RRC ID 59773
Author Métais JY, Doerfler PA, Mayuranathan T, Bauer DE, Fowler SC, Hsieh MM, Katta V, Keriwala S, Lazzarotto CR, Luk K, Neel MD, Perry SS, Peters ST, Porter SN, Ryu BY, Sharma A, Shea D, Tisdale JF, Uchida N, Wolfe SA, Woodard KJ, Wu Y, Yao Y, Zeng J, Pruett-Miller S, Tsai SQ, Weiss MJ.
Title Genome editing of HBG1 and HBG2 to induce fetal hemoglobin.
Journal Blood Adv
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.
Volume 3(21)
Pages 3379-3392
Published 2019-11-12
DOI 10.1182/bloodadvances.2019000820
PII 422725
PMID 31698466
PMC PMC6855127
MeSH Anemia, Sickle Cell / genetics Animals Base Sequence CRISPR-Cas Systems Disease Models, Animal Erythropoiesis / genetics Fetal Hemoglobin / genetics* Gene Editing* Gene Expression Regulation Gene Targeting Hematopoietic Stem Cell Transplantation Hematopoietic Stem Cells / metabolism Hemoglobinopathies / genetics Heterografts Humans Immunophenotyping Mice Models, Biological Mutation Promoter Regions, Genetic RNA, Guide, Kinetoplastida Sequence Deletion gamma-Globins / genetics*
IF 4.91
Times Cited 4
Human and Animal Cells HUDEP-2(RCB4557)