An evolved AAV variant enables efficient genetic engineering of murine T cells

William A Nyberg; Jonathan Ark; Angela To; Sylvanie Clouden; Gabriella Reeder; Joseph J Muldoon; Jing-Yi Chung; William H Xie; Vincent Allain; Zachary Steinhart; Christopher Chang; Alexis Talbot; Sandy Kim; Alan Rosales; L Patrick Havlik; Harold Pimentel; Aravind Asokan; Justin Eyquem

doi:10.1016/j.cell.2022.12.022

An evolved AAV variant enables efficient genetic engineering of murine T cells

Cell. 2023 Jan 19;186(2):446-460.e19. doi: 10.1016/j.cell.2022.12.022. Epub 2023 Jan 12.

Authors

William A Nyberg¹, Jonathan Ark², Angela To¹, Sylvanie Clouden³, Gabriella Reeder⁴, Joseph J Muldoon¹, Jing-Yi Chung¹, William H Xie⁵, Vincent Allain⁶, Zachary Steinhart¹, Christopher Chang⁷, Alexis Talbot⁶, Sandy Kim⁸, Alan Rosales⁹, L Patrick Havlik⁹, Harold Pimentel¹⁰, Aravind Asokan¹¹, Justin Eyquem¹²

Affiliations

¹ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA.
² Department of Molecular Genetics & Microbiology, Duke University School of Medicine, Durham, NC 27710, USA.
³ Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA.
⁴ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94131, USA.
⁵ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94131, USA.
⁶ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Université de Paris Cité, INSERM UMR976, Hôpital St-Louis, Paris, France.
⁷ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94131, USA; Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA 94131, USA.
⁸ Bioinformatics Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA.
⁹ Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.
¹⁰ Bioinformatics Interdepartmental Program, University of California, Los Angeles, Los Angeles, CA 90095, USA; Howard Hughes Medical Institute, Sloan Foundation, Departments of Computational Medicine, Human Genetics, University of California, Los Angeles, Los Angeles, CA 90095, USA.
¹¹ Department of Molecular Genetics & Microbiology, Duke University School of Medicine, Durham, NC 27710, USA; Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA; Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA. Electronic address: [email protected].
¹² Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143, USA; Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA; UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA. Electronic address: [email protected].

Abstract

Precise targeting of large transgenes to T cells using homology-directed repair has been transformative for adoptive cell therapies and T cell biology. Delivery of DNA templates via adeno-associated virus (AAV) has greatly improved knockin efficiencies, but the tropism of current AAV serotypes restricts their use to human T cells employed in immunodeficient mouse models. To enable targeted knockins in murine T cells, we evolved Ark313, a synthetic AAV that exhibits high transduction efficiency in murine T cells. We performed a genome-wide knockout screen and identified QA2 as an essential factor for Ark313 infection. We demonstrate that Ark313 can be used for nucleofection-free DNA delivery, CRISPR-Cas9-mediated knockouts, and targeted integration of large transgenes. Ark313 enables preclinical modeling of Trac-targeted CAR-T and transgenic TCR-T cells in immunocompetent models. Efficient gene targeting in murine T cells holds great potential for improved cell therapies and opens avenues in experimental T cell immunology.

Keywords: AAV; CAR-T cell; CRISPR/Cas9; Gene Editing; Gene targeting; Genome wide CRISPR screen; Immunology; T cell; Trac-CAR.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
CRISPR-Cas Systems / genetics
Dependovirus* / genetics
Gene Targeting
Genetic Engineering* / methods
Mice
T-Lymphocytes*

Abstract

Publication types

MeSH terms

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