Abstract
The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. This study represents the first evidence of implementing a gene editing strategy in a murine safe harbor locus site that phenotypically corrects primary cells from a mouse model of Fanconi anemia A. By means of the co-delivery of transcription activator-like effector nucleases and a donor therapeutic FANCA template to the Mbs85 locus, we achieved efficient gene targeting (23%) in mFA-A fibroblasts. This resulted in the phenotypic correction of these cells, as revealed by the reduced sensitivity of these cells to mitomycin C. Moreover, robust evidence of targeted integration was observed in murine wild type and FA-A hematopoietic progenitor cells, reaching mean targeted integration values of 21% and 16% respectively, that were associated with the phenotypic correction of these cells. Overall, our results demonstrate the feasibility of implementing a therapeutic targeted integration strategy into the mMbs85 locus, ortholog to the well-validated hAAVS1, constituting the first study of gene editing in mHSC with TALEN, that sets the basis for the use of a new safe harbor locus in mice.
Highlights
The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination
The AAVS1 locus has been defined as a bona-fide safe harbor locus in humans[34], in which an exogenous gene could be efficiently expressed in various cell lines and iPSCs28–34,48–52
We nucleofected a pair of transcription activator-like effector nucleases (TALEN) in combination with a homologous donor template, in the form of plasmid DNA, in both Mouse embryonic fibroblasts (MEFs) and hematopoietic stem and progenitor cells (HSPC) from wt or Fanconi anemia (FA)-A mice
Summary
The promising ability to genetically modify hematopoietic stem and progenitor cells by precise gene editing remains challenging due to their sensitivity to in vitro manipulations and poor efficiencies of homologous recombination. ✉e-mail: www.nature.com/scientificreports vitro, we explored a similar strategy in the context of a mouse model of FA-A by integrating a therapeutic FANCA expression cassette into the murine Mbs[85] gene, the ortholog of the human AAVS136–39 This mouse Mbs[85] locus is located in the reverse strand of chromosome 7. The gene is located >2.5 kilobases (kb) from the 5′ end of any coding region, and the target site has been designed outside any coding region, in an intron site whose ablation is well tolerated[41] These observations, together with the stable expression of fluorescent and therapeutic transgene inserted in this locus, and the phenotype correction demonstrated in human FA cells, would set the basics of the requirements of a safe harbor locus to be used for the gene therapy (GT) of FA. Our study demonstrates the feasibility of conducting a targeted gene therapy approach in embryonic fibroblasts and hematopoietic progenitors from a mouse model of FA-A using TALE nucleases as a gene editing platform, and highlights the potential of using for the first time the Mbs[85] locus as a murine safe harbor for targeted integration, opening a new platform for in vitro and in vivo gene therapy applications in different disease models
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