title: Targeted genome editing in human repopulating haematopoietic stem cells.
abstract: 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.
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Pietro
authors.initials: P
authors.lastname: Genovese
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.|Vita Salute San Raffaele University, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Schiroli
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.|Vita Salute San Raffaele University, Milan, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Escobar
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Tiziano Di
authors.initials: TD
authors.lastname: Tomaso
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Firrito
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Calabria
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Davide
authors.initials: D
authors.lastname: Moi
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Roberta
authors.initials: R
authors.lastname: Mazzieri
authors.affiliation: Experimental Hematology Unit, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Chiara
authors.initials: C
authors.lastname: Bonini
authors.affiliation: Sangamo BioSciences Inc., Richmond, CA, United States.
authors.forename: Michael C
authors.initials: MC
authors.lastname: Holmes
authors.affiliation: Sangamo BioSciences Inc., Richmond, CA, United States.
authors.forename: Philip D
authors.initials: PD
authors.lastname: Gregory
authors.affiliation: Dept. of Immunology Erasmus MC, University Medical Center, Rotterdam, The Netherlands.
authors.forename: Mirjam
authors.initials: M
authors.lastname: van der Burg
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.|Vita Salute San Raffaele University, Milan, Italy.
authors.forename: Bernhard
authors.initials: B
authors.lastname: Gentner
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Eugenio
authors.initials: E
authors.lastname: Montini
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.|Vita Salute San Raffaele University, Milan, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Lombardo
authors.affiliation: TIGET, San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, Milan, Italy.|Vita Salute San Raffaele University, Milan, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Naldini
mesh_terms: D000818:Animals; D018952:Antigens, CD34 / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D004720:Endonucleases / Q000378:metabolism; D005312:Fetal Blood / Q000166:cytology / Q000378:metabolism / Q000637:transplantation; D018390:Gene Targeting / Q000379:methods*; D015894:Genome, Human / Q000235:genetics*; D006410:Hematopoiesis / Q000235:genetics; D018380:Hematopoietic Stem Cell Transplantation; D006412:Hematopoietic Stem Cells / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D053631:Interleukin Receptor Common gamma Subunit / Q000235:genetics; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics; D052416:Targeted Gene Repair / Q000379:methods*; D053632:X-Linked Combined Immunodeficiency Diseases / Q000235:genetics* / Q000628:therapy
chemical_list: D018952:Antigens, CD34; D018076:DNA, Complementary; C507941:IL2RG protein, human; D053631:Interleukin Receptor Common gamma Subunit; D004720:Endonucleases