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Targeted genome editing by artificial nucleases has brought the goal of gene correction within the reach of gene therapy. A candidate disease for HSPC gene correction is SCID-X1, because gene therapy with early generation integrating vectors showed robust clinical efficacy even from few corrected cells but also the occurrence of adverse events due to insertional mutagenesis and unregulated transgene expression. We recently reported a strategy that enabled targeted integration of a corrective cDNA into the IL2RG gene in 6% of human HSPC with high specificity. Gene corrected HSPC generated polyclonal lymphoid cells that express the IL2RG protein and have a selective growth advantage over those carrying disruptive IL2RG mutations (Genovese, Nature, 2014). Here, to model SCID-X1 disease correction, we developed a mouse model carrying the IL2RG human gene including a common disease-causing mutation in place of the murine Il2rg gene, allowing to use the same reagents utilized for gene correction of human cells. These mice have impairment in lymphoid development which phenocopies that reported for Il2rg-/- mice. To assess the minimal level of corrected HSPC required to achieve immune reconstitution we first performed competitive transplants with wild-type (WT) and Il2rg-/- HSPC and found that 1% of WT cells are sufficient to reconstitute at least in part the T and B cell compartments. We then developed a protocol to obtain gene correction in murine Lin- HSPC based on the delivery of donor DNA template by IDLVs followed by transfection of ZFN mRNAs. This protocol was associated with high on-target nuclease activity (40%) and a mean of 6% transgene integration by HDR, but also with high levels of acute cytotoxicity (65% cell loss). The surviving cells remained capable of expansion in culture and preserved their clonogenic potential. Importantly, upon transplant into lethally irradiated mice, only the gene corrected cells were able to generate lymphoid lineages (B and T cells), showing a clear selective advantage over un-corrected cells. These data indicate functional correction of the IL2RG gene by our strategy. Yet, measuring percentage of correction within myeloid cells at long-term we found that it was almost undetectable. Despite the lack of HSC marking, gene corrected lymphoid cells stably persisted in the mice up to 7 months post transplantation within all the hematopoietic organs. Furthermore, upon challenging the transplanted mice with a murine pathogen (LCMV Arm.) we observed viral-specific γIFN production by CD8+ gene corrected cells at a similar extent as for WT mice, proving in vivo the functionality of corrected T cells. These results suggest that our protocol achieves biologically relevant levels of gene correction in progenitors that sustain long-term lymphopoiesis but is limited in multipotent HSC. Ongoing studies aim to improve murine HSC gene targeting and to compare safety and efficacy of gene correction vs gene replacement in our disease model.

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.