Fig 2: Establishment and validation of stable eVLP-producing cell lines. (a) Schematic representation of Stable eVLP generation. (b) Schematic diagram of SIN-CD7 gRNA plasmid delivery into precursor monoclonal cells via retroviral vectors. (c) Detection of positive transduction rates in precursor monoclonal cell lines following four rounds of SIN-CD7 gRNA retroviral transduction. (d) Supernatants collected from different SIN-CD7 eVLP cell lines after equal culture duration in 6-well plates were applied under identical conditions to Jurkat cells for knockout validation. (e) The SIN-CD7 eVLP monoclonal cell line (Clone 28) was expanded, and eVLPs were harvested and concentrated 25-fold using PEG 8K, followed by gradient knockout validation in Jurkat cells with increasing doses (0.5μl, 1μl, 5μl, 10μl, 25μl).

Fig 3: Generation and selection of precursor monoclonal cell lines for eVLP production. (a) Schematic representation of the precursor cells used for producing eVLP vectors. (b) Schematic representation of plasmid constructs used for establishing precursor monoclonal cells (top) and gRNA plasmid targeting the CD7 locus for functional validation (bottom). (c) CD7 gRNA plasmids were transfected into monoclonal cell lines selected in (b) to produce eVLPs, and knockout efficiency was subsequently validated in Jurkat cells using the generated eVLPs.

Fig 4: Generation and Functional Validation of 1×10^8 CD7-KO/CD7-CAR-T Cells Derived from Healthy-Donor PBMCs. (a) Schematic diagram of CD7 KO + CD7 CAR-T cell preparation. (b) Flow cytometry analysis of knockout efficiency and CAR expression in CD7 KO + CD7 CAR-T cells. (c) Comparison of cytotoxicity curves against Jurkat cells at different effector-to-target (E:T) ratios between naturally selected CD7 CAR-T cells and CD7 KO + CD7 CAR-T cells. (n=3) (d) At an E:T ratio of 1:1, secretion levels of Granzyme B, TNFα, and IFNγ were compared between the two CAR-T cell groups after 16 h co-culture with Jurkat cells. (n=3) (e) The CD4 and CD8 ratios of CD7 CAR-T cells and CD7 KO + CD7 CAR-T cells. (n=3) (f) Memory phenotype detection of CD7 CAR-T cells and CD7 KO + CD7 CAR-T cells. (g) Depletion phenotype TIM3 detection of CD7 CAR-T cells and CD7 KO + CD7 CAR-T cells. (h) Detection of TIGIT expression in exhausted phenotypes of CD7 CAR-T cells and CD7 KO + CD7 CAR-T cells. c, (d) Values were expressed as the means ± SD, unpaired t test was performed. ***p<0.001; **p<0.01; *p<0.05.