In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313

William A Nyberg¹, Charlotte H Wang², Jonathan Ark³, Chang Liu⁴, Sylvanie Clouden⁵, Anita Qualls⁶, Sofia Caryotakis⁷, Elina Wells⁸, Katherine Simon⁵, Celeste Garza⁹, Pierre-Louis Bernard⁴, Maya Lopez-Ichikawa², Zhongmei Li¹⁰, Jin Seo¹⁰, Gabriella R Kimmerly², Joseph J Muldoon⁴, Peixin Amy Chen², Mingcheng Li⁴, Hong-Erh Liang⁹, Kelly Kersten¹¹, Alan Rosales¹², Nicholas Kuhn¹¹, Chun Jimmie Ye¹³, James M Gardner¹⁴, Ari Molofsky⁸, Roberto R Ricardo-Gonzalez¹⁵, Aravind Asokan¹⁶, Justin Eyquem¹⁷

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. Electronic address: william.nyberg@ki.se.
² 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 94143, USA.
³ Department of Molecular Genetics & Microbiology, 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.
⁵ Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA.
⁶ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ Department of Dermatology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA.
¹¹ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.
¹³ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Arc Institute, Palo Alto, CA 94304, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA.
¹⁴ Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁵ Department of Dermatology, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub San Francisco, San Francisco, CA 94158, USA.
¹⁶ 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: aravind.asokan@duke.edu.
¹⁷ 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 94143, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, 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: justin.eyquem@ucsf.edu.

PMID: 39909036
DOI: 10.1016/j.immuni.2025.01.009

Free article

In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313

William A Nyberg et al. Immunity. 2025.

Free article

. 2025 Feb 11;58(2):499-512.e7.

doi: 10.1016/j.immuni.2025.01.009. Epub 2025 Feb 4.

Authors

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. Electronic address: william.nyberg@ki.se.
² 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 94143, USA.
³ Department of Molecular Genetics & Microbiology, 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.
⁵ Department of Surgery, Duke University School of Medicine, Durham, NC 27710, USA.
⁶ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
⁷ Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁸ Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA 94143, USA.
⁹ Department of Dermatology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁰ Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA.
¹¹ Department of Pathology, University of California, San Francisco, San Francisco, CA 94143, USA.
¹² Department of Biomedical Engineering, Duke University, Durham, NC 27710, USA.
¹³ Department of Medicine, University of California, San Francisco, San Francisco, CA 94143, USA; Gladstone-UCSF Institute of Genomic Immunology, San Francisco, CA 94158, USA; Arc Institute, Palo Alto, CA 94304, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, USA.
¹⁴ Department of Surgery, University of California, San Francisco, San Francisco, CA 94143, USA.
¹⁵ Department of Dermatology, University of California, San Francisco, San Francisco, CA 94143, USA; Chan Zuckerberg Biohub San Francisco, San Francisco, CA 94158, USA.
¹⁶ 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: aravind.asokan@duke.edu.
¹⁷ 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 94143, USA; Parker Institute for Cancer Immunotherapy, San Francisco, CA 94143, USA; Institute for Human Genetics, University of California, San Francisco, San Francisco, CA, 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: justin.eyquem@ucsf.edu.

PMID: 39909036
DOI: 10.1016/j.immuni.2025.01.009

Abstract

Genetic engineering of T cells in mouse models is essential for investigating immune mechanisms. We aimed to develop an approach to manipulate T cells in vivo using an evolved adeno-associated virus (AAV) capsid named Ark313. Delivery of a transient transgene expression cassette was feasible using Ark313, and this serotype outperformed natural serotypes. A single intravenous injection of a Cre recombinase-expressing Ark313 in the Ai9 fluorescent reporter mouse model achieved permanent genetic modifications of T cells. Ark313 facilitated in vivo gene editing in both tissue-resident and splenic T cells and validation of immunotherapy targets in solid tumor models. Ark313 delivered large DNA donor templates to T cells in vivo and integrated transgenes in primary CD4⁺ and CD8⁺ T cells, including naive T cells. Ark313-mediated transgene delivery presents an efficient approach to target mouse T cells in vivo and a resource for the interrogation of T cell biology and for immunotherapy applications.

Keywords: AAV; Ark313; T cell engineering; gene targeting; immune therapy; in vivo delivery; in vivo gene editing; knockin; mouse T cells.

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Conflict of interest statement

Declaration of interests J.A., W.A.N., J.E., and A.A. are co-inventors on a patent application filed on the subject matter of this study. A.A. is a cofounder and board member at Torque Bio and Lucidigm Therapeutics. He serves as a scientific advisor to Atsena Therapeutics, Ring Therapeutics, Sphere Gene Therapies, Coave Therapeutics, and Nvelop Therapeutics. J.E. is a compensated co-founder at Mnemo Therapeutics and Azalea Therapeutics. J.E. owns stocks in Mnemo Therapeutics, Azalea Therapeutics, and Cytovia Therapeutics. J.E. has received a consulting fee from Casdin Capital, Resolution Therapeutics, and Treefrog Therapeutics. The J.E. lab has received research support from Cytovia Therapeutics, Mnemo Therapeutics, and Takeda Pharmaceutical Company.

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In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313

Affiliations

In vivo engineering of murine T cells using the evolved adeno-associated virus variant Ark313

Authors

Affiliations

Abstract

Conflict of interest statement

MeSH terms

Substances

Grants and funding

LinkOut - more resources

Full Text Sources

Molecular Biology Databases

Research Materials