Journal: Nature
Article Title: In vivo site-specific engineering to reprogram T cells
doi: 10.1038/s41586-026-10235-x
Figure Lengend Snippet: a , Activated T cells were treated with either AAV6 or AAV-hT7 carrying a GFP transcript for transient expression. The cells were cultured in either serum free media (SF) or media supplemented with 10% fetal bovine serum (FBS) or human serum (HS) and GFP expression was measured by flow cytometry. Plotted values are GFP + cells relative to the SF value in each MOI condition in order to highlight changes in transduction efficiency when serum is present. Results are the mean ± SEM from three donors (n = 3). Significance was assessed using two-way ANOVA and Dunnett’s multiple comparisons test. b , Activated T cells were treated with either AAV6 or AAV-hT7 carrying a GFP transcript for transient expression at three different MOI. The cells were cultured in either serum free media. These values are used to calculate the relative GFP expression in (Fig. ). Results are the mean from two donors (n = 2). c , Activated T cells from three donors were electroporated with an RNP targeting the TRAC locus and treated with either AAV6 or AAV-hT7 carrying an HDR template to knock in a CAR at TRAC . The cells were cultured in either serum free media. These values are used to calculate the relative GFP expression in (Fig. ). Results are the mean ± SEM from three donors (n = 3). Significance was assessed using multiple two-tailed unpaired t -tests and the Holm-Šidák multiple comparisons test. d , TRAC -CAR-T cells expressing a 1928z-1XX were generated with a combination of EDV and AAV and functionally validated. TRAC -CAR-T cells were co-culture NALM6 cells at three effector cells to tumour cell ratios (E:T) for 24 h. Cytotoxicity was calculated by a luminescence read-out. Results are the mean ± SEM from four technical replicates (n = 4). e , Schematic of a genome-wide knockout screen to identify genes associated with AAV-hT7 uptake and processing in primary T cells. T cells were isolated from PBMCs, activated with CD3/CD28 beads, and transduced with the lentiviral sgRNA library, followed by Cas9 electroporation (SLICE approach). 3 days later, T cells were re-activated for 48 h and transduced with AAV6 or AAV-hT7 expressing GFP. At 48 h after AAV-hT7 transduction cells were sorted into four bins based on GFP expression. Genomic DNA was extracted from cells in each bin, and amplicon libraries were prepared and sequenced to determine sgRNA enrichment. f , g , Volcano plot displaying genome-wide KO screen results for AAV6 ( f) and AAV-hT7 ( g ), p-values determined using MAGeCK RRA one-sided tests and methods. h , PBMC-humanized were injected with either AAV6 (n = 6) or AAV-hT7 (n = 6) carrying a ss-CAG-GFP cargo, or PBS (n = 3). DNA was isolated from different organs harvested one week after injection. Viral genomes were quantified through qPCR and normalized by the DNA input. Results are the mean ± SEM.
Article Snippet: After thawing, T lymphocytes were purified using the EasySep Human T cell isolation kit (StemCell Technologies, 17951) and activated with Dynabeads Human T Expander CD3/CD28 at a 1:1 bead-to-cell ratio (Gibco, 11141D) in X-VIVO 15 medium (Lonza, BP04-744Q) supplemented with human serum (5%, Gemini Bioproducts, 100-512), IL-7 (5 ng ml −1 , Miltenyi Biotec, 130-095-367) and IL-15 (5 ng ml −1 , Miltenyi Biotec, 130-095-760) at a density of 1 × 10 6 cells per ml.
Techniques: Expressing, Cell Culture, Flow Cytometry, Transduction, Knock-In, Two Tailed Test, Generated, Co-Culture Assay, Genome Wide, Knock-Out, Isolation, Electroporation, Amplification, Injection
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