Journal: Frontiers in Immunology

Article Title: A universally applicable toolbox for single-molecule quantification of chimeric antigen receptors using linker-resolved d STORM microscopy

doi: 10.3389/fimmu.2026.1897225

Figure Lengend Snippet: Direct quantification of CAR surface expression by linker-specific staining using flow cytometry and d STORM. (A) Antibody titration for linker-specific detection of CAR constructs by spectral flow cytometry. Representative histograms showing staining of EGFRt-sorted CAR-T cells using commercially available Alexa Fluor 647-conjugated antibodies targeting the (G4S)3- (left, blue; Cell Signaling Technology, E7O2V) or Whitlow (right, green; Miltenyi Biotec, REA1400) linker. Representative histograms are shown from one donor. (B) Quantification of linker-positive CAR-T cells by spectral flow cytometry. Bar graph showing the percentage of linker-positive cells in CAR-T cell products or untransfected T cells (UTD) following staining with (G4S)3- or Whitlow-specific antibodies at the selected final working concentrations (2.5 μg/mL and 15 μg/mL respectively). Representative graphs are shown from one donor. (C, D) Antibody titration for linker-specific CAR detection by d STORM super-resolution microscopy. Untransfected (UTD) and CAR-T cells were stained with increasing concentrations of (G4S)3- ( (C) , blue; Cell Signaling Technology, E7O2V) or Whitlow-specific ( (D) , green; Miltenyi Biotec, REA1400) antibodies. Each point represents an individual cell from one representative donor. For each antibody concentration, 21–33 cells were analyzed in (C) and 16–30 cells were analyzed in (D) . Box plots indicate the median and interquartile range, with whiskers showing the minimum to maximum values. Statistical analysis was performed using Brown-Forsythe and Welch ANOVA with Welch correction. Right panels: representative d STORM images of CAR-T positive cells stained with various concentrations. Scale bars = 5 µm. Significance indicated as: ****P ≤ 0.0001; *P ≤ 0.05; ns = P > 0.05.

Article Snippet: For example, one clone (Miltenyi Biotec; REA1400) yielded quantifiable signals in d STORM even at lower concentrations than flow cytometry, whereas another clone (Cell Signaling; E3U7Q) performed well with both techniques but usage by flow cytometry was limited by availability or concentration.

Techniques: Expressing, Staining, Flow Cytometry, Titration, Construct, Super-Resolution Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Structural models of receptor-binding-deficient PIRYV (ePIRYV RBD ) engineered with K182A and Y352A mutations to eliminate low-density lipoprotein receptor (LDL-R) binding while preserving fusogenic capacity. Receptor-interacting region is highlighted in magenta b, Close up view of the two LDL-R contact residues, glycoprotein (green sticks) form hydrogen bond (dashed lines) with LDL-R (cyan) whereas in the mutant complex, substitutions abolish these bonds. c, Complete structural model of the engineered PIRYV RBD glycoprotein in complex with LDL-R depicting ablation of LDL-R recognition, alongside the four immunogenicity-reducing residues. d , Schematic of lentiviral production and T cell isolation. e, Representative flow cytometry plots of transduction efficiency (%GFP + ) in CD3 + T cells at MOI 2.5 with vector control (VC). ePIRYV wt . ePIRYV RBD or ePIRYV RBD+nbC3/7 . f , Bar graph showing the mean fluorescence intensity (MFI) of GFP + cells. Representative of three independent experiments (n=6 biologically independent samples). g, Humanized scFv design for bispecific BCMA/CD19 CAR construct showing complementarity-determining regions (CDRs) in heavy and light chains. h, Representative contour plots showing comparative CAR expression (PE) in transduced CD4 + (APC, top) and CD8 + (APC, bottom) T cell subsets in activated primary CD3 + T cells from healthy donors (n = 6). Bar graph quantifies the percentage of CAR + cells (using an anti-G4S linker antibody) among CD4 + and CD8 + T cells. i, PBMCs were either left unstimulated (None) or activated with anti-CD3/CD28 (CD3/CD28) Dynabeads before transduction with PIRVYwt or ePIRYRBD +nbC3/7 , followed by analysis of activation. j, T cell activation markers (CD69) measured as MFI following transduction with PIRYV wt or ePIRYV RBD+nbC3/7 at MOI 1 and MOI2.5 after 24 h post-transduction. Each point represents one donor (n = 8). k, l, In vitro cytotoxicity of CAR-T cells generated with PIRY wt or ePIRY RBD+nbC3/7 against BCMA + multiple myeloma targets MM.1S (k) and MM.1R (l), expressed as percentage target-cell survival across the indicated effector-to-target (E:T) ratios after 24 h coculture. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis with n = 3 biologically independent samples). See Supplementary Figures 7-11 for extended nanobody characterization and CAR construct optimization .

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Preserving, Mutagenesis, Immunopeptidomics, Cell Isolation, Flow Cytometry, Transduction, Plasmid Preparation, Control, Fluorescence, Construct, Expressing, Activation Assay, In Vitro, Generated

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Experimental timeline of in vivo CAR-T delivery in humanized xenograft model. NSG mice received intravenous engraftment of human hematopoietic stem cells (HSCs) on days −28 with prior busulfan treatment. viroVbot1 (ePIRYV RBD+nbC3/7 encoding bi-CAR transgene) was administered intravenously at doses ranging from 0.25 × 10 6 to 1 × 10 6 TU/mouse on day 0. Blood, was collected at days −4, 7, 14, 21, and 28 post-vector administration and bone marrow (BM), and spleen at the end of the experiment. b , Kinetics of CAR-T cell expansion in peripheral blood. Percentage of BCMA-CAR + CD3 + T cells over time (Day −4 to 28). c , d, CAR-T cell frequency in (c) bone marrow and (d) spleen at Day 28. Each point represents pooled samples from 15 animals (n = 5 from 3 pooled in each group). e, f, CAR-T cell transduction efficiency in peripheral blood cells determined by flow cytometry by collecting the samples at day 7 and day 28. g , Flow cytometry analysis of CD19 + cells among hCD45 + cells showing rapid B cell depletion over time. h, i, Similarly in bone marrow and spleen. j, Representative IHC images of major organs (spleen, liver, lungs, and kidneys) showing CAR expression as indicated by red arrowheads with quantitative analysis (n=6 images); Bar graph shows CAR+ cells per 1×10 3 cells by tissue in visceral tissues. k, Schematic of vector production of the two lentiviral vectors used: (left) ePIRY wt with MHC-I −/− modification and bispecific BCMA/CD19 CAR; (right) viroVbot1 with TcrBM-detargeted envelope (ePIRY RBD+nbC3/7 ), with the same bispecific CAR. Both vectors were used to transduce a panel of 65 distinct human-derived cell lines to assess CAR expression at MOI 2.5. l, CAR expression profile across 65 cell lines (ex vivo transduction assay). Dot plot showing CAR fluorescence (n=3 biologically independent samples). Data represents mean ± SEM. **p < 0.01; *p < 0.05. A non-parametric t-test was used for statistical analysis between groups. Scale bar; d: 200 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: In Vivo, Plasmid Preparation, Transduction, Flow Cytometry, Expressing, Modification, Derivative Assay, Ex Vivo, Fluorescence

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic showing receptor-mediated CAR uptake by B cells with wild-type (CD19 wt Raji, BCMA wt multiple myeloma (MM) phenotype and CRISPR-Cas9 knockout (BCMA/CD19 −/− MM, CD19 −/− Raji) phenotype. Cell lines were transduced with viroVbot1 or wild type PIRYV b, Flow cytometry showing ex vivo CAR transduction the wild type and knockout cells (n=6). c, Schematic of receptor competition assay showing that pre-incubation of viroVbot1 with recombinant BCMA (BCMAR) and CD19 (CD19R) extracellular domain proteins before transduction of wild-type Raji and MM.1S cells. d, Transduction efficiency (% CAR + cells) in Raji and MM.1S cells, with viroVbot1 incubated with vehicle (PBS) or viroVbot1 incubated with CD19R or BCMAR (n=8 biologically independent samples). e, Schematic showing CAR glycoprotein displayed on wild-type producer cell line (left) is incorporated onto the lentiviral envelope in contrast to CAR-TRAP producer cell (right) where CD19-ECD-KDEL retains the CAR in ER lumen rather than plasma membrane. f , Representative Immunofluorescence image of HEK293T control cells (left), show robust CD19-CAR (green) throughout the cell surface; CAR-TRAP-expressing cells (right) show minimal surface CD19-CAR fluorescence. Bottom panel is quantification of CD19-CAR intensity density (integrated intensity per cell area) in HEK293T versus CAR-TRAP (n=9 cells). g, Contour plots of CAR (FITC-CD19) surface expression under non-permeabilising conditions with quantitative analysis shown as MFI (n=6), h, Representative Immunofluorescence image showing intracellular ER-localized CAR (anti-CD19-CAR, green) colocalized with calnexin (ER marker, red) in CAR-TRAP cells, along with the lines scans (LS) showing co-localisation of ER signal with CD19 CAR. i, Illustration of viroVbot1 particle produced from wild type producer cell and viroVbot1.1 particle produced from CAR-TRAP cells with bi-CAR as transgene. j, Dot plots showing % BCMA or % CD19 CAR expression in Raji wt or MM.1S wt cells with LVV obtained from HEK293T or CAR-TRAP cells. Quantification bar graphs (right) under same conditions (n= 8). k, BCMA CAR expression in patient multiple myeloma samples (PMM-1 to PMM-12) showing % of BCMA/CAR + cells after transduction with viroVbot1 versus viroVbot1.1. l-m, Similarly, BCMA CAR expression in patient-derived leukemia (PLK-1 to PLK-10) and lymphoma (PLM-1 to PLM-15) samples. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups. Scale bar; f: 50 μm, n: 10 μm.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: CRISPR, Knock-Out, Transduction, Flow Cytometry, Ex Vivo, Competitive Binding Assay, Incubation, Recombinant, Clinical Proteomics, Membrane, Immunofluorescence, Control, Expressing, Fluorescence, Marker, Produced, Derivative Assay

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of miR-122-mediated hepatocyte-specific transgene silencing strategy incorporating five tandem miR-122 target sequences (5×miR-122T) in the 3’ untranslated region (UTR) downstream of CD3ζ costimulatory domain of bi-CAR transgene (viroVbot2). b, Flow cytometry analysis of % CAR expression (BCMA CAR-PE) in Huh-7 hepatoma cells transduced with LVV containing miR-122 (viroVbot2) or non-targeting control shRNA (NTC) with bi-CAR as transgene. c, Schematics of CD47 overexpression strategy in CAR-TRAP producer cells for macrophage evasion (viroVbot2.1). d, Flow cytometry analysis of % CAR expression in THP1 cells transduced with either viroVbot2 or viroVbot2.1. e, Schematic flow diagram of PromoterForge pipeline (details of the pipeline are provided in methods) . f, Candidate synthetic promoters fused upstream with GFP reporter cassette in reporter-plasmid format. Each synthetic promoter represents a unique combination of core promoter elements, TFBS motifs and enhancer arrangements. g, Flow cytometric analysis of GFP expression as MFI in primary human T cells transduced with constructs in which GFP is driven by the EF-1α promoter or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15) (n=5 biologically independent samples). h, Schematic of the dual-luciferase reporter assay used to validate promoter activity. i, Relative luciferase activity in T cells transfected with reporter constructs containing EF-1α or the indicated synthetic promoters (Syn-T3, Syn-T8, Syn-T15), normalized to the CMV control (n=6 biologically independent samples). j, Schematic of the viroVbot2.2 construct and LVV design. The transfer vector encodes bi-CAR under the Syn-T8 promoter with mir-122. Producer cells co-express the CAR-TRAP system and CD47, generating viroVbot2.2 particles displaying CD47 on the envelope. k, Flow cytometry analysis of CAR expression on CD3 + T cells in PBMCs from six healthy donors (HD-1 to HD-6) following ex vivo transduction with viroVbot2.1 (top row) and viroVbot2.2 (bottom row). Numbers in gates indicate the percentage of CAR-FITC + cells. The bar graph shows the %CAR + of CD3 + cells per donor. l, Scheme of the humanized mouse model. NCG mice were engrafted with human PBMCs (day −5), inoculated with 1×10 6 MM.1S multiple myeloma cells (day −4), and treated with a single dose of viroVbots (day 0). Peripheral blood was collected and BLI performed on days 7, 14, 21, 28, and 56; all organs were harvested at endpoint (day 56). m Representative BLI images of MM.1S tumor burden in mice treated with PBS control (MM.1S), viroVbot2.1, or viroVbot2.2 at the indicated time points. Red “X” denotes deceased animals. n, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each treatment group. Each line represents an individual mouse (n=5 per group). o, Percentage of circulating MM.1S tumor cells per 100 µl of blood over time in mice treated with viroVbot2.1 (orange) or viroVbot2.2 (blue). Data shown as mean ± SEM. p, Percentage of CAR + T cells in peripheral blood over time in the same treatment groups, demonstrating in vivo expansion kinetics and contraction of CAR-T cells. q, Kaplan-Meier survival curves of mice bearing MM.1S tumors and treated with PBS (red), viroVbot2.1 (orange), or viroVbot2.2 (blue); n = mice per group, log-rank test. Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Flow Cytometry, Expressing, Transduction, Control, shRNA, Over Expression, Plasmid Preparation, Construct, Luciferase, Reporter Assay, Activity Assay, Transfection, Ex Vivo, In Vivo

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, Schematic of the screening strategy for selecting a synthetic IL-7R agonist. Three synthetic binding proteins (sBC-P1, sBC-P2, sBC-P3) were paired with full-length IL-7R or minimized IL-7R variants (min1-, min2-, min3-IL7R), expressed in T cells, and assessed for downstream STAT5 phosphorylation using the PathScan Phospho-STAT5 assay. b, Representative flow cytometry histograms of phospho-STAT5 (p-STAT5) in T cells expressing the indicated constructs, compared with isotype and non-transduced (NTP) controls. Anti-p-STAT5 antibody was used followed by Alexafluor 488 and acquired in FITC, channel. c, Quantification of pSTAT5 MFI across conditions, showing comparable STAT5 activation by sBCP3-IL7R and the minimized sBCP3_min3-IL7R receptor relative to NTP-IL7R control (n=8 biologically independent samples). d, Dose-response curves of ligand-induced receptor activation measured by ELISA across a concentration range (mM) for NTP-IL7R, sBCP3-IL7R, and sBCP3_min3-IL7R (n=5 biologically independent samples). e, f Schematic of the viroVbot3 transfer vector and producer cell design. The bi-CAR (BCMA/CD19) cassette is driven by the Syn-Tp promoter, linked via T2A, and detargeted from hepatocytes by 5× miR-122 target sites in the 3′ UTR to co-express the synthetic sBCP3-min3-IL7R (sBCP-mIL7R) cytokine receptor module. g, In vitro cytotoxicity assay showing % MM.1S tumor cell survival at increasing effector-to-target (E:T) ratios following co-culture with untransduced T cells (UT), viroVbot2.2-, or viroVbot3-generated CAR-T cells (n=5 biologically independent samples) after 24 h. h, IL-2 secretion (pg/mL) by CAR-T cells co-cultured with MM.1S target cells at the indicated E:T ratios (n=5 biologically independent samples). i, Similarly, intracellular Granzyme B expression (MFI) in CAR-T cells across the same E:T ratios (n=5). j, Schematic of the serial tumor-rechallenge assay. k, Percentage of Patient MM (PMM) cell survival over time during serial rechallenge in co-cultures with UT, viroVbot2.2, or viroVbot3 CAR-T cells. l, Absolute CAR-T cell counts (5×10 4 ) during serial rechallenge, demonstrating superior expansion and persistence of viroVbot3-generated CAR-T cells. m, Bar graph of frequency of PD1 low (gray) versus PD1 high (orange) populations within CD8 + effector memory (T EM ) cells at days 14, 21, and 28 of co-culture for the three groups. n, Similarly, CD8 + central memory (T cm ) cells at the same time points (n=5 biologically independent samples). o, Memory subset distribution (% of CD8 + T cells); naive/stem-cell memory (T n /T scm ), effector (T eff ), effector memory (T em ), and central memory (T cm ), across UT, viroVbot2.2, and viroVbot3 groups. p, Pie charts showing the relative proportions of T n (gray) and T scm (blue) compartments within CD8 + T cells (n=5 biologically independent samples). Data represents mean ± SEM. ****p < 0.0001. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Binding Assay, Phospho-proteomics, Flow Cytometry, Expressing, Construct, Activation Assay, Control, Enzyme-linked Immunosorbent Assay, Concentration Assay, Plasmid Preparation, In Vitro, Cytotoxicity Assay, Co-Culture Assay, Generated, Cell Culture

Journal: bioRxiv

Article Title: Safe Redosable Low-Immunogenic In Vivo CAR-T Therapy for B Cell Malignancies and Solid Tumors

doi: 10.64898/2026.06.30.735484

Figure Lengend Snippet: a, NCG mice received MM.1S cells (day −7), human PBMCs (day −5), and viroVbot3 (day 0). On day 45, mice were rechallenged with BCMA negative but expressing GPRC5D (GR) MM.1S (GR + /BCMA − /CD19 − ) and given a second PBMC infusion plus viroVbot-CO or viroVbot-VS on day 47. Blood and BLI were collected at indicated time points; organs were harvested at day 90. b, Representative BLI images of tumor burden over time (days 10-90) in five treatment groups as indicated (n=5 mice in each group). c, Quantification of whole-body BLI radiance (p/sec/cm 2 /sr) over time for each group; each line represents an individual mouse. d, Kaplan-Meier survival curves of the five groups (Group-1 to Group-5) across 12 weeks. Log-rank test (n=10 mice in each group). e, Longitudinal flow cytometric quantification of BCMA + CAR-T cells (% of T cells) in peripheral blood from days 0-90 across Groups 2-5, showing initial expansion and contraction kinetics of the first-line CAR-T population. f, Frequency of GPRC5D + CAR-T cells (% of T cells) in peripheral blood across Groups 2, 4, and 5, demonstrating expansion of the second-line viroVbot-CO/VS-derived CAR-T cells following antigen-loss rechallenge. g, Quantification of BCMA + (left axis) and GPRC5D + (right axis) CAR-T cells in bone marrow (BM) at endpoint (day 90) for Groups 2, 4, and 5. ND, not detected. h, Similarly in spleen. Data represents mean ± SEM. A non-parametric t-test was used for statistical analysis between groups.

Article Snippet: Cells were analyzed on BD FACS Aria, Accuri, Lyric, or Beckman Coulter CytoFLEX instruments and data analyzed using FlowJo v10 or CytExpert software as described previously., CAR transgene expression was detected using CAR-specific detection reagents: CD19 CAR detection (Miltenyi Biotec #130-129-550), BCMA CAR detection (Miltenyi Biotec #130-126-727), anti-4-1BB monoclonal antibodies for 4-1BB-containing constructs, anti-G4S linker antibody for GPRC5D (#62405, Cell Signaling Technologies) and CLDN18.2 CARs, or Protein L-APC (CST #29480) for CD20 CAR.

Techniques: Expressing, Derivative Assay

Journal: Molecular Therapy. Nucleic Acids

Article Title: Engineered mRNA backbones for gene expression in human T cells

doi: 10.1016/j.omtn.2026.102913

Figure Lengend Snippet: T cell-specific UTRs for optimized CAR expression, reactivity, and tonic signaling (A). Representative flow cytometry analysis of PBMC-derived T cells electroporated with CD19-CAR-encoding constructs using various 5′ UTRs. (B) Quantification of percentages of CD19-CAR + cells in (A). Data are normalized to the HBA1 UTR condition and presented as mean ± SEM from four independent experiments ( n = 4). Kruskal-Wallis test revealed no statistically significant difference ( p = 0.29). (C) MFI of CD19-CAR + cells in (A) normalized to the HBA1 UTR. Data represent four independent experiments ( n = 4). Kruskal-Wallis test indicated a significant difference among groups ( p < 0.005); Dunn’s post hoc test revealed significant reduction in the TNF-UTR group compared to HBA1 ( p = 0.0022). (D) Secreted IFN-γ levels in co-culture supernatants of PBMC-derived T cells electroporated with CD19-CAR mRNA using different 5′ UTRs and CD19 + NALM6 target cells. Data from two healthy donors (D29 and D40) are shown at various effector-to-target (E:T) ratios. (E) Left: interferon gamma ELISA in media taken from co-cultures of PBMC-derived T cells electroporated with CD19-CAR mRNA using different UTRs, either together with CD19 + (filled) or CD19 − (NALM6 KO, empty) at an E:T ratio of 4:1. Right: delta of interferon gamma secretion of E between the co-culture of electroporated T cells with CD19 + vs. CD19 − NALM6 cells. Bars represent mean ± SEM, n = 5 (CD19 + co-cultures) and n = 2 (CD19 − co-cultures) per construct. (F) Flow cytometry analysis of virus-specific T cells (VSTs) electroporated with mRNA constructs encoding for CD19-CAR using various UTRs. Note that cells are grown without target cells to demonstrate tonic signaling. (G) Pie charts of PD-1/TIM-3 population distribution of (F). Data are representative of two independent experiments.

Article Snippet: Cells were harvested 24–48 h post-electroporation, washed in FACS buffer (PBS with 2% FBS), and stained with the following fluorochrome-conjugated antibodies: CD19 CAR Detection Reagent, Biotin (Miltenyi Biotec, #130-129-550), followed by secondary staining with Anti-Biotin-APC (Miltenyi Biotec, REAfinity #130-113-854); TIM-3 APC-Cy7 (BioLegend, #345025); 4-1BB PE-Cy7 (BioLegend, #309820); Viability Dye eFluor 506/AmCyan (Thermo Fisher Scientific, #65-0866-14).

Techniques: Expressing, Flow Cytometry, Derivative Assay, Construct, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Virus

Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Polyfunctional editing strategy for the epi-silencing of B2M and TET2 and insertion of the CD19–28ζ CAR:ΔLNGFR construct into TRAC . B Left: percentages of the indicated T cell populations at day 14 after poly-editing (mean ± SD of 7 blood donors). TCR + : TCR-positive cells (green bar). TCR - : TCR-negative cells (light blue bar). TCR - /ΔLNGFR + : cells negative for endogenous TCRs and positive for ΔLNGFR (lilac bar). B2M - : B2M-negative cells (gray bar). B2M + : B2M-positive cells (white bar). Right: representative flow cytometry dot plots of poly-edited T cells showing expression of the TCRs and ΔLNGFR (left plot) and, within the TCR - /ΔLNGFR + cells, of B2M (right plot). C Fold-change in TET2 (light blue bars) and B2M (gray bars) expression in poly-edited vs . mock-treated cells (mean ± SD of 7 blood donors). D Schematic of the in vivo experiment. NSG mice were injected with GLuc.NALM-6 cells and, 7 days later, with the indicated T cell populations. Peripheral blood was collected at the indicated time points to measure tumor growth, phenotype human T cells, and quantify cytokine release. Mock: mock-transfected T cells. CAR-T Δ LNGFR : T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC . Poly-edited cells: T cells expressing CD19–28ζ CAR:ΔLNGFR from TRAC with epi-silencing of B2M and TET2 . E Tumor growth (left; mean ± SEM) and survival (right) curves over 20 days post-transplantation of the indicated T cell populations. n = 6 mice for Mock; n = 12 mice for each other group. ** p = 0.0075, *** p = 0.0008 by Mantel-Cox (log-rank) test. F Circos plots from CAST-Seq analyses of triple KO (left) and poly-edited (right) T cells ( n = 2 experimental replicates). Triple-KO cells were transfected with mRNA encoding Cas9 and the TRAC gRNA, together with gRNA B#6 and gRNA TE#19 . For poly-editing, the TRAC gRNA and the selected guide combinations for B2M and TET2 were used. Aberrations at the TRAC locus are in violet; translocations between TRAC and either B2M or TET2 are in blue; translocations between TRAC and OT sites of all gRNAs are in gray. G Number of unique CAST-Seq reads corresponding to either aberrations (violet bars) or translocations (gray bars) at the TRAC locus in the indicated treatments. Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Construct, Flow Cytometry, Expressing, In Vivo, Injection, Transfection, Transplantation Assay, Generated, Software

Journal: Nature Communications

Article Title: Simultaneous orthogonal cell engineering by a single CRISPR-Cas9 polyfunctional editor

doi: 10.1038/s41467-026-72846-2

Figure Lengend Snippet: A Scatter plot comparing whole-transcriptome analyses of T cells treated with the tripartite ETR and either full-length or truncated gRNAs targeting CD3D (left) or TGFBR2 (right) ( n = 2 experimental replicates). Data are expressed as log 2 TPM of mapped reads. B Polyfunctional editing strategy used to epi-silence CD3D and TGFBR2 and insert the scHLA-E:CD19–28ζ CAR cassette into exon 1 of B2M . C Left: percentages of the indicated T cell populations as measured by flow cytometry 14 days after polyfunctional editing (mean ± SD of 7 blood donors). HLA-ABC + : HLA-ABC-positive cells (light yellow bars); HLA-ABC - : HLA-ABC-negative cells (blue bars); CD19-CAR - : cells negative for the expression of the CD19–28ζ CAR (gray bars); CD19-CAR + : cells positive for the expression of the CD19–28ζ CAR (lilac bars); CD3 + : CD3-positive cells; CD3 - : CD3-negative cells; HLA-E - : HLA-E-negative cells; HLA-E + : HLA-E-positive cells. Right: representative flow cytometry dot plots of poly-edited T cells showing the expression levels of the HLA-ABC and CD3 (left plot) and, within the double-negative cells, of HLA-E and CD19-CAR (right plot). D Fold-change in the expression levels of CD3D (green bars) and TGFBR2 (orange bars) in poly-edited cells relative to mock-treated cells, 14 days post-editing (mean ± SD of 7 blood donors). Graphs were generated using GraphPad Prism (GraphPad Software). Source data are provided as a file.

Article Snippet: LNGFR; 130-113-422, Miltenyi), CD3 (345763, BD Biosciences), HLA-ABC (565332, BD Biosciences), HLA-E (130-117-402, Miltenyi), and CD19 CAR FMC63 Idiotype (130-127-342, Miltenyi).

Techniques: Flow Cytometry, Expressing, Generated, Software