Figure 6.

Capsid-Independent and Widespread ON-Bipolar Cell-Specific Expression Driven by Promoter 770En_454P(hGRM6) in HREs

(A) Flat mount transduced with scAAV2(7m8)-770En_454P(hGRM6)-mCitrine, with the application site indicated with blue arrowheads pointing toward pipette marks. mCitrine indicates that the drop of viral solution remained locally at the application site and, consequently, accessed the inner nuclear layer (INL) predominantly through the inner limiting membrane. Red arrowhead indicates blood vessel. Scale bar, 500 μm. (B–F) The ON-bipolar cell (OBC) expression efficacy, given in percentages, remained approximately constant throughout the explant (B–E), throughout the inner edge (C), middle (D) and outer edge (E) of the INL, and in explants from different donors (A–E versus F). IPL, inner plexiform layer; OPL, outer plexiform layer. Scale bars: 100 μm in (B) and 50 μm in (C)–(F). Staining in (B)–(F) was done with anti-Gao and anti-mCitrine. (G) Transduction fingerprint shown to be independent of the viral capsid used. AAV2(7m8) (n = 3) and AAV2(Y252,272,444,500,700,730F) (n = 3) produced virtually identical expression profiles with a high OBC specificity of ∼88%. Off-target INL includes OFF-bipolar cells and amacrine cells. GCL, ganglion cell layer; ONL, outer nuclear layer. (H) Somatic fluorescence intensities produced by 770En_454P(hGRM6) quantified in immunolabeled cryosections (n = 3). The fluorescence intensities in rOBCs and cOBCs were significantly higher than the intensities in off-target populations. Statistical analysis by one-way ANOVA with post hoc Tukey HSD test: ∗p < 0.05; ∗∗p < 0.01. Data are represented as mean ± SD of biological replicates.