Fig 1: Designed AAV_ITGs were well-produced and improved transduction via αVβ6 binding.A AAV titers of different AAV variants in bulked small-scale production in suspension 3-day post-triple-transfection (2 ml production, 6 biological replicates, one-way ANOVA followed by FDR correction). B Western blot of VP proteins from purified AAVs showed similar VP ratios for designed AAV_ITGs capsids compared to AAV9 and AAV9rh74, suggesting successful capsid assembly. C, D VCN (C) and luciferase activity (D) of 293_αVβ6 after AAV infection (3 and 4 biological replicates, one-way ANOVA followed by FDR correction). Both the two designed AAV_ITGs showed enhanced VCN and luciferase activities compared to AAV9rh74 and AAV9. E Inhibition of cell entry of designed AAV_ITGs, but not for AAV9 or AAV9rh74, in 293_αVβ6 cells by αVβ6 recombinant protein. AAVs were preincubated with human αVβ6 recombinant protein (r.ITGAV-B6) for 30 min at 37 °C before infection (4 biological replicates, two-way ANOVA followed by FDR correction, 1 µg protein per 5E9vg AAV, 2E5 vg per cell). The same condition treated with recombinant SGCA protein (r.SGCA) was used as the control. F–J Enhanced transduction of AAV_ITGs in in vitro human differentiated myotubes, but not in myoblasts. F Representative images of the GFP signal of myotubes 48 h post-infection (scale bar: 400 µm). G–J VCN and luciferase activities of AAV_ITGs in comparison with AAV9 and AAV9rh74 in myoblasts (G, I, 3 and 3/4 biological replicates, respectively) and myotubes (H, J, 3 and 3/4/6 biological replicates, respectively) (one-way ANOVA followed by FDR correction). Data are presented as mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.
Fig 2: LICA1 showed conserved interaction with αVβ6 from multiple species.A Structure of human αVβ6 binding to human TGF-β3-derived motif (pdb code: 4um9). The binding interface, defined as all amino acid in αVβ6 with distance to the binding motif of < 8 Å, is highlighted. B Aligment of ITGAV (upper panel) and ITGB6 (lower panel) protein sequence around the binding interface from multiple species. The binding interface defined in A is highlighted, and is identical across species being examined. Amino acids with distance to the binding motif of < 6 Å, are in bold and boxed. The sequence mismatches, only found outside the binding interface, are colored in red. C Transduction efficiency, measured by luciferase activity, of AAV_ITGs but not AAV9 was inhibited by pre-incubating AAVs before infection with recombinant αVβ6 protein from both human, rat, and mouse. 2E8vg AAVs were incubated with different αVβ6 concentrations (0–120 nM) for 1 h at 37 °C before added directly into cell medium (dose: 1E4 vg/cell, 96-well plate, duration: 24 h, cell line: 293_WT, n = 3 biological replicates). Same incubation conditions using 120 nM of recombinant SGCA protein were used as the control, which showed no significant difference with 0 nM αVβ6 condition. Data are presented as mean ± SEM. The statistics were performed to compare with the condition of no αVβ6 protein during incubation (0 nM αVβ6) by using two-way ANOVA (~AAV serotypes * αVβ6 treatments) followed by FDR correction. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; ns not significant. Source data are provided as a Source Data file.
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