Fig. 2: T cell-evolved AAV capsid and CD3-targeted EDVs improve delivery specificity.
From: In vivo site-specific engineering to reprogram T cells
a, Schematic of AAV6 capsid library evolution through three selection cycles on activated human T cells cultured in human serum; parental and evolved libraries were analysed by NGS. b, NGS analysis of parental versus evolved libraries. Each variant is plotted by the fold enrichment in the evolved library versus parental (y axis) and the percentage of total evolved reads (x axis). Variants with >100-fold enrichment and >0.1% reads are highlighted. c, Sequence logo of the conserved 7-mer motif among top variants from b. d, Activated human T cells were electroporated with TRAC-targeting RNPs and treated with AAV6 or AAV-hT7 delivering TRAC CAR HDRT, in serum-free or human-serum-supplemented medium. CAR expression was measured using flow cytometry. Values are normalized to serum-free conditions within each MOI. Data are mean ± s.e.m. n = 3 donors. Statistical analysis was performed using multiple two-tailed unpaired t-tests with Holm–Šidák correction. e, Genome-wide KO screen for AAV-hT7 transduction, displaying cell surface genes. Correlation was assessed using the Spearman test with Benjamini–Hochberg-adjusted P values. FC, fold change. f,g, Activated T cells were electroporated with RNPs targeting KIAA0319L, SLC35B2 or CD7, reactivated for 48 h, then transduced with AAV6 or AAV-hT7 encoding sc-CAG-GFP. The GFP mean fluorescence intensity (MFI) was measured using flow cytometry 48 h later and normalized to the untreated controls. f, Relative GFP MFI data are mean ± s.e.m.; n = 2 donors. g, Representative flow cytometry plots. h, Schematic of EDVs pseudotyped with WT VSVG (yellow) or mutated VSVG plus anti-CD3 scFv (anti-CD3, red). i,j, Cells were treated with VSVG-WT or anti-CD3 EDVs (Cas9–sgCLTA) and AAV6 or AAV-hT7 (CLTA exon 1 sfGFP HDRT) at 3 × 105 sgRNA per cell and 5 × 105 vg per cell. GFP indicates correct integration and was assessed using flow cytometry ≥72 h after treatment; integration was confirmed by dPCR. i, Schematic. For j, n = 3 (primary T cells), n = 2 (NK cells), n = 3 (CD34+ HSCs), n = 3 (macrophages) and n = 1 (four B cell lines). The heat map shows the relative knock-in normalized to VSVG-WT + AAV6 for which all cell types were positive for GFP signal (plotted above).
