paper n a pggaselect new england biolabs rrid addgene 195714 hdr template igp2 230 tag  (Addgene inc)

Journal: Blood

Article Title: Integration of ζ -deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

doi: 10.1182/blood.2023020973

Figure Lengend Snippet: Integration of a truncated CD19-specific CAR into CD3ζ , but not TRAC , conveys cytotoxicity in conventional T cells toward CD19 + leukemia cells. (A) full-length second-generation CAR protein (left) and virus-free knock-in strategies to integrate a full-length CAR into TRAC or a truncated CAR ( trunc CAR) into TRAC or CD3ζ . (B) Flow cytometry dot plots after knock-in. Transgene integration into TRAC or CD3ζ disrupts expression of the TCR/CD3 complex. (C) Relative cytotoxicity in coculture with (CD19 + ) Nalm-6 target cells and CD19 KO Nalm-6 control cells (VITAL assay). Calculation of relative cytotoxicity according to formula stated in methods section. (n = 2 biological replicates each in 2 technical replicates; ordinary one-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (D-G) Functional testing of CD3ζ trunc CAR, T cells in comparison to TRAC CAR and LV CAR-T cells. (D) Mean fluorescence intensity (MFI) determined by flow cytometry as a measure of cellular CAR expression and normalized to each donor’s mean CAR MFI in the TRAC condition. (n = 7 biological replicates each in 2-5 technical replicates; mixed-effects analysis with Geisser-Greenhouse correction + Holm-Šídák multiple comparison test with individual variances computed for each comparison). (E) Relative cytotoxicity towards CD19 + cells assessed in a 6-hour VITAL assay. (mock-E′: mock-electroporated controls without ribonucleoproteins/HDR templates) (n = 4 biological replicates each in 1-3 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance (F) Changes in CAR-expression levels (MFI normalized to start) after target cell encounter. ( TRAC and LV in 4 biological replicates; CD3ζ in 2 biological replicates). (G) Acute lymphoblastic leukemia xenograft mouse model using luciferase-labeled Nalm-6 (CD19 + ) tumor cells. 4 days post Nalm-6 administration, 1 × 10 6 cryopreserved, 14-day expanded TCR-deleted CAR + T cells were injected systemically. Tumor burden was assessed via bioluminescence imaging. (n = 5-6; 2-way ANOVA with Geisser-Greenhouse correction of log-transformed bioluminescence imaging data followed by Holm-Šídák multiple comparison test, with individual variances computed for each comparison). Asterisks in this and all further figures represent different P values calculated in the respective statistical tests (not significant [ns], P > .05; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001).

Article Snippet: Plasmids encoding CD3ζ -HDR-templates will be distributed through Addgene (pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene ID: 215758; pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene-ID: 215759).

Techniques: Virus, Knock-In, Flow Cytometry, Expressing, Control, Comparison, Functional Assay, Fluorescence, Luciferase, Labeling, Injection, Imaging, Transformation Assay

Journal: Blood

Article Title: Integration of ζ -deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

doi: 10.1182/blood.2023020973

Figure Lengend Snippet: Evaluation of an optimized CD3ζ trunc CAR transgene and its impact on CAR-T cell function in vitro. (A) dsDNA templates for targeted delivery of a CAR or trunc CAR respectively into TRAC (left) or CD3ζ (middle), as in Figure 1 A, and for targeted delivery of a GSG-P2A-linker-modified trunc CAR into CD3ζ (right). (B) Top: Mean fluorescence intensity (MFI) determined by flow cytometry at steady state (n = 4 biological replicates in 4-6 technical replicates in 2 independent experiments, data normalized to mean of TRAC for each donor; mixed-effects analysis with Geisser-Greenhouse correction followed by Holm-Šídák multiple comparison test, with individual variances computed for each comparison). Bottom: dynamics of CAR MFI after CAR-stimulation using CD19 + Nalm-6 tumor cells. (n = 3-4 biological replicates in 1-2 technical replicates). (C) Relative cytotoxicity assessed in a 6-hour VITAL assay (similar to Figure 1 C, n = 4 biological replicates in 3 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance.). (D) Cytokine expression in CAR + cells in response to control (CD19 − ) cell or target (CD19 + ) cell encounter (n = 3 biological replicates). (E-H) CAR-T-cell rechallenge in serial cocultures with Nalm-6 target cells. (E) Top: CAR MFI normalized to TRAC condition at steady state (n = 2 biological replicates in 4 technical replicates; statistics as in B). Bottom: dynamics of CAR MFI after target cell engagement (n = 2-4 biological replicates in 1-2 technical replicates). (F) 6-hour VITAL assay. (n = 3 biological replicates in 3-4 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance.). (G) Top: relative expansion of CAR + T cells. Bottom: CAR + frequency within T-cell products. (n = 4 biological replicates). (H) Cell surface expression of inhibitory receptors (LAG-3, PD-1, TIM-3; means of n = 4 biological replicates). (I) In vivo CAR-T-cell efficacy tested in Nalm-6 acute lymphoblastic leukemia xenograft mouse model (n = 5-6 mice/group; multiple log-rank tests).

Article Snippet: Plasmids encoding CD3ζ -HDR-templates will be distributed through Addgene (pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene ID: 215758; pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene-ID: 215759).

Techniques: Cell Function Assay, In Vitro, Modification, Fluorescence, Flow Cytometry, Comparison, Expressing, Control, In Vivo

Journal: Blood

Article Title: Integration of ζ -deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

doi: 10.1182/blood.2023020973

Figure Lengend Snippet: CD3ζ trunc CAR-integration facilitates CAR expression in different nonconventional T-cell subtypes and NK cells. (A) HLA-A2 CAR-integration in regulatory T-cells, n = 3 biological replicates. (B) CD19-CAR integration in TCR γ/δ T cells. TRAC integration generates CAR + /TCR γ/δ + double positive T cells, n = 2 biological replicates. (C) Integration of a CD19-CAR in primary human NK cells, n = 6 biological replicates.

Article Snippet: Plasmids encoding CD3ζ -HDR-templates will be distributed through Addgene (pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene ID: 215758; pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene-ID: 215759).

Techniques: Expressing

Journal: Blood

Article Title: Integration of ζ -deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

doi: 10.1182/blood.2023020973

Figure Lengend Snippet: CD3ζ -disuption does not impede canonical NK cell functions in vitro. (A) CD3ζ editing outcomes assessed by flow cytometry. (B) Cytotoxicity of primary CD3ζ disrupted NK cells in simple 16 hours coculture assay with K562 cells, Nalm-6 cells or allogeneic PBMC (n = 3 biological replicates). (C) Degranulation of primary CD3ζ disrupted NK cells assessed by flow cytometry (n = 3 biological replicates; two-way ANOVA followed by Dunnett’s multiple comparison test with a single pooled variance). (D) Expression of CD16 and CD3ζ in wild-type NK-92 cells and primary NK cells after CD3ζ -disruption. (E) ADCC of primary CD3ζ -disrupted NK cells against CD20 + bGal − Jeko-1 cells at different concentrations of antibodies specific for CD20 (rituximab) or bGal (n = 3 biological replicates, each in 3 technical replicates).

Article Snippet: Plasmids encoding CD3ζ -HDR-templates will be distributed through Addgene (pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene ID: 215758; pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene-ID: 215759).

Techniques: In Vitro, Flow Cytometry, Co-culture Assay, Comparison, Expressing, Disruption

Journal: Blood

Article Title: Integration of ζ -deficient CARs into the CD3 ζ gene conveys potent cytotoxicity in T and NK cells

doi: 10.1182/blood.2023020973

Figure Lengend Snippet: CD3ζ -editing enables redirection of NK cells with CARs and does not impede canonical NK cell functions in vitro. CAR editing in primary NK cells via LV CAR transfer, TRAC -CAR or CD3ζ-trunc CAR-integration: (A) CAR + frequencies after editing (n = 6 biological replicates, mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (B) mean CAR expression (MFI) normalized to CD3ζ -truncCAR integrated NK cells and robust coefficient of variation in CAR + cells (n = 6 biological replicates; t test). (C) CAR-dependent cytotoxicity detected in a VITAL assay (data normalized to mock-electroporated (wildtype) NK cells; n = 6 biological replicates each in 3-4 technical replicates; 2-way ANOVA followed by Tukey’s multiple comparison test with a single pooled variance). (D) Degranulation as indicator of NK effector function via flow cytometric detection of CD107a (n = 6 biological replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance). (E) ADCC of primary (CAR) NK cells against CD20 + bGal − Jeko-1 cells. Bars represent killing for each condition in the presence of a CD20-targeting monoclonal antibody (0.5 μg/mL) normalized to the respective condition without supplemented antibody (n = 5 biological replicates; mixed-effects analysis with Geisser-Greenhouse correction followed by Tukey’s multiple comparison test with individual variances computed for each comparison). (F-H) CD19-CAR (2) transfer to NK-92 cells via AAVS1 integration of a CMV promotor-controlled, full-length CAR or CD3ζ integration of a trunc CAR. CAR + fractions were enriched using MACS. (F) CAR expression in flow cytometry histograms. (G) CAR-dependent cytotoxicity in a 4-hour VITAL-assay (n = 6 technical replicates; two-way ANOVA with Tukey’s multiple comparison test with a single pooled variance. (H) CAR-independent cytotoxicity towards the MHC I deficient, CD19 − K562 (control) cell line (n = 15 technical replicates; two-way ANOVA followed by Holm-Šídák multiple comparison test with a single pooled variance).

Article Snippet: Plasmids encoding CD3ζ -HDR-templates will be distributed through Addgene (pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene ID: 215758; pUC19-HDRT- CD3ζ-trunc CAR GSG Addgene-ID: 215759).

Techniques: In Vitro, Comparison, Expressing, Flow Cytometry, Control