Using linkers to increase PHP.B peptide flexibility does not enable Ly6a binding in AAV1-PHP.B and removes Ly6a binding in AAV9-PHP.B. (A and B) AAV1-PHP.B linker variants (A) or AAV9-PHP.B linker variants (B) were tested alongside WT control vectors in HEK293 and HEK293-Ly6a cells. All vectors packaged a CMV-ffLuc reporter gene cassette. Results show transduction efficiency normalized to the WT capsid transduction efficiency for each cell type. Transduction efficiencies for AAV1, AAV1-PHP.B, AAV9, and AAV9-PHP.B are included as reported in Fig. 1B for comparison. Horizontal bars represent group averages; SDs are reported as error bars; n = 3. Groups were compared using ANOVA followed by post hoc two-sample, two-sided t tests (version R.4.0.0) for specific pairwise comparisons. The # symbols represent statistical significance for comparisons between Ly6a (−/+) cell lines; the * symbols represent statistical significance for comparisons between engineered vectors and WT vectors in a given cell type: #/*, P ≤ 0.05; ##/**, P ≤ 0.01; ###/***, P ≤ 0.001; ns, not significant. NL, N-terminal linker; CL, C-terminal linker; WT, wild type.