Figure 4.

Retinal abnormalities and ON-bipolar transduction with AAV8BP2 in a retinal degeneration dog model. (a and b) Representative near infrared reflectance (NIR-REF) en-face images (column a) and horizontal 9-mm-long spectral domain optical coherence tomography (SD-OCT; column b) cross-sections in a representative normal dog around 5 years old (top row) compared with images from the retina nasal (middle row) and temporal (bottom row) to the optic nerve head (ONH) of a 5-year-old crd1 dog. This crd1 dog was injected subretinally in the temporal retina with AAV8BP2-4XGrm6-CatCh-eGFP at a titer of 1E + 11 vg. SD-OCT scans covered approximately 18 mm of retina along a horizontal line crossing just above the ONH (thin green arrow in column a) along the boundary between tapetal and non-tapetal retina; shown are 7-mm-long SD-OCT images cropped to match location of the normal and crd1 dog nasal retinas. The external limiting membrane (ELM) is a thin line that lies just above a hyperreflective retinal pigment epithelium (RPE). Vertical yellow arrows point to regions of transitions to hyper-reflective, lighter regions on NIR-REF images, which co-localized with delaminated thinned retina on SD-OCT. Large regions of well-laminated retina nasal to the ONH (middle row) shows a thinned ONL in the crd1 dog (23 μm) compared with that of the normal dog (86 μm; top row). The crd1 eye also shows patchy depigmentation on NIR-REF images (middle row, to the left of yellow arrow) co-localizing with severe retinal thinning, particularly around the ONH. Photoreceptor degeneration is associated with a relatively thickened inner retina (middle row, INL and retina superficial to it) with epiretinal membrane formation at the surface (bottom row, short diagonal arrow) and intraretinal hypo-reflective images (asterisk) that suggest intraretinal edema. At the transition zone, there can be an elevation of the ELM just before approximating the RPE in areas of transition to total photoreceptor (ONL) loss resembling a stage of outer retinal tubulation (white diagonal arrow). T, temporal; N, nasal retina. Histologic sections (c–e) of the crd1 temporal retina reveals eGFP (green) 1 month post injection. In (c), co-localization of eGFP with ON-bipolar cells was confirmed with immunohistochemistry using anti-Goα antibodies (red). The boxed region in (c) is magnified in (d) with arrows indicating transduced ON-bipolar cells in regions of retinal thinning. Panel (e) is a region of thicker retina with more outer nuclei with eGFP (green), ON-bipolar cells (red) and nuclei (blue) labeled. Scale bars represent 50 μm. Color images available online at www.liebertpub.com/hum