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

Nyberg WA, Ark J, To A, Clouden S, Reeder G, Muldoon JJ, Chung JY, Xie WH, Allain V, Steinhart Z, Chang C, Talbot A, Kim S, Rosales A, Havlik LP, Pimentel H, Asokan A, Eyquem J
Source: Cell
Publication Date: (2023)
Issue: 186(2): 446-460
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Basic Research
Molecular Biology
Regenerative medicine
Cells used in publication:
T cell, human stim.
Species: human
Tissue Origin: blood
T cell, mouse - C57BL/6
Species: mouse
Tissue Origin: blood
T cell, mouse, stim
Species: mouse
Tissue Origin: blood
Culture Media:
4D-Nucleofector® 96-well Systems

Human T cells were isolated from leukopaks with obtained from STEMCELL Technologies (# 70500.1). T lymphocytes were then purified using the EasySep Human T cell isolation kit (STEMCELL Technologies #17951) and activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher #11131D) in X-VIVO 15 medium (Lonza #BP04-744Q) supplemented with human serum (5%:Gemini Bioproducts #100-512), IL-7 (5 ng/ml: Miltenyi Biotec #130-095-367), and IL-15 (5 ng/ml: Miltenyi Biotec #130-095-
760) at a density of 1x10^6 cells per ml.

After 24 h of T cell activation, CD3/CD28 Dynabeads were magnetically removed, and T cells were nucleofected in P3 buffer (Lonza #V4SP-3096) with ribonucleoprotein (RNP) using a 4D-Nucleofector 96-well unit (Lonza #AAF-1003S). An amount of RNP for one reaction was generated by incubating 60 pmol Cas9 protein (QB3 MacroLab) and 120 pmol sgRNA (Synthego) at 37°C. 2x10^6 cells were electroporated with RNP per well. Lonza program code DN-100 was used for murine T cells, and EH-115 was used for human T cells. After nucleofection, cells were diluted in culture medium and incubated (37°C, 5% CO2). For making knock-ins, AAV was added to the culture between 30–60 min after nucleofection at the indicated MOI, and the culture was incubated overnight. The next day, the medium was exchanged for fresh T cell medium, and cells were expanded using standard culture conditions and maintained at a density of approximately 2x10^6 cells per ml.


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.