Fig 1.
Comparisons of ERGs at two time-points following AAV vector treatment.
As a mouse model of achromatopsia, cpfl5 mice show selective loss of cone ERG responses. Scotopic (A) and photopic ERGs (B) were recorded at 1 and 3 months following treatment with AAV8 (Y447, 733F + T494V) (red), AAV2 (Y272, 444, 500, 730F + T491V) (green), or AAV8 (Y447, 733F) (blue). Recovered photopic ERG (cone-mediated) amplitudes were compared among groups (C). (D) Photopic-ERG dose response of the AAV-vector dilutions. The cpfl5 mice were treated in one eye at P14 and evaluated at 1 month after subretinal injection. Age-matched WT and untreated cpfl5 eyes were used as controls (n = 6 mice). P, postnatal day. *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001, NS = no statistical difference.
Fig 2.
Long-term (9 months) electroretinographic assessment of treated cpfl5 eyes.
Cpfl5 eyes were treated with AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3 at P14. An ERG recording was repeated at 9 months following treatment. (A) Scotopic ERG elicited in treated cpfl5 eyes (red), compared to WT and untreated cpfl5 eyes (black). (B) Scotopic b-wave amplitudes elicited at 0 log cd-s/m2 intensity in the age-matched WT, treated, and untreated cpfl5 eyes (n = 6). (C) Photopic ERG elicited in treated cpfl5 eyes (red) compared to WT and untreated cpfl5 eyes (black). (D) Photopic b-wave amplitudes elicited at 1.0 log cd-s/m2 intensity in age-matched WT, treated, and untreated cpfl5 eyes (n = 6). P, postnatal day. **indicates P < 0.01, ***indicates P < 0.001, NS = no statistical difference.
Fig 3.
Immuno-staining of retinal CNGA3 at 3 months post-injection.
CNGA3 expression (red) was detected primarily in the photoreceptor OS following treatment with AAV8 (Y447, 733F + T494V), AAV2 (Y272, 444, 500, 730F + T491V), or AAV8 (Y447, 733F). Age-matched WT and untreated cpfl5 retinas were used as controls. OS, outer segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer.
Fig 4.
Long-term retinal CNGA3 expression mediated by AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3 treatment.
At 9 months following subretinal injection, retinal cryosections were immunostained with anti-CNGA3 antibody. CNGA3 expression (red) was detected primarily in the photoreceptor OS (arrows) in the AAV8 (Y447, 733F + T494V)-treated cpfl5 retinas and age-matched WT controls, but not in the untreated cpfl5 retinas. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue).
Fig 5.
Long-term preservation of retinal M-opsin and CNGA3 after treatment with AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3.
At 9 months after P14 treatment, cpfl5 retinal immunostaining revealed normal expression and the cone OS distribution (merge) of M-opsin, compared with WT controls. In untreated cpfl5 eyes, the inferior retinas had little M-opsin and cone specific PNA staining, whereas the superior retinas showed mislocalization of M-opsin within residue cones (arrows). In WT and treated cpfl5 retinas, M-opsin was located in the cone OS. Red: M-opsin or CNGA3 staining; Green: cone-specific PNA staining; Blue: nuclei staining with DAPI (4′,6-diamidino-2-phenylindole). P, postnatal day.
Fig 6.
Long-term preservation of retinal S-opsin after treatment with AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3.
At 9 months after treatment of P14 mice, cpfl5 retinal immunostaining revealed normal expression (red) and the cone OS distribution (merge) of S-opsin compared with WT controls. S-opsin was not detected in untreated cpfl5 retinas. In WT and treated cpfl5 retinas, S-opsin was located in the cone OS. Red: S-cone opsin staining; Green: cone-specific PNA staining; Blue: nuclei staining with DAPI (4′,6-diamidino-2-phenylindole). P, postnatal day.
Fig 7.
M-cone and S-cone opsins preservation in retinal whole mounts after treatment.
At 3 and 9 months after P14 treatment with AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3, M-cone opsins (red) and S-cone opsins (white) were imaged (A) and counted (B, C) in the same field of ventral nasal retina (n = 6). WT and untreated cpfl5 mice were used as controls. D, dorsal; V, ventral; T, temporal; N, nasal. P, postnatal day; M, months. *indicates P < 0.05, **indicates P < 0.01, ***indicates P < 0.001, NS = no statistical difference.
Fig 8.
Cone-mediated visually guided behavioral test after treatment with AAV8 (Y447, 733F + T494V)-IRBP/GNAT2-Cnga3.
#Statistical analysis indicates a significant difference in performance (P < 0.01) in the WT mice with unilateral eyelid suture compared to untreated cpfl5 mice and the treated cpfl5 mice when the treated eye was sutured. *Statistical analysis indicates a significant difference in performance (P < 0.05) in the treated cpfl5 mice compared to untreated cpfl5 mice and treated cpfl5 mice with the treated eye sutured. No statistical difference in performance was found between the WT mice with unilateral eyelid suture and treated cpfl5 mice or between untreated cpfl5 mice and treated cpfl5 mice when the treated eye was sutured (n = 6).