Targeted gene addition in human CD34+ hematopoietic cells for correction of X-linked chronic granulomatous disease.

De Ravin SS¹, Reik A², Liu PQ², Li L³, Wu X⁴, Su L⁴, Raley C⁴, Theobald N¹, Choi U¹, Song AH², Chan A², Pearl JR², Paschon DE², Lee J¹, Newcombe H¹, Koontz S¹, Sweeney C¹, Shivak DA², Zarember KA¹, Peshwa MV⁴, Gregory PD², Urnov FD², Malech HL¹.

Author information

• ¹Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
• ²Sangamo BioSciences, Inc., Richmond, California, USA.
• ³MaxCyte, Inc., Gaithersburg, Maryland, USA.
• ⁴Cancer Research Technology Program, Leidos Biomedical Research, Inc., Frederick, Maryland, USA.

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.