Targeted gene therapy into a safe harbor site in human hematopoietic progenitor cells

Directed gene therapy mediated by nucleases has become a new alternative to lead targeted integration of therapeutic genes in specific regions in the genome. In this work, we have compared the efficiency of two nuclease types, TALEN and meganucleases (MN), to introduce an EGFP reporter gene in a specific site in a safe harbor locus on chromosome 21 in an intergenic region, named here SH6. The efficiency of targeted integration mediated by SH6v5-MN and SH6-TALEN in HEK-293H cells was up to 16.3 and 15.0%. A stable expression was observed both in the pool of transfected cells and in established pseudoclones, with no detection of off-target integrations by Southern blot. In human hematopoietic stem and progenitor CD34⁺ cells, the nucleofection process preserved the viability and clonogenic capacity of nucleofected cells, reaching up to 3.1% of specific integration of the transgene in colony forming cells when the SH6-TALEN was used, although no expression of the transgene could be found in these cells. Our results show the possibility to specifically integrate genes at the SH6 locus in CD34⁺ progenitor cells, although further improvements in the efficacy of the procedure are required before this approach could be used for the gene editing of hematopoietic stem cells in patients with hematopoietic diseases.