| Summary: | Gene addition strategies to correct inherited diseases are showing promise for treatment in the clinic, but an improved approach to treat both dominant and recessive genetic disorders would be to repair the mutant gene by homologous recombination-mediated gene targeting. Recently, gene targeting frequencies have dramatically increased through the development of (i) improved designer nuc1eases, including zinc finger nucleases (ZFN), able to introduce specific double-strand breaks at their target locus; and (ii) efficient DNA delivery tools, including integration-deficient lentiviral vectors (IDL Vs). Even when using these state-of-the-art systems, gene repair frequencies in stem cells are moderate. To obtain proof-of-principle of phenotypic rescue in the haematopoietic system, a model in which corrected cells had selective advantage would be optimum. An ex vivo strategy to correct the classical scid mouse, a model of human DNA-dependent protein kinase catalytic subunit (DNA-PKcs, PRKDC) deficiency, is hereby presented. Donor templates to correct the scid point mutation and a ZFN were produced, optimised and incorporated into IDLVs. Specific ZFN activity in mouse scid fibroblasts and haematopoietic progenitors was demonstrated by Cel-I assay (which detects modifications introduced at the target site upon repair by non-homologous end- joining) and by deep sequencing. ZFN- and template-mediated gene repair of the scid mutation was demonstrated via the incorporation of a selection cassette and/or a diagnostic restriction site from the donor template into the targeted locus. In scid fibroblasts, gene repair led to DNA-PKcs activity rescue and increased resistance to DNA damage. In scid haematopoietic progenitors, gene correction was demonstrated only when the ZFN genes were delivered by integrating lentiviral vectors. Following an optimised ex vivo protocol, transplantation of potentially corrected scid haematopoietic progenitors into irradiated scid recipients has been carried out. Preliminary results of the ex vivo gene repair and transplantation experiment have indicated potential rescue of the T -cell compartment in a fraction of the scid transplant recipients. The results presented in this work highlight the potential of gene repair for future therapy in the haematopoietic system.
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