558. Targeted Gene Therapy in CD34+ Cells from Healthy Donors and Fanconi Anemia Patients

Gene targeting is rapidly progressing thanks to the development of improved nucleases and donor constructs, and represents a new gene therapy-based strategy for the treatment of inherited diseases, such as Fanconi anemia (FA). Because FA can be generated by multiple mutations in up to 19 different genes, we focused our interests in the insertion of therapeutic FA genes in safe harbor loci, since this approach could serve as a platform for treating all FA subtypes and pathogenic mutations. In previous studies we demonstrated the feasibility of correcting the phenotype of hematopoietic progenitors through the genome editing and reprogramming of fibroblasts from FA patients. Because of the limited repopulating properties so far reported for iPSC-derived hematopoietic precursor cells, here we aimed to investigate the possibility of conducting a similar gene targeting approach using human primary CD34+ cells. In a first set of experiments we investigated the efficiency of targeting the AAVS1 locus in cord blood HSPCs. Pre-stimulated CD34+ cells were transduced with integrase-defective lentiviral vectors (IDLV) encoding an EGFP reporter gene under the control of the PGK promoter, flanked by sequences homologous to the AAVS1 target site. The cells were then nucleofected with AAVS1 -specific ZFN mRNAs. The average targeting efficiencies ranged from 10-20% both in in vitro cultured cells and NSG mice-repopulating cells. In subsequent experiments, the EGFP reporter gene was replaced by the hFANCA therapeutic gene. Gene targeting experiments conducted in lymphoblast cell lines and primary CD34+ cells from FA-A patients reverted the characteristic hypersensitivity of FA cells to mitomycin C (MMC), and also restored the formation of FANCD2 foci after DNA damage, evidencing the phenotypic correction of these cells. Our data confirm the efficacy of AAVS1-gene targeting in hHSPCs and demonstrate the feasibility of conducting these approaches in primary CD34+ cells from FA patients.