Site-Specific Gene Editing of Human Hematopoietic Stem Cells for X-Linked Hyper-IgM Syndrome
Caroline Y. Kuo,
Joseph D. Long,
Beatriz Campo-Fernandez,
Satiro de Oliveira,
Aaron R. Cooper,
Zulema Romero,
Megan D. Hoban,
Alok V. Joglekar,
Georgia R. Lill,
Michael L. Kaufman,
Sorel Fitz-Gibbon,
Xiaoyan Wang,
Roger P. Hollis,
Donald B. Kohn
Affiliations
Caroline Y. Kuo
Division of Allergy & Immunology, Department of Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095, USA
Joseph D. Long
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Beatriz Campo-Fernandez
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Satiro de Oliveira
Division of Hematology & Oncology, Department of Pediatrics, David Geffen School of Medicine at the University of California, Los Angeles, Los Angeles, CA 90095, USA
Aaron R. Cooper
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Zulema Romero
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Megan D. Hoban
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Alok V. Joglekar
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Georgia R. Lill
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Michael L. Kaufman
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Sorel Fitz-Gibbon
Department of Molecular, Cell, and Developmental Biology, University of California, Los Angeles, Los Angeles, CA 90095, USA
Xiaoyan Wang
Department of General Internal Medicine and Health Services Research, University of California, Los Angeles, Los Angeles, CA 90095, USA
Roger P. Hollis
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
Donald B. Kohn
Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA
X-linked hyper-immunoglobulin M (hyper-IgM) syndrome (XHIM) is a primary immunodeficiency due to mutations in CD40 ligand that affect immunoglobulin class-switch recombination and somatic hypermutation. The disease is amenable to gene therapy using retroviral vectors, but dysregulated gene expression results in abnormal lymphoproliferation in mouse models, highlighting the need for alternative strategies. Here, we demonstrate the ability of both the transcription activator-like effector nuclease (TALEN) and clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) platforms to efficiently drive integration of a normal copy of the CD40L cDNA delivered by Adeno-Associated Virus. Site-specific insertion of the donor sequence downstream of the endogenous CD40L promoter maintained physiologic expression of CD40L while overriding all reported downstream mutations. High levels of gene modification were achieved in primary human hematopoietic stem cells (HSCs), as well as in cell lines and XHIM-patient-derived T cells. Notably, gene-corrected HSCs engrafted in immunodeficient mice at clinically relevant frequencies. These studies provide the foundation for a permanent curative therapy in XHIM.
