Canine Retina Has a Primate Fovea-Like Bouquet of Cone Photoreceptors Which Is Affected by Inherited Macular Degenerations

Retinal areas of specialization confer vertebrates with the ability to scrutinize corresponding regions of their visual field with greater resolution. A highly specialized area found in haplorhine primates (including humans) is the fovea centralis which is defined by a high density of cone photoreceptors connected individually to interneurons, and retinal ganglion cells (RGCs) that are offset to form a pit lacking retinal capillaries and inner retinal neurons at its center. In dogs, a local increase in RGC density is found in a topographically comparable retinal area defined as the area centralis. While the canine retina is devoid of a foveal pit, no detailed examination of the photoreceptors within the area centralis has been reported. Using both in vivo and ex vivo imaging, we identified a retinal region with a primate fovea-like cone photoreceptor density but without the excavation of the inner retina. Similar anatomical structure observed in rare human subjects has been named fovea-plana. In addition, dogs with mutations in two different genes, that cause macular degeneration in humans, developed earliest disease at the newly-identified canine fovea-like area. Our results challenge the dogma that within the phylogenetic tree of mammals, haplorhine primates with a fovea are the sole lineage in which the retina has a central bouquet of cones. Furthermore, a predilection for naturally-occurring retinal degenerations to alter this cone-enriched area fills the void for a clinically-relevant animal model of human macular degenerations

Recombinant AAV-Mediated <i>BEST1</i> Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

<div><p>Mutations in the <i>BEST1</i> gene constitute an underlying cause of juvenile macular dystrophies, a group of retinal disorders commonly referred to as bestrophinopathies and usually diagnosed in early childhood or adolescence. The disease primarily affects macular and paramacular regions of the eye leading to major declines in central vision later in life. Currently, there is no cure or surgical management for <i>BEST1</i>-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (<i>cmr</i>), recapitulates a full spectrum of clinical and molecular features observed in human bestrophinopathies and offers a valuable model system for development and testing of therapeutic strategies. In this study, the specificity, efficiency and safety of rAAV-mediated transgene expression driven by the human VMD2 promoter were assessed in wild-type canine retinae. While the subretinal delivery of rAAV2/1 vector serotype was associated with cone damage in the retina when <i>BEST1</i> and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or <i>BEST1</i> transgene under control of human VMD2 promoter was safe, and enabled specific transduction of the RPE cell monolayer that was stable for up to 6 months post injection. These encouraging studies with the rAAV2/2 vector lay the groundwork for development of gene augmentation therapy for human bestrophinopathies.</p></div

Specificity and stability of rAAV-mediated RPE transduction regulated by hVMD2 in the canine retina.

<p>The rAAV2/2 vector construct carrying GFP reporter gene under control of human VMD2 promoter specifically and exclusively target transgene expression to the RPE cells. Native (green) or anti-GFP probed (red) GFP expression was analyzed on frozen retinal cross-sections 2- (9.11×10¹⁰ vg), 4- (1.21×10¹¹ vg), 6- (9.11×10¹⁰ vg) weeks, and 6 months (9.11×10¹⁰ vg) post injection. Immunohistochemical staining confirmed the gradual increase of the transgene expression level over the first 6 weeks p.i. that remained stable up to 6 months after vector administration. DAPI stain was used to detect cell nuclei; vg: vector genomes injected; p.i.: post injection; scale bar: 40 µm.</p

rAAV-mediated <i>BEST1</i> transfer to the retinal pigment epithelium and cone toxicity associated with rAAV2/1 vector serotype. (A-B)

<p>Co-expression of endogenous canine and human Best1 transgene <i>in vivo</i>. Histological and immunohistochemical analysis of wild-type canine retinae injected with rAAV2/1-hVMD2-h<i>BEST1</i> (1.16×10¹² vg) and a spike-in of corresponding vector expressing GFP (1.04×10⁹) <b>(A)</b> and rAAV2/2-hVMD2-h<i>BEST1</i> (8.82×10¹¹ vg) <b>(B)</b> at 4 weeks post injection. No structural abnormalities were seen by H&E staining in any of the samples. In the retina transduced with rAAV2/1 capsid serotype (A), individual photoreceptor cells emitted green autofluorescence (arrowheads) as shown on the photomicrographs probed with anti-Best1, anti-L/M & S opsin and anti-hCAR (red) (A). Cone-specific labeling revealed loss of cone photoreceptors (arrows) only in the areas transduced with rAAV2/1 vector serotype <b>(A)</b>. Co-expression of endogenous canine bestrophin1 and human <i>BEST1</i> transgene was well tolerated when injected with rAAV2/2 serotype and no abnormalities were noted <b>(B)</b>. <b>(C–D)</b> Comparison of rAAV2/1- and rAAV2/2-mediated c<i>BEST1</i> transgene expression in the <i>cmr1</i> (C₇₃T/R₂₅X) carrier retina. Histological and immunohistochemical evaluation of <i>cmr1</i>^+/− retinae injected with rAAV2/1-hVMD2-c<i>BEST1</i> (1.92×10¹¹ vg) and a spike-in of corresponding vector expressing GFP (1.74×10⁹) <b>(C)</b> and rAAV2/2-hVMD2-c<i>BEST1</i> (4.44×10¹¹ vg) <b>(D)</b> at 4 weeks post injection. No morphological abnormalities were detected by H&E staining between the two capsid serotypes <b>(C–D)</b>. Sporadic autofluorescent cells (green) were observed in IS and ONL layers (<b>C</b>, arrowheads), and cone-specific immunolabeling (L/M & S opsin and anti-hCAR in red) revealed focal loss of cone photoreceptors (arrows) within the rAAV2/1-hVMD2-c<i>BEST1</i>-injected area <b>(C)</b>. rAAV2/2-mediated c<i>BEST1</i> transfer to the <i>cmr1</i>^+/− retina results in bestrophin1 overexpression in the RPE (<b>D</b>, Best1 in red) with no adverse effects in the retinal tissue <b>(D)</b>. RPE: retinal pigment epithelium, OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer. Cell nuclei were stained with DAPI; vg: vector genomes injected; scale bar: 40 µm and applies to all panels.</p

Evaluation of rAAV2/1- and rAAV2/2-mediated <i>BEST1</i> transgene expression in the canine retina.

<p>(<b>A</b>) Comparison of two normal canine eyes that received subretinal injection of rAAV2/1 (1.94×10¹¹ vg) and a spike-in of corresponding vector expressing GFP (3.81×10⁹ vg) or rAAV2/2 (3.92×10¹¹ vg) expressing canine <i>BEST1</i> under control of human VMD2 promoter. The outlines of the injected areas detectable in NIR mode and more evident in AF mode for rAAV2/1 (arrowheads) corresponded to the bleb formed immediately after injection (insets). The arrows indicate retinotomy sites. (<b>B</b>) Retinal thickness profiling done by manual segmentation across the bleb boundaries revealed no significant changes 4 weeks p.i. with either vector construct. High-resolution OCT images were obtained using a 30° lens; NIR and AF images were captured using a 55° lens; vg: vector genomes injected; p.i.: post injection.</p

Bestrophin1 overexpression induced by rAAV2/2 in the wild-type canine retina.

<p>Confocal photomicrographs illustrating Best1 expression (red) in the wild-type canine RPE six months p.i. The endogenous expression of Best1 (boxed area left and corresponding magnification) was limited to the basolateral plasma membrane while the transgene protein was also observed in the cell cytoplasm as a result of overexpression mediated by rAAV2/2-hVMD2-c<i>BEST1</i> (3.92×10¹¹ vg) (boxed area right and corresponding magnification). Cell nuclei were stained with DAPI; vg: vector genomes injected; p.i.: post injection; scale bar: 40 µm.</p

Consequences of rAAV2/1- and rAAV2/2-induced <i>BEST1</i> transgene expression <i>in vivo</i>.

<p>Histological and immunohistochemical evaluation of wild-type canine retinae injected with rAAV2/1-hVMD2-c<i>BEST1</i> (2.63×10¹¹ vg) and a spike-in of corresponding vector expressing GFP (2.5×10⁹ vg) or rAAV2/2-hVMD2-c<i>BEST1</i> (4.44×10¹¹ vg) in comparison to the non-injected control. H&E staining did not reveal any histological changes with either vector serotype. Both vectors induced bestrophin1 overexpression in the RPE cells 4 weeks post injection (Best1, red). While no abnormalities were observed in rAAV2/2-transduced retina, the rAAV2/1 serotype caused fluorescence in individual photoreceptor cells (green), occasional mislocalization of cone and rod opsins (arrowheads) and patchy loss of cone photoreceptors (arrows) in the rAAV2/1-hVMD2-c<i>BEST1</i>-injected area. RPE: retinal pigment epithelium, OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer. Cell nuclei were stained with DAPI; vg: vector genomes injected; scale bar: 40 µm and applies to all panels.</p

Comparison of rAAV2/1- and rAAV2/2-mediated GFP expression in the wild-type canine retina.

<p>Immunohistochemical assessment of rAAV2/1-hVMD2-GFP (2.63×10¹¹ vg) (<b>A</b>) and rAAV2/2-hVMD2-GFP (9.11×10¹⁰ vg) (<b>B</b>) injected retinas 6 weeks post injection. GFP expression (native expression  =  green; anti-GFP antibody  =  red) is shown only in the first row of images; selected retinal and RPE proteins were evaluated by antibody labeling. RPE cells expressed Best1 and RPE65 proteins; the structure of cone photoreceptors was demonstrated by hCAR and L/M & S opsin labeling, while rods were assessed based on Rho localization. In all cases, protein expression was normal, specific and comparable to the non-injected eyes (data not shown), irrespective of the recombinant vector serotype used. Preservation of the retinal structure is demonstrated by H&E. RPE: retinal pigment epithelium, OS: photoreceptor outer segments; IS: photoreceptor inner segments; ONL: outer nuclear layer; INL: inner nuclear layer; cell nuclei were stained with DAPI; vg: vector genomes injected; scale bar: 40 µm.</p

Photoreceptor layer lamination in wildtype dogs and in naturally-occuring genetic diseases primarily affecting the canine fovea-like area.

<p>(a,d,f) <i>En face</i> infrared view of representative wildtype (a), <i>BEST1</i>-mutant (d) and <i>RPGR</i>-mutant (f) dogs. *, fovea-like area. Arrows, locations of cross-sectional OCT scans shown below each panel. Outer photoreceptor nuclear layer (ONL) and retinal pigment epithelium (RPE) are highlighted for visibility on OCT scans. (b) ONL thickness topography in a 22-wk-old wildtype dog displayed in pseudo-color. There is a distinct localized region of ONL thinning supero-temporal (ST) to the optic nerve head (black circle) corresponding to the fovea-like area. Major blood vessels are overlaid. (c) Diagonal profiles of ONL thickness (along arrow shown in b) in individual wildtype dogs (lines; ages: 7 wks –8 yrs; n = 13). 95% confidence interval shown (gray area). The break in the axis corresponds to the optic nerve head which lacks photoreceptors. F, fovea-like area. (e) Topographic localization of the sites (*) of the early macular lesions in <i>BEST1</i>-mutant dogs (ages: 10–62 wks; n = 7, left) correspond to the localization of the fovea-like area in wildtype dogs (ages: 7 wks –8 yrs; n = 13, right). (g) ONL thickness topography in an 11-wk-old <i>RPGR</i>-mutant dog displayed in pseudo-color. *, fovea-like area. (h) Diagonal profiles of ONL thickness in young <i>RPGR</i>-mutant dogs (lines; age: 11 wks; n = 6 eyes of 3 dogs) shows abnormal thinning corresponding to the fovea-like area and its immediate surrounds compared to WT (gray area). All eyes are shown as left eyes (temporal retina to the right).</p

High density of cone photoreceptors at the wildtype canine fovea-like area.

<p>(a,b) Central areas of specialization (white) are avoided by retinal blood vessels in human and canine eyes. (c) Retinal ganglion cell (RGC) density map across the canine retina and peak density at the center of the area of specialization. Brn3a: brain-specific homeobox/POU domain protein 3A (d) Retinal cross-section (H&E stained) through the fovea-like area of a 6 week-old dog shows a focal elevation on the retinal surface, thickening of the ganglion cell layer (GCL), and thinning of the outer nuclear layer (ONL). Immunohistochemistry at 7 weeks shows focal high density of cones (red), markedly reduced density of rods (green), elongated inner segments (IS), outer segments (OS) and multiple layers of RGCs in GCL. CA: cone arrestin; Rho: Rhodopsin. ONL is stained with DAPI (blue). (e) Abrupt increase of cone density associated with an abrupt decrease of rod density in 4 eyes (at different postnatal ages for later comparison with mutant dogs). Rod and cone nuclei are highlighted in enlarged insets with blue and yellow, respectively, for visibility. (f) Two-photon microscopy imaging of the fovea-like area and immediate surrounding region. <i>En face</i> view (XY) is an overlay of two Z scans taken at different depths (shown as dotted lines on the orthogonal XZ view) to illustrate the cone IS densities at the fovea-like area and surrounding regions. Insets illustrate the abrupt increase in central peak cone IS density within the canine fovea-like area. vWF: von Willebrand factor VIII; (g) Comparison of peak cone densities in dogs to that reported for macaques and humans measured by adaptive optics imaging, or histology Filled symbols are mean±sd.</p