Targeted genome editing in human repopulating haematopoietic stem cells

• Published in: Nature (London) Vol. 510; no. 7504; pp. 235 - 240
• Main Authors: Genovese, Pietro, Schiroli, Giulia, Escobar, Giulia, Di Tomaso, Tiziano, Firrito, Claudia, Calabria, Andrea, Moi, Davide, Mazzieri, Roberta, Bonini, Chiara, Holmes, Michael C., Gregory, Philip D., van der Burg, Mirjam, Gentner, Bernhard, Montini, Eugenio, Lombardo, Angelo, Naldini, Luigi
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 12.06.2014; Nature Publishing Group
• Subjects: 13/31; 38/23; 38/77; 42/100; 42/109; 42/41; 42/44; 631/61/201/2110; 64/60; Animals; Antigens, CD34 - metabolism; Apoptosis; Bone marrow; Cytokines; Deoxyribonucleic acid; DNA; DNA repair; DNA, Complementary - genetics; Endonucleases - metabolism; Fetal Blood - cytology; Fetal Blood - metabolism; Fetal Blood - transplantation; Gene Targeting - methods; Gene therapy; Genome, Human - genetics; Genomics; Hematopoiesis - genetics; Hematopoietic Stem Cell Transplantation; Hematopoietic stem cells; Hematopoietic Stem Cells - cytology; Hematopoietic Stem Cells - metabolism; Humanities and Social Sciences; Humans; Interleukin Receptor Common gamma Subunit - genetics; Male; Mice; multidisciplinary; Mutation - genetics; Repopulation; Science; Stem cells; Targeted Gene Repair - methods; Transplantation; X-Linked Combined Immunodeficiency Diseases - genetics; X-Linked Combined Immunodeficiency Diseases - therapy
• ISSN: 0028-0836; 1476-4687; 1476-4687
• DOI: 10.1038/nature13420

Targeted genome editing by artificial nucleases has brought the goal of site-specific transgene integration and gene correction within the reach of gene therapy. However, its application to long-term repopulating haematopoietic stem cells (HSCs) has remained elusive. Here we show that poor permissiveness to gene transfer and limited proficiency of the homology-directed DNA repair pathway constrain gene targeting in human HSCs. By tailoring delivery platforms and culture conditions we overcame these barriers and provide stringent evidence of targeted integration in human HSCs by long-term multilineage repopulation of transplanted mice. We demonstrate the therapeutic potential of our strategy by targeting a corrective complementary DNA into the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). Gene-edited HSCs sustained normal haematopoiesis and gave rise to functional lymphoid cells that possess a selective growth advantage over those carrying disruptive IL2RG mutations. These results open up new avenues for treating SCID-X1 and other diseases. The feasibility of targeted genome editing in human haematopoietic stem cells is demonstrated; the study overcomes previously existing barriers by tailoring delivery platforms and culture conditions. Gene editing of human stem cells Gene addition technology has been used to treat some inherited diseases but toxicity remains a major problem. This makes gene repair, targeting the defective gene only, an attractive alternative. Here Luigi Naldini and colleagues report successful targeted genome editing in human haematopoietic stem cells (HSCs). They overcome previously existing barriers by tailoring delivery platforms and culture conditions. The therapeutic potential of the strategy is demonstrated by the insertion of a corrective cDNA into a mutational hotspot of the IL2RG gene of HSCs from healthy donors and a subject with X-linked severe combined immunodeficiency (SCID-X1). The gene-edited HSCs gave rise to functional lymphoid cells with a selective growth advantage over those carrying disruptive IL2RG mutations.