CRISPR-Cas9-AAV versus lentivector transduction for genome modification of X-linked severe combined immunodeficiency hematopoietic stem cells
Year: 2023
Type: article
Abstract: Introduction Ex vivo 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 IL2RG transgene for functional correction of HSPCs from patients with X-linked Severe Combined Immunodeficiency (SCID-X1 or XSCID); delivery via current clinical lentivector (LV)- IL2RG versus targeted insertion (TI) of IL2RG via homology-directed repair (HDR) when using an adeno-associated virus (AAV)- IL2RG donor following double-strand DNA break at the endogenous IL2RG locus. Results and discussion In vitro 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- IL2RG HSPCs with efficient T cell development following TI- IL2RG 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 ( in vitro ) or following transplant, in stark contrast to LV’s non-targeted vector integration sites. Together, the improved efficiency and safety of IL2RG correction via CRISPR-Cas9-based TI approach provides a strong rationale for a clinical trial for treatment of XSCID patients.
Source: Frontiers in Immunology
Authors: 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
Institutions: National Institutes of Health, National Institute of Allergy and Infectious Diseases, Leidos Biomedical Research Inc. (United States), Stanford Medicine, Stanford University, Office of Research Services, National Cancer Institute, Center for Cancer Research, Harvard University, Massachusetts General Hospital, St. Jude Children's Research Hospital
Language: English
Subfield: Genetics
Domain: Life Sciences
Open Access status: gold
Estimated APC paid: $3,295
Funders: U.S. Department of Health and Human Services, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, St. Jude Children's Research Hospital, American Lebanese Syrian Associated Charities, National Institutes of Health, National Heart, Lung, and Blood Institute, National Cancer Institute, National Institute of Allergy and Infectious Diseases, NHLBI Division of Intramural Research
Awards: Gene Therapy Basic Research to Treat Inherited Immune Deficiencies, G4091324117, G4934656353, Basic Research on Hematopoietic Adult Human Stem Cells, Optimizing Cancer Immunotherapy Safety and Efficacy using Genome Editing, A novel human T-cell platform to define biological effects of genome editing, G7764947648, G9000050519, G9000310734, Hematopoiesis in Cardiovascular Disease
OpenAlex ID: W4313584909