Journal: STAR Protocols

Article Title: Protocol to generate human stem cell-derived CD70-directed allogeneic CAR-NKT cells for treating renal cell carcinoma

doi: 10.1016/j.xpro.2025.104340

Figure Lengend Snippet: Generation of Allo CAR70-NKT cells from HSPCs using a clinically guided culture method (A and B) Schematics showing the generation of Allo CAR70-NKT cells (A) and the design of Lenti/iNKT-CAR70-IL-15 lentivector (B). (C) FACS detection of NKT TCR expression in lentivector-transduced CD34 + HSPCs. (D) Quantification of (C) (n = 5; n indicates different cord blood donors). (E) FACS monitoring of the generation of Allo CAR70-NKT cells during the 6-week culture. (F) Percentage of Allo CAR70-NKT cells in total live cells during the 6-week culture (n = 5; n indicates different cord blood donors). (G) Yield of Allo CAR70-NKT cells (n = 5; n indicates different cord blood donors). (H) CAR70 expression on Allo CAR70-NKT cells (n = 5; n indicates different cord blood donors). (I) ELISA measurements of IL-15 production by Allo CAR70-NKT cells with or without αGC stimulation (n = 4). (J) CD4/CD8 subpopulations of Allo CAR70-NKT cells. Data generated from 5 cord blood donors are shown. SP, single-positive; DP, double-positive; DN, double-negative. (K–M) Antigen responses of Allo CAR70-NKT cells. Allo CAR70-NKT cells were stimulated with/without αGC-loaded PBMCs for 1 week. (K) Experimental design. (L) Growth curve of Allo CAR70-NKT cells (n = 4). (M) ELISA measurements of effector cytokine levels in the culture supernatants collected on day 5 (n = 4). Representative of over 5 experiments. Data are presented as the mean ± SEM. ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001 by Student’s t test (I and M).

Article Snippet: Human CD34 MicroBeads Kit , Miltenyi Biotec , CAT#130-046-703.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Generated

Journal: Molecular Therapy. Nucleic Acids

Article Title: Complex HBB gene editing outcomes revealed by a fluorescent reporter cell model

doi: 10.1016/j.omtn.2026.102854

Figure Lengend Snippet: CRISPR editing of HBB delays erythroid maturation in HSPCs from SCD patients Sickle-patient-derived CD34 + HSPCs from two donors were edited at HBB using HiFi SpCas9 RNPs with sgRNAs R-66S or R-02, delivered by electroporation ± ssODN donor. (A) HBB exon 1 sequence showing the sickle mutation (red). Protospacers for R-66S and R-02 are shown with PAMs underlined (blue). (B) Allele frequency quantified by NGS and ddPCR. ddPCR measured allelic-drop off as a surrogate for large deletion (LD) resulting from loss of the primer/probe binding site, while NGS quantified HDR and small indels on intact alleles. NGS values were normalized to ddPCR to true allelic fractions across all HBB alleles (intact and drop-off). Efficient editing was achieved with R-66S and R-02 in n = 2 biological replicates (independent donors), each with two technical replicates (independent electroporation). (C) Erythroid maturation assays showed a significant delay after R-66S or R-02 RNP versus mock ( p < 0.0001 and p = 0.0001, respecitvely using a 2-sided Welch t test, evidenced by increased CD36 + /CD235a + immature cells in donor 1 (15-day differentiation) and donor 2 (13-day differentiation). Adding the ssODN donor restored maturation to near-mock levels. (D) In a third donor, edited cells were differentiated for 10 days, sorted into immature and mature fractions, and allele frequencies were measured by LongAmp-seq. Across both gRNAs, frameshift indels were enriched in the immature fraction, whereas in-frame indels and unmodified alleles were enriched in the mature fraction, implicating frameshift indels in the observed maturation delay ( n = 1 experiment).

Article Snippet: CD34+ cells were extracted from the mononuclear fraction by immunomagnetic separation using CD34 Microbeads Kit (Miltenyi Biotech, CD34 MicroBead Kit UltraPure, human) according to the manufacturer’s instructions.

Techniques: CRISPR, Derivative Assay, Electroporation, Sequencing, Mutagenesis, Binding Assay