Canine Best disease as a translational model

Abstract

In this review, we summarize the findings of several pre-clinical studies in the canine BEST1 disease model. To this end, client-owned and purpose bred dogs that were compound heterozygotes or homozygotes, respectively, for two or one of 3 different mutations in BEST1 were evaluated by ophthalmic examination, cSLO/sdOCT imaging, and retinal immunohistochemistry to characterize the clinical and microanatomic features of the disease. Subsequently AAV-mediated gene therapy was done to transfer the BEST1 transgene to the RPE under control of a hVMD2 promoter. We demonstrated that canine bestrophinopathies are an RPE-photoreceptor interface disease with underdeveloped RPE apical microvilli that invest rod and cone outer segments. This leads to microdetachments which later progress to clinically evident RPE-retinal separation and a spectrum of disease stages, ranging from vitelliform to vitelliruptive/atrophic lesions, similar to Best Vitelliform Macular Dystrophy (BVMD). Gene therapy corrects the microdetachments and reverses large lesions when delivered at the pseudohypopyon stage of disease. Because of the similar clinical and microstructural abnormalities between the canine model and BVMD, and positive response to gene therapy, the canine model is a valuable translational model for developing gene and other therapies for BVMD.

Fig. 1. Clinical stages of canine BEST1 disease.

A Pre-vitelliform stage (Stage I). Near infrared (NIR) fundus image by cSLO (left), and single OCT b-scan showing a very small RPE-retinal separation (white arrow) not visible on fundus examination. B Vitelliform stage (Stage II). Near infrared (NIR) fundus image by cSLO (left), and single OCT b-scan showing a distinct RPE-retinal separation with debris accumulating over the photoreceptors. C Pseudohypopyon stage (Stage III). Fundus autofluorescence (FAF) with blue light image by cSLO shows expansion of the lesion and autofluorescent material settling to the inferior border of the large retinal lesion, and single OCT b-scan showing the debris material that is intermeshed with the photoreceptor layer. D Vitelliruptive stage (Stage IV). Fundus autofluorescence (FAF) with blue light image by cSLO shows that autofluorecent material is now redistributed and irregular, and single OCT b-scan shows a reduction of the debris material, and slight thinning of the ONL.

Fig. 2. Treatment outcome following subretinal gene therapy with AAV2/2-hVMD2-cBEST1.

OD. Eye treated at 12 months of age examined 33 months post injection. Near infrared (NIR) imaging shows a focal retinotomy scar (white arrow) and no evidence of the pseudohypopyon lesion present following treatment. OS. Fundus photographs (12 and 15 months of age), and fundus autofluorescence images (22 and 45 months) of the untreated fellow eye. By 45 months, of age the vitelliruptive stage has changed to atrophic stage (Stage V). At this stage, the outer nuclear layer has been markedly reduced in thickness in the fovea-like area and its immediate surrounding area centralis region.

Fig. 3. cSLO and sdOCT imaging done in the conventional imaging position (sternal) followed by rotating the dog to a dorsal position and repeating the scan.

A_1–3 Blue autofluorescent imaging (A₁) shows autofluorescent material settled at the inferior border of the pseudohypopyon lesion. The single OCT b-scan shows that the debris material accumulates at the site of the autofluorescent material, and also covers the photoreceptor layer (A_2-3). B_1–4, C_{1, 2} When rotated to a dorsal scanning position, the autofluorescent material slowly redistributes through a funnel-shaped channel to the new inferior portion of the lesion. This process is slow and incomplete during the 25 min of scanning. This redistribution is observed in the single OCT b-scan image (C₂).

Fig. 4. Abnormal RPE-photoreceptor interface in mutants is corrected by gene therapy.

Immunohistochemistry (IHC) of the wild-type (A) and mutant (B, cmr1) RPE layer labeled with antibodies against BEST1 (red) and monocarboxylate transporter SLC16A1 (green). In the normal retina, BEST1 clearly is localized basally, and SLC16A1 is labeled throughout the RPE, and is especially prominent in the finger-like projections of the RPE-cone outer segment sheath. The mutant retina lacks BEST1 labeling, and the RPE appears to be disorganized and has no apical SLC16A1 labeled extensions. This is seen at higher magnification in an untreated area of a treated eye (D). C_1,2 Following gene therapy, BEST1 is now expressed in the RPE cells, and Ezrin, another marker for RPE cells and the finger-like projections of the RPE-cone outer segment sheath, are clearly seen. E. Treated region of cmr1 mutant shows BEST1 expression and intense labeling of RPE cells and RPE-cone outer segment sheath with SLC16A1 antibody. These projections invest the cone outer segments for most of their length.

Fig. 5. Fundus photographs showing a pseudohypopyon lesion in the area centralis of a dog at 1 year of age before injection, and at several time points following treatment.

By 2 weeks post injection (p.i.), the retinal lesion is markedly decreased in size and elevation, and is flattened by 12 weeks p.i. The retina remains normal, and the retinotomy scar (white arrow) remains unchanged over an extended p.i. observation period.