title: Targeted gene editing restores regulated CD40L function in X-linked hyper-IgM syndrome.
abstract: Loss of CD40 ligand (CD40L) expression or function results in X-linked hyper-immunoglobulin (Ig)M syndrome (X-HIGM), characterized by recurrent infections due to impaired immunoglobulin class-switching and somatic hypermutation. Previous attempts using retroviral gene transfer to correct murine CD40L expression restored immune function; however, treated mice developed lymphoproliferative disease, likely due to viral-promoter-dependent constitutive CD40L expression. These observations highlight the importance of preserving endogenous gene regulation in order to safely correct this disorder. Here, we report efficient, on-target, homology-directed repair (HDR) editing of the CD40LG locus in primary human T cells using a combination of a transcription activator-like effector nuclease-induced double-strand break and a donor template delivered by recombinant adeno-associated virus. HDR-mediated insertion of a coding sequence (green fluorescent protein or CD40L) upstream of the translation start site within exon 1 allowed transgene expression to be regulated by endogenous CD40LG promoter/enhancer elements. Additionally, inclusion of the CD40LG 3'-untranslated region in the transgene preserved posttranscriptional regulation. Expression kinetics of the transgene paralleled that of endogenous CD40L in unedited T cells, both at rest and in response to T-cell stimulation. The use of this method to edit X-HIGM patient T cells restored normal expression of CD40L and CD40-murine IgG Fc fusion protein (CD40-muIg) binding, and rescued IgG class switching of naive B cells in vitro. These results demonstrate the feasibility of engineered nuclease-directed gene repair to restore endogenously regulated CD40L, and the potential for its use in T-cell therapy for X-HIGM syndrome.
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Hubbard
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: David
authors.initials: D
authors.lastname: Hagin
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: Karen
authors.initials: K
authors.lastname: Sommer
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: Yumei
authors.initials: Y
authors.lastname: Song
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: Iram
authors.initials: I
authors.lastname: Khan
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and.
authors.forename: Courtnee
authors.initials: C
authors.lastname: Clough
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and Department of Pediatrics and.
authors.forename: Hans D
authors.initials: HD
authors.lastname: Ochs
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and Department of Pediatrics and Department of Immunology, University of Washington, Seattle, WA.
authors.forename: David J
authors.initials: DJ
authors.lastname: Rawlings
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and Department of Pediatrics and Department of Immunology, University of Washington, Seattle, WA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.affiliation: Center for Immunity and Immunotherapies and Program for Cell and Gene Therapy, Seattle Children's Research Institute, Seattle, WA; and Department of Pediatrics and.
authors.forename: Troy R
authors.initials: TR
authors.lastname: Torgerson
mesh_terms: D020413:3' Untranslated Regions / Q000276:immunology; D000818:Animals; D001402:B-Lymphocytes / Q000276:immunology*; D023201:CD40 Ligand / Q000235:genetics / Q000276:immunology; D004742:Enhancer Elements, Genetic / Q000276:immunology; D005260:Female; D000072669:Gene Editing / Q000379:methods*; D005786:Gene Expression Regulation / Q000276:immunology*; D006801:Humans; D053307:Hyper-IgM Immunodeficiency Syndrome, Type 1 / Q000235:genetics / Q000276:immunology / Q000628:therapy; D017578:Immunoglobulin Class Switching / Q000235:genetics / Q000276:immunology; D008297:Male; D051379:Mice; D016688:Mice, Inbred NOD; D016513:Mice, SCID; D027041:Somatic Hypermutation, Immunoglobulin / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D052416:Targeted Gene Repair / Q000379:methods*
chemical_list: D020413:3' Untranslated Regions; D023201:CD40 Ligand