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

Targeted genetic correction of hematopoietic stem cells is applied to X-linked chronic granulomatous disease. 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.