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dc.contributor.authorMetais, Jean-Yves
dc.contributor.authorLuk, Kevin
dc.contributor.authorWolfe, Scot A.
dc.contributor.authorTsai, Shengdar Q.
dc.contributor.authorWeiss, Mitchell J.
dc.date2022-08-11T08:09:54.000
dc.date.accessioned2022-08-23T16:48:23Z
dc.date.available2022-08-23T16:48:23Z
dc.date.issued2019-11-12
dc.date.submitted2019-12-09
dc.identifier.citation<p>Blood Adv. 2019 Nov 12;3(21):3379-3392. doi: 10.1182/bloodadvances.2019000820. <a href="https://doi.org/10.1182/bloodadvances.2019000820">Link to article on publisher's site</a></p>
dc.identifier.issn2473-9529 (Linking)
dc.identifier.doi10.1182/bloodadvances.2019000820
dc.identifier.pmid31698466
dc.identifier.urihttp://hdl.handle.net/20.500.14038/41264
dc.description<p>Full author list omitted for brevity. For the full list of authors, see article.</p>
dc.description.abstractInduction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-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/Il2rgamma-/-/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.
dc.language.isoen_US
dc.relation<p><a href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&list_uids=31698466&dopt=Abstract">Link to Article in PubMed</a></p>
dc.rightsCopyright The American Society of Hematology. Publisher PDF posted as allowed by the publisher's copyright and author rights information at https://ashpublications.org/bloodadvances/pages/copyright.
dc.subjectGene Therapy
dc.subjectHematopoiesis and Stem Cells
dc.subjectRed Cells
dc.subjectIron
dc.subjectand Erythropoiesis
dc.subjectcd34 antigens
dc.subjectfetal hemoglobin
dc.subjecttransplantation
dc.subjectheterologous
dc.subjectgenome editing
dc.subjectdonors
dc.subjectchild
dc.subjectglobins
dc.subjectdna
dc.subjecttransplantation
dc.subjectgenes
dc.subjectAmino Acids, Peptides, and Proteins
dc.subjectCancer Biology
dc.subjectCell Biology
dc.subjectComputational Biology
dc.subjectCongenital, Hereditary, and Neonatal Diseases and Abnormalities
dc.subjectGenetics and Genomics
dc.subjectHematology
dc.subjectHemic and Lymphatic Diseases
dc.subjectNucleic Acids, Nucleotides, and Nucleosides
dc.titleGenome editing of HBG1 and HBG2 to induce fetal hemoglobin
dc.typeJournal Article
dc.source.journaltitleBlood advances
dc.source.volume3
dc.source.issue21
dc.identifier.legacyfulltexthttps://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=5069&amp;context=oapubs&amp;unstamped=1
dc.identifier.legacycoverpagehttps://escholarship.umassmed.edu/oapubs/4050
dc.identifier.contextkey15956220
refterms.dateFOA2022-08-23T16:48:23Z
html.description.abstract<p>Induction of fetal hemoglobin (HbF) via clustered regularly interspaced short palindromic repeats/Cas9-mediated disruption of DNA regulatory elements that repress gamma-globin gene (HBG1 and HBG2) expression is a promising therapeutic strategy for sickle cell disease (SCD) and beta-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/Il2rgamma-/-/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.</p>
dc.identifier.submissionpathoapubs/4050
dc.contributor.departmentDepartment of Molecular, Cell and Cancer Biology
dc.source.pages3379-3392


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