Highly efficient therapeutic gene editing of human hematopoietic stem cells

Nat Med. 2019 May;25(5):776-783. doi: 10.1038/s41591-019-0401-y. Epub 2019 Mar 25.

Abstract

Re-expression of the paralogous γ-globin genes (HBG1/2) could be a universal strategy to ameliorate the severe β-globin disorders sickle cell disease (SCD) and β-thalassemia by induction of fetal hemoglobin (HbF, α2γ2)1. Previously, we and others have shown that core sequences at the BCL11A erythroid enhancer are required for repression of HbF in adult-stage erythroid cells but are dispensable in non-erythroid cells2-6. CRISPR-Cas9-mediated gene modification has demonstrated variable efficiency, specificity, and persistence in hematopoietic stem cells (HSCs). Here, we demonstrate that Cas9:sgRNA ribonucleoprotein (RNP)-mediated cleavage within a GATA1 binding site at the +58 BCL11A erythroid enhancer results in highly penetrant disruption of this motif, reduction of BCL11A expression, and induction of fetal γ-globin. We optimize conditions for selection-free on-target editing in patient-derived HSCs as a nearly complete reaction lacking detectable genotoxicity or deleterious impact on stem cell function. HSCs preferentially undergo non-homologous compared with microhomology-mediated end joining repair. Erythroid progeny of edited engrafting SCD HSCs express therapeutic levels of HbF and resist sickling, while those from patients with β-thalassemia show restored globin chain balance. Non-homologous end joining repair-based BCL11A enhancer editing approaching complete allelic disruption in HSCs is a practicable therapeutic strategy to produce durable HbF induction.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anemia, Sickle Cell / blood
  • Anemia, Sickle Cell / genetics
  • Anemia, Sickle Cell / therapy
  • Base Sequence
  • CRISPR-Cas Systems
  • Carrier Proteins / genetics
  • Enhancer Elements, Genetic
  • Erythroid Precursor Cells / metabolism
  • Fetal Hemoglobin / biosynthesis
  • Fetal Hemoglobin / genetics
  • Gene Editing / methods*
  • Hematopoietic Stem Cell Transplantation
  • Hematopoietic Stem Cells / metabolism*
  • Humans
  • INDEL Mutation
  • Nuclear Proteins / genetics
  • RNA, Guide / genetics
  • Repressor Proteins
  • beta-Thalassemia / blood
  • beta-Thalassemia / genetics
  • beta-Thalassemia / therapy
  • gamma-Globins / biosynthesis
  • gamma-Globins / genetics

Substances

  • BCL11A protein, human
  • Carrier Proteins
  • Nuclear Proteins
  • RNA, Guide
  • Repressor Proteins
  • gamma-Globins
  • Fetal Hemoglobin