CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells.
Julie Brault, Taylor Liu, Siyuan Liu, Amanda Lawson, Uimook Choi, Nikita Kozhushko, Vera Bzhilyanskaya, Mara Pavel-Dinu, Ronald J Meis, Michael A Eckhaus, Sandra S Burkett, Marita Bosticardo, Benjamin P Kleinstiver, Luigi D Notarangelo, Cicera R Lazzarotto, Shengdar Q Tsai, Xiaolin Wu, Gary A Dahl, Matthew H Porteus, Harry L Malech, Suk See De Ravin
Author Information
Julie Brault: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Taylor Liu: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Siyuan Liu: Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD, United States.
Amanda Lawson: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Uimook Choi: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Nikita Kozhushko: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Vera Bzhilyanskaya: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Mara Pavel-Dinu: Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University, Palo Alto, CA, United States.
Ronald J Meis: Cellscript LLC Inc., Madison, WI, United States.
Michael A Eckhaus: Division of Veterinary Resources, Office of Research Services, National Institutes of Health, Bethesda, MD, United States.
Sandra S Burkett: Molecular Cytogenetic Core Facility, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, United States.
Marita Bosticardo: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Benjamin P Kleinstiver: Center for Genomic Medicine and Department of Pathology, Massachusetts General Hospital, Boston, MA, United States.
Luigi D Notarangelo: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Cicera R Lazzarotto: Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, United States.
Shengdar Q Tsai: Department of Hematology, St. Jude Children's Research Hospital, Memphis, TN, United States.
Xiaolin Wu: Cancer Research Technology Program, Leidos Biomedical Research Inc., Frederick, MD, United States.
Gary A Dahl: Cellscript LLC Inc., Madison, WI, United States.
Matthew H Porteus: Department of Pediatrics, Division of Stem Cell Transplantation and Regenerative Medicine, Stanford University, Palo Alto, CA, United States.
Harry L Malech: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Suk See De Ravin: Laboratory of Clinical Immunology and Microbiology, National Institute of Allergy and Infectious Diseases (NIAID), National Institutes of Health (NIH), Bethesda, MD, United States.
Introduction: gene therapy for treatment of Inborn errors of Immunity (IEIs) have demonstrated significant clinical benefit in multiple Phase I/II clinical trials. Current approaches rely on engineered retroviral vectors to randomly integrate copy(s) of gene-of-interest in autologous hematopoietic stem/progenitor cells (HSPCs) genome permanently to provide gene function in transduced HSPCs and their progenies. To circumvent concerns related to potential genotoxicities due to the random vector integrations in HSPCs, targeted correction with CRISPR-Cas9-based genome editing offers improved precision for functional correction of multiple IEIs. Methods: We compare the two approaches for integration of transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery current clinical lentivector (LV)- versus targeted insertion (TI) of homology-directed repair (HDR) when using an adeno-associated virus (AAV)- donor following double-strand DNA break at the endogenous locus. Results and discussion: differentiation of LV- or TI-treated XSCID HSPCs similarly overcome differentiation block into Pre-T-I and Pre-T-II lymphocytes but we observed significantly superior development of NK cells when corrected by TI (40.7% versus 4.1%, p = 0.0099). Transplants into immunodeficient mice demonstrated robust engraftment (8.1% and 23.3% in bone marrow) for LV- and TI- HSPCs with efficient T cell development following TI- in all four patients' HSPCs. Extensive specificity analysis of CRISPR-Cas9 editing with rhAmpSeq covering 82 predicted off-target sites found no evidence of indels in edited cells before () or following transplant, in stark contrast to LV's non-targeted vector integration sites. Together, the improved efficiency and safety of correction CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.
