title: Targeted gene addition in human CD34(+) hematopoietic cells for correction of X-linked chronic granulomatous disease.
abstract: Gene therapy with genetically modified human CD34(+) hematopoietic stem and progenitor cells (HSPCs) may be safer using targeted integration (TI) of transgenes into a genomic 'safe harbor' site rather than random viral integration. We demonstrate that temporally optimized delivery of zinc finger nuclease mRNA via electroporation and adeno-associated virus (AAV) 6 delivery of donor constructs in human HSPCs approaches clinically relevant levels of TI into the AAVS1 safe harbor locus. Up to 58% Venus(+) HSPCs with 6-16% human cell marking were observed following engraftment into mice. In HSPCs from patients with X-linked chronic granulomatous disease (X-CGD), caused by mutations in the gp91phox subunit of the NADPH oxidase, TI of a gp91phox transgene into AAVS1 resulted in ∼15% gp91phox expression and increased NADPH oxidase activity in ex vivo-derived neutrophils. In mice transplanted with corrected HSPCs, 4-11% of human cells in the bone marrow expressed gp91phox. This method for TI into AAVS1 may be broadly applicable to correction of other monogenic diseases.
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Suk See
authors.initials: SS
authors.lastname: De Ravin
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Andreas
authors.initials: A
authors.lastname: Reik
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Pei-Qi
authors.initials: PQ
authors.lastname: Liu
authors.affiliation: MaxCyte, Inc., Gaithersburg, Maryland, USA.
authors.forename: Linhong
authors.initials: L
authors.lastname: Li
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.
authors.forename: Xiaolin
authors.initials: X
authors.lastname: Wu
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.
authors.forename: Ling
authors.initials: L
authors.lastname: Su
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.
authors.forename: Castle
authors.initials: C
authors.lastname: Raley
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Narda
authors.initials: N
authors.lastname: Theobald
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Uimook
authors.initials: U
authors.lastname: Choi
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Song
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Andy
authors.initials: A
authors.lastname: Chan
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Jocelynn R
authors.initials: JR
authors.lastname: Pearl
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: David E
authors.initials: DE
authors.lastname: Paschon
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Lee
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Hannah
authors.initials: H
authors.lastname: Newcombe
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Sherry
authors.initials: S
authors.lastname: Koontz
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Colin
authors.initials: C
authors.lastname: Sweeney
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: David A
authors.initials: DA
authors.lastname: Shivak
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Kol A
authors.initials: KA
authors.lastname: Zarember
authors.affiliation: Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.
authors.forename: Madhusudan V
authors.initials: MV
authors.lastname: Peshwa
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: Sangamo BioSciences, Inc., Richmond, California, USA.
authors.forename: Fyodor D
authors.initials: FD
authors.lastname: Urnov
authors.affiliation: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Harry L
authors.initials: HL
authors.lastname: Malech
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000737:chemistry*; D002478:Cells, Cultured; D015316:Genetic Therapy / Q000379:methods*; D006105:Granulomatous Disease, Chronic / Q000628:therapy*; D018380:Hematopoietic Stem Cell Transplantation / Q000379:methods*; D006412:Hematopoietic Stem Cells / Q000166:cytology*; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic
chemical_list: D018952:Antigens, CD34