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Blood
05 13, 2021;137 (19): 2598-2608.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Suk See De Ravin, Julie Brault, Ronald J Meis, Siyuan Liu, Linhong Li, Mara Pavel-Dinu, Cicera R Lazzarotto, Taylor Liu, Sherry M Koontz, Uimook Choi, Colin L Sweeney, Narda Theobald, GaHyun Lee, Aaron B Clark, Sandra S Burkett, Benjamin P Kleinstiver, Matthew H Porteus, Shengdar Tsai, Douglas B Kuhns, Gary A Dahl, Stephen Headey, Xiaolin Wu, Harry L Malech
Author Information
  1. Suk See De Ravin: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. ORCID
  2. Julie Brault: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. ORCID
  3. Ronald J Meis: CELLSCRIPT, LLC, Madison, WI.
  4. Siyuan Liu: Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD.
  5. Linhong Li: MaxCyte Inc., Gaithersburg, MD.
  6. Mara Pavel-Dinu: Department of Pediatrics, Stanford University, School of Medicine, Stanford, CA.
  7. Cicera R Lazzarotto: Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.
  8. Taylor Liu: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. ORCID
  9. Sherry M Koontz: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
  10. Uimook Choi: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
  11. Colin L Sweeney: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. ORCID
  12. Narda Theobald: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
  13. GaHyun Lee: Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.
  14. Aaron B Clark: Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD.
  15. Sandra S Burkett: Molecular Cytogenetic Core Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD. ORCID
  16. Benjamin P Kleinstiver: Center for Genomic Medicine and Department of Pathology, Massachusetts General Hospital, Boston, MA. ORCID
  17. Matthew H Porteus: Department of Pediatrics, Stanford University, School of Medicine, Stanford, CA. ORCID
  18. Shengdar Tsai: Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN.
  19. Douglas B Kuhns: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD.
  20. Gary A Dahl: CELLSCRIPT, LLC, Madison, WI.
  21. Stephen Headey: School of Science, RMIT University, Melbourne, VIC, Australia. ORCID
  22. Xiaolin Wu: Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD. ORCID
  23. Harry L Malech: Genetic Immunotherapy Section, Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD. ORCID
PMID: 33623984 DOI: 10.1182/blood.2020008503

Abstract

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

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MeSH Term

Animals
Bacterial Proteins
Caspase 9
Cells, Cultured
DNA Repair
Dependovirus
Exons
Gene Editing
Genetic Therapy
Genetic Vectors
Granulomatous Disease, Chronic
Hematopoietic Stem Cell Transplantation
Hematopoietic Stem Cells
Heterografts
Humans
Male
Mice
Mice, Inbred NOD
Mice, SCID
NADPH Oxidase 2
Phagocytes
RNA, Messenger
Reactive Oxygen Species
Ribonucleoproteins
Sequence Deletion
Streptococcus pyogenes
Tumor Suppressor p53-Binding Protein 1

Chemicals

Bacterial Proteins
RNA, Messenger
Reactive Oxygen Species
Ribonucleoproteins
TP53BP1 protein, human
Tumor Suppressor p53-Binding Protein 1
CYBB protein, human
NADPH Oxidase 2
Caspase 9
Journal Article Research Support, N.I.H., Intramural
Article has an altmetric score of 11

Word Cloud

Created with Highcharts 10.0.0cellsproteinlong-termrepairgeneX-CGDapproachdonorCD34+hematopoietichomology-directedhighlyefficientLentivectortherapyX-linkedchronicgranulomatousdiseaseprovenviablerandomvectorintegrationsubnormalproductionexogenouspromoterstransducedremainconcerningsafetyefficacypreviousgenomeediting-basedusingStreptococcuspyogenesCas9mRNAoligodeoxynucleotidegeneticmutationsshowedcapabilityrestorephysiologicalexpressionlackedsufficientefficiencyquiescentclinicaltranslationreporttransientinhibitionp53-binding153BP1significantlyincreased23-foldachieve80%gp91phox+comparedhealthycontrolsubjectscorrectionEnhancededitingstem/progenitor

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