, MA.
3
Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA.
4
Department of Pediatrics, Harvard Medical School, Boston, MA.
5
Harvard Stem Cell Institute, Cambridge, MA.
6
Broad Institute of MIT and Harvard, Cambridge, MA.
7
Molecular and Clinical Hematology Branch, National Heart, Lung, and Blood Institute, Bethesda, MD.
8
Department of Molecular, Cell and Cancer Biology, University of Massachusetts Medical School, Worcester, MA; and.
9
Division of Orthopedics.
10
Flow Cytometry and Cell Sorting Shared Resource.
11
Department of Cell and Molecular Biology, and.
12
Department of Bone Marrow Transplantation and Cellular Therapy, St. Jude Children's Research Hospital, Memphis, TN.

Abstract

Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress γ-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and β-thalassemia, although the optimal technical approaches and limiting toxicities are not yet fully defined. We disrupted an HBG1/HBG2 gene promoter motif that is bound by the transcriptional repressor BCL11A. Electroporation of Cas9 single guide RNA ribonucleoprotein complex into normal and SCD donor CD34+ hematopoietic stem and progenitor cells resulted in high frequencies of on-target mutations and the induction of HbF to potentially therapeutic levels in erythroid progeny generated in vitro and in vivo after transplantation of hematopoietic stem and progenitor cells into nonobese diabetic/severe combined immunodeficiency/Il2rγ-/-/KitW41/W41 immunodeficient mice. On-target editing did not impair CD34+ cell regeneration or differentiation into erythroid, T, B, or myeloid cell lineages at 16 to 17 weeks after xenotransplantation. No off-target mutations were detected by targeted sequencing of candidate sites identified by circularization for in vitro reporting of cleavage effects by sequencing (CIRCLE-seq), an in vitro genome-scale method for detecting Cas9 activity. Engineered Cas9 containing 3 nuclear localization sequences edited human hematopoietic stem and progenitor cells more efficiently and consistently than conventional Cas9 with 2 nuclear localization sequences. Our studies provide novel and essential preclinical evidence supporting the safety, feasibility, and efficacy of a mechanism-based approach to induce HbF for treating hemoglobinopathies.

Supplemental Content

Full text links

Icon for Silverchair Information Systems Icon for PubMed Central
Loading ...
Support Center