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

William A Nyberg; Charlotte H Wang; Jonathan Ark; Chang Liu; Sylvanie Clouden; Anita Qualls; Sofia Caryotakis; Elina Wells; Katherine Simon; Celeste Garza; Pierre-Louis Bernard; Maya Lopez-Ichikawa; Zhongmei Li; Jin Seo; Gabriella R Kimmerly; Joseph J Muldoon; Peixin Amy Chen; Mingcheng Li; Hong-Erh Liang; Kelly Kersten; Alan Rosales; Nicholas Kuhn; Chun Jimmie Ye; James M Gardner; Ari Molofsky; Roberto R Ricardo-Gonzalez; Aravind Asokan; Justin Eyquem

doi:10.1016/j.immuni.2025.01.009

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

Immunity. 2025 Feb 11;58(2):499-512.e7. doi: 10.1016/j.immuni.2025.01.009. Epub 2025 Feb 4.

Authors

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

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.

MeSH terms

Animals
CD4-Positive T-Lymphocytes* / immunology
CD8-Positive T-Lymphocytes* / immunology
Capsid Proteins / genetics
Dependovirus* / genetics
Dependovirus* / immunology
Gene Editing / methods
Genetic Engineering / methods
Genetic Vectors / genetics
Humans
Integrases / genetics
Mice
Mice, Inbred C57BL
Mice, Transgenic
T-Lymphocytes* / immunology
Transgenes / genetics

Substances

Integrases
Capsid Proteins