Fig 1.
Self-complementarity increased transduction efficiency of AAV2-mediated GFP expression in the organ of Corti.
(A) A representative image from a neonatal cochlear explant treated with ssAAV2-CMV-GFP (SS-Ctrl) with GFP shown in green and parvalbumin (PVALB) shown in magenta. (B) A representative image from a neonatal cochlear explant treated with scAAV2-CMV-GFP which shows a large number of cells in the organ of Corti as being GFP positive. (C–E) Show images from explants treated with ssAAV2-CMV-GFP virions that were either packaged with Lipofectamine 2000 reagent (C) or pretreated with EERI (D) or Tyr23 (E). Post-hoc comparisons of each treatment group to the control group showed a significant effect of self-complementarity (F) on total numbers of GFP positive cells in the organ of Corti, and similarly a significant effect of self-complementary AAV on the numbers of GFP positive hair cells specifically (G). Error bars represent ±1 SEM, *** denotes p < 0.001, and all scale bars = 20 μm.
Fig 2.
Self-complementarity increased transduction efficiency of AAV1-mediated GFP expression in the organ of Corti.
(A) A representative image from a neonatal cochlear explant treated with ssAAV1_CMV_GFP (SS-Ctrl) with GFP shown in green and parvalbumin (PVALB) shown in magenta. (B) A representative image from a neonatal cochlear explant treated with scAAV1-CMV-GFP. (C–E) Show images from explants treated with ssAAV1-CMV-GFP virions that were either packaged with Lipofectamine 2000 reagent (C) or pretreated with EERI (D) or Tyr23 (E). Post-hoc comparisons of each treatment group to the control showed a significant effect of self-complementarity (F) on total numbers of GFP positive cells in the organ of Corti, but no effect of any treatment condition on the numbers of GFP positive hair cells (G). Error bars represent 1 SEM, * denotes p < 0.05, and all scale bars = 20 μm.
Fig 3.
Self-complementary AAV2 transduces more cochlear cells than single stranded vectors following inner ear injections in vivo.
(A, A’) Numerous cells in the cochlear duct, including supporting cells and MYO7A positive hair cells (magenta) express GFP (green) following semicircular canal injection of a self-complementary AAV2 vector (scAAV2-GFP). (B, B’) In contrast, injection of a single stranded AAV2 vector (ssAAV2-GFP) at the same dose (6.6 x 108 vg) resulted in only a small number of cells expressing GFP. (C) Treatment with scAAV2-GFP (black dots) results in significantly more GFP positive inner hair cells and more GFP positive outer hair cells in both the apical and basal turns of the cochlea as compared to ears treated with ssAAV2-GFP (red dots). (D) Treatment with scAAV2-GFP (black dots) results in significantly more GFP positive inner phalangeal cells (IPhC) and significantly more GFP positive pillar and Deiters cells (PCDC) in both the apical and basal turns of the cochlea as compared to ears treated with ssAAV2-GFP (red dots). Scale bars represent 50 μm. Asterisks indicate significance of corrected p values: *p < 0.05, **p < 0.01.
Fig 4.
Self-complementary AAV vectors administered via round window membrane injection demonstrate robust GFP expression in vivo.
(A-D) Representative confocal images of the organ of Corti 1–2 weeks after neonatal injection with self-complimentary vectors encoding eGFP driven by a CBh promoter packaged into AAV serotype capsids 1 (A), 8 (B), or 9 (C). Merged images are separated into their individual channels with AAV vector derived eGFP in the middle column (green) and Alexa-546 conjugated phalloidin in the right column (magenta). Mice were injected with 1μl of vector via the round window membrane at age P0-P3 with a total dose of 2–4 x 109 viral genomes per ear. Scale bar = 100 μm.
Table 1.
Table 2.