Recombinant AAV-Mediated \u3cem\u3eBEST1\u3c/em\u3e Transfer to the Retinal Pigment Epithelium: Analysis of Serotype-Dependent Retinal Effects

Mutations in the BEST1 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 BEST1-associated disorders. The recently characterized human disease counterpart, canine multifocal retinopathy (cmr), 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 BEST1 and GFP were co-expressed, the rAAV2/2 vector serotype carrying either GFP reporter or BEST1 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

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

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

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

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

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

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