Journal: Gene Therapy

Article Title: Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells

doi: 10.1038/s41434-021-00220-6

Figure Lengend Snippet: a Schematic of gene targeting strategy. Integration of a CAR–RQR8 construct is targeted to exon 2 of the CD52 locus. Gene targeting by homology-direct repair is mediated by a CRISPR-Cas9 nuclease in combination with an AAV6 donor vector that harbors the CAR–RQR8 expression cassette flanked by ≈500 bp long homology arms. EFS, elongation factor 1α short promoter; CD19-CAR, CD19-targeting CAR; P2A, 2A self-cleaving peptide of porcine teschovirus; RQR8, see schematic; polyA, poly-adenylation signal. b Genotyping by T7E1 assay. The percentage of cleaved products is indicated. c Genotyping by NGS analysis. Pie charts show fractions of alleles with indels. d Genotyping by in–out junction PCR. Primer binding sites for 5′ and 3′-junction PCRs as well as expected product lengths are indicated in panel ( a ). Indel, insertion/deletion mutation; UT, untreated T cells; AAV, AAV-transduced T cells; RNP, RNP-electroporated T cells; RNP + AAV, electroporated with RNP and transduced with 1 × 10 4 , 3 × 10 4 , and 5 × 10 4 genome copies/cell from left to right, respectively.

Article Snippet: To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus.

Techniques: Construct, CRISPR, Plasmid Preparation, Expressing, Binding Assay, Mutagenesis, Transduction

Journal: Gene Therapy

Article Title: Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells

doi: 10.1038/s41434-021-00220-6

Figure Lengend Snippet: a , b Sensitivity to alemtuzumab or rituximab. Complement-dependent cytotoxicity (CDC) assay was performed with media only (N), complement only (C), antibody only (AB), or antibody plus complement (AB + C). Dead cells were identified by 7-AAD staining. UT, untreated T cells; AAV, AAV-transduced T cells; RNP, RNP-electroporated T cells; RNP + AAV, electroporated with RNP and transduced with 5 × 10 4 genome copies/cell. c , d Cytotoxicity assays. Effector esCAR T cells or untreated T cells (UT) were co-cultured with NALM6-GFP (CD19 + ) or K562-GFP (CD19 – ) target cells at effector-to-target (E:T) ratios of 1:1. Percentages of viable GFP + cells are shown for 24 and 48 h time points. Fraction of GFP + cells was normalized to the target-only sample (NALM6, K562). e Secreted factors. Supernatants of cytotoxicity assays ( c ) at 24 h of co-culture were used to assess the release of IFN-γ, granzyme B, or GM-CSF by cytometric bead array. f Enrichment of RQR8-positive cells. esCAR T cells or untreated T cells (UT) were co-cultured with irradiated NALM6 cells for up to 9 days. The percentages of RQR8 + /CAR + T cells were determined at indicated time points. *, **, and *** indicate P < 0.05, P < 0.01, P < 0.001, respectively. The center values reported are the mean and error bars represent standard deviation (SD) of at least three biological independent experiments ( N > 3).

Article Snippet: To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus.

Techniques: CDC Assay, Staining, Transduction, Cell Culture, Co-Culture Assay, Irradiation, Standard Deviation

Journal: Gene Therapy

Article Title: Targeted multi-epitope switching enables straightforward positive/negative selection of CAR T cells

doi: 10.1038/s41434-021-00220-6

Figure Lengend Snippet: a Viability. Viabilities of treated cells are shown for different time points. b CD52 expression. The percentages of CD52 + cells were determined by flow cytometry at different time points. c CAR expression. The percentages of CAR + /CD52 − T cells were determined at indicated time points. d RQR8 expression. The percentages of RQR8 + /CD52 − T cells were determined at indicated time points. UT_US, untreated T cells unstained; UT, untreated T cells; RNP, RNP-electroporated T cells; AAV, AAV-transduced T cells RNP + AAV, RNP-electroporated T cells transduced with 1 × 10 4 , 3 × 10 4 , and 5 × 10 4 genome copies/cell. ** and *** indicate P < 0.01 and P < 0.001, respectively. The center values reported are the mean and error bars represent standard deviation (SD) of at least three independent experiments ( N > 3).

Article Snippet: To address these limitations, we integrated RQR8, a multi-epitope molecule harboring a CD34 epitope and two CD20 mimotopes, alongside a CD19-targeting CAR, into the CD52 locus.

Techniques: Expressing, Flow Cytometry, Transduction, Standard Deviation