Fig. 6: In vivo delivery and activation of FUS-CRISPR enables synNotch CAR-T cell therapy.
From: Ultrasound Control of Genomic Regulatory Toolboxes for Cancer Immunotherapy
a Schematic illustration of FUS-CRISPR-mediated synNotch CAR-T activation. Priming of synNotch CAR-T cells by FUS-CRISPR-induced tCD19 enables the killing of PSMA + PC3 cells. b Principle of FUS-CRISPR-mediated tCD19 expression. The tCD19 gene is split by tandem repeated sequences flanking a Cas9 cutting site, which can be recombined into functional tCD19 after Cas9 cutting and single-strand annealing (SSA). c FUS-CRISPR-mediated tCD19 expression in PC3 cells quantified by anti-CD19 antibody staining. N = 3 biological repeats. d Cell death (%) of PC3 cells in (c) without (w/o T) or with (w/ T) co-culture with αCD19-synNotch PSMACAR-T cells. P = 2.85 × 10−5 for w/ T. N = 3 technical replicates representative of two independent experiments. e Timeline of in vivo experiment in NSG mice. f BLI images showing tumor aggressiveness. g Tumor aggressiveness quantified by the total flux of the tumor from BLI measurement. P = 1.49 × 10−6 at 29, 1.14 × 10−11 at 32. N = 5 mice. h Survival curves of the tumor-bearing mice. N = 5 mice. i Immunofluorescence images of the tumor sections. GFP: PC3 cells; mCherry: synNotch CAR-T cells. j Quantification of CAR-T cell percentage in the tumors on Day 28 via flow cytometry. N = 4 mice. In (c, d), CT: without HS; HS: with 15 min HS. FUS-: no FUS treatment. FUS + 10 min FUS stimulation. Error bars and error bands represent s.e.m. Two-tailed unpaired t test was used in (c). Two-way ANOVA followed by Sidak’s multiple comparisons test was used in (d, g). Log-rank (Mantel-Cox) test was used in (h). Two-tailed paired t test was used in (j). Source data are provided as a Source Data file. Created in BioRender. Liu, L. (2024) https://BioRender.com/o33v949.
