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

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access 23 January 2024 INTRODUCTION Mutations in the Bestrophin 1 gene (_BEST1_, _VMD2_) cause several phenotypically distinct monogenic retinal disorders in man, the prototypical one being

autosomal dominant Best Vitelliform Macular Dystrophy (BVMD) [1]. This form of Best disease has a characteristic macular lesion that progresses from a small elevated fried egg-like

(vitelliform) lesion of the macula to a larger focal lesion that accumulates autofluorescent (AF) lipofuscin pigment, before undergoing the degenerative vitelliruptive and atrophic phases

with loss of central acuity [2, 3]. While there is no question that mutations in _BEST1_ cause retinal disorders, the specific function of the protein, and some of the pathways leading from

mutant gene to disease, are not well understood. BEST1 is proposed as a Ca++-activated chloride channel [4], but this hypothesis has been challenged based on studies in _BEST1_ knockout (KO)

mice that found that Ca++-activated chloride channel currents are not abolished [5]. The problem with basing _BEST1_-mechanistic studies on mice is that the currently available KO mouse

models fail to represent the essential features of the disease. These mice have no ocular phenotype, do not recapitulate the human disease, have no gross visual deficits or pathology, and

have normal Cl- currents in the RPE where the protein is expressed (see ref. [2] for review and ref. [5]). A major limitation of the _BEST1_ KO models is in translational applications, i.e.

going from the cage to the bedside, using gene and other therapies. While it is possible to test therapeutic gene therapy vectors to establish specific expression in the target tissue, and

stability of expression of the therapeutic transgene, it is not possible to determine if treatment prevents disease, or reverses disease once established, or, as well, how effective is long

term correction of the disease following treatment as the KO mice have no retinal disease phenotype. Hence there is a need for a model that recapitulates the essential features of the

patient disease. The canine bestrophinopathy models fill this void and their translational potential are summarized in the following sections. SUBJECTS, RESULTS, DISCUSSION Findings

presented in this study come from examinations carried out in client-owned dogs seen at our clinic, and, primarily, from detailed clinical, structural and functional studies carried out in

purpose bred research dogs maintained at the Retinal Disease Studies Facility of the University of Pennsylvania. Details of those studies, including the breeding, housing, and care of the

dogs have been presented in several prior publications [6,7,8,9,10]. All protocols and studies have been approved by the University of Pennsylvania IACUC. BEST1 DISEASE MODELS AND CLINICAL

PHENOTYPES There are 3 Best disease canine models. Until the mutant gene and breed-specific mutations were identified, these autosomal recessive diseases were referred to as canine

multifocal retinopathy (cmr1-cmr3) [11,12,13]. The first one, cmr1, represents a founder mutation in the English mastiff breed that is common in most large-sized dog breeds that originated

from mastiffs [13]. The other two, cmr2 and 3, are ‘private’ mutations that are limited, respectively, to the Coton de Tulear and Lapponian herder breeds [12, 13]. Although these _Best1_

mutations involve different domains of the protein {involving exons 2 (cmr1), 5 (cmr2) or 10 (cmr3)}, it is somewhat surprising that the resultant retinal phenotypes are similar (Table 1).

Following the clinical staging classification for BVMD in man [3, 14], we have used similar phenotypic characteristics for staging the canine disease (Fig. 1). Retinal abnormalities are not

found on routine ophthalmoscopic examination in the pre-vitelliform stage, although distinct focal separation, i.e. microdetachments, of the apical RPE from the photoreceptor outer segments

is found by OCT (Fig. 1A). A distinct separation of the subretinal space is clearly evident in the vitelliform stage (Fig. 1B) which involves the canine fovea-like region [15] and extra

foveal lesions [8, 12]. Blue autofluorescent (AF) material, presumably lipofuscin, accumulates in the pseudohypopyon stage (Fig. 1C), and this settles to the inferior border of the cystic

appearing foveomacular lesion. In dogs, the fovea-like area and the immediately surrounding area centralis are analogous to the human foveomacular region. This AF material becomes more

dispersed in the vitelliruptive stage with slight thinning of the outer nuclear layer (Fig. 1D). Marked thinning of the outer nuclear layer with disruption of photoreceptor layer and

decreased AF material are present in the atrophic stage (Fig. 2, lower panel-OS). CHARACTERIZATION OF SUBRETINAL LESION; FLUID OR GEL MATRIX? While the early microdetachments appear to be

modulated by light, ie. expanding in the light and contracting in the dark [8, 10], larger vitelliform and pseudohypopyon lesions remain unchanged. Expansion/contraction of lesions with

light would suggest that the subretinal material in the small lesions is liquid and is readily transported into or out of the subretinal space. In contrast, it seems unlikely that the

unchanged larger lesions have a fluid component. Because gene therapy would involve subretinal injections directed at the fovea-like region and surrounding area centralis, we felt important

to characterize this material, at least clinically, as a means of informing therapeutic approaches and interpretation of outcome measures. To this end, we carried sdOCT/cSLO imaging with the

dog placed in the conventional sternal recumbency position, followed by a repeat of the procedure in the same imaging session with the dog in dorsal recumbency. In the sternal position,

autofluorescence imaging of a pseudohypyon lesion showed that the AF material accumulated in the inferior border of the cystic lesion (Fig. 3A1); OCT imaging shows the accumulated material

mainly clustered in the lower quadrant, but also distributed throughout the subretinal space in close association with the photoreceptor layer (Fig. 3A2, 3). Sequential autofluorescence

scans taken after the dog is placed in dorsal recumbency shows a very slow redistribution of the AF material (Fig. 3B1–4); it did not cascade down, as in a snow globe, but ‘trickled’ through

a channel in a more solid gel or matrix. Even after 25 min in dorsal recumbency, the AF material is not fully distributed to the new inferior border of the lesion and continues to flow

through the same channel (Fig. 3C1, 2). STRUCTURAL ABNORMALITIES IN THE RPE-PHOTORECEPTOR INTERFACE Canine bestrophinopathies represent an RPE-photoreceptor interface disease. The

interphotoreceptor space is complex. It is bordered by 4 cellular elements which include the RPE apical microvilli, rod and cone inner/outer segments, and Müller cell fiber baskets. This

space also contains soluble and insoluble components among which, respectively, include, IRBP and the insoluble rod and cone matrix [16, 17]. In mutant dogs, we find abnormal pan-retinal

RPE-photoreceptor interface with an apparent loss of cone-ensheathing RPE apical processes and compromised cone-associated insoluble interphotoreceptor matrix (IPM) [9, 18]. The apical

microvilli of rods are shorter, and the multilayered RPE-cone outer segment sheath [19] is abnormally short, and fails to envelop most of the cone outer segments. Studies in young affected

dogs at the end of postnatal retinal differentiation showed that the RPE rod apical microvilli and the cone sheath failed to develop (ref. [18] and Fig. 4A, B). Furthermore, peanut

agglutinin (PNA) labeling of the insoluble cone extracellular matrix sheath showed that the close association between the outer segment/RPE-cone sheath/PNA cone insoluble matrix was

compromised as the intervening RPE cone sheath was absent [18]. In vitro studies of human induced PSC-RPE carried out in David Gamm’s laboratory showed that autosomal recessive

bestrophinopathy cells had a paucity of rod microvilli, and those present were short [9]. As well, BVMD hiPSC-RPE cells examined by electron microscopy show a reduced number and marked

shortening of the RPE apical microvilli. These studies support the strong phenotypic similarities between the canine and human diseases. BEST1 GENE AUGMENTATION REVERSES DISEASE AND CORRECTS

STRUCTURAL ABNORMALITIES Because of the success of AAV-mediated gene therapy for another primary RPE disease, _RPE65_-LCA [20], we examined if AAV-mediated _BEST1_ gene augmentation would

reverse the ongoing retinal abnormalities, and if expression/overexpression of the therapeutic transgene in the RPE was safe. Initially, we tested the AAV2/2 serotype used in _RPE65_ therapy

studies along with AAV2/1 serotype which in dogs was shown to effectively target RPE cells in the _RPE65_ mutant dogs [21]. In this pilot study, AAV2/1-human VMD2-cBest which was

co-injected with an AAV expressing GFPcaused severe and specific damage to cone cells in the treated area and was not considered further [22]. In contrast, subretinal administration of

_AAV2/2-hVMD2-cBEST_ to the wild type retina resulted in intense BEST1 expression in the RPE. Instead of localizing to the basolateral membranes of the RPE cells as the endogenous protein,

the expressed transgene protein was also was located diffusely through the cytoplasm without any adverse effects to the RPE and neuroretina in a 4-6 week/6 month observation period [22].

Additionally, in the pilot study the _AAV2/2-hVMD2-cBEST_ vector was tested in a heterozygote (cmr1+/−) dog without any adverse effects [22]. These pilot studies led to more formal studies

examining the therapeutic efficacy of gene therapy for Best disease. For the formal proof of concept efficacy studies, subretinal injections of the _AAV2/2-hVMD2_ vector with either the

canine or human _BEST1_ cDNA were carried out as previously detailed [8], and the injections were directed mainly to the fovea-like area and surrounding area centralis region. In all cases,

the surgical bleb flattened within 24–48 hours post injection (p.i.). Untreated control eyes, or those injected with balanced salt solution (BSS) showed progression of the lesions. In

contrast, mutant eyes treated with the therapeutic vector showed reversal of the lesions. Figure 5 shows one such dog injected at 12 months of age at the pseudohypopyon stage of disease. By

2 wks p.i, the large foveo-macular lesion decreased in size and the contents, present in the inferior border, also were diminished. Correction of the lesion was stable over several years as

the retina in the treated area remained normal and attached with no evidence of any subretinal elevations or abnormalities. Only the focal retinotomy scar remained unchanged. In contrast,

the retinal lesion in the untreated fellow eye continued to progress, and by 45 months of age the outer nuclear layer had thinned (atrophic stage) (Fig. 2). The development and progression

of the fovea-like area and surrounding area centralis lesion over time in the untreated OS eye is illustrated in ref. [8], Fig. S4. Following gene therapy, there is correction of the

RPE-photoreceptor interface disease. There is expression of BEST1 protein in the RPE, both cytoplasmic and in basolateral membranes, and formation of new RPE cone outer segment sheaths which

extend from the apical RPE to invest most of the cone outer segments (Fig. 4C1, C2 and E). Accompanying the correction the RPE-photoreceptor interface disease with gene therapy, the retinal

microdetachments resolve [8]. Studies of canine BEST1 disease emphasize its value as a translational model. Even though the canine disease is autosomal recessive, and heterozygous dogs show

no clinically evident phenotype, it is similar to human BVMD in terms of retinal phenotype and disease progression in the fovea-like area, and to autosomal recessive bestrophinopathy in

extra foveal regions. The diseases in both species demonstrate many similarities in studies in which the same assessment methods are used. The dramatic and stable response to subretinal gene

therapy, even in cases with large pseudohypopyon lesions, indicate that administering an injection to this area causes no damage, and lesion reversal occurs without adverse effects. It is

remarkable that the retina in the foveo-macular region, which is already distended because of the lesion, has sufficient elasticity to accommodate a subretinal fluid injection without

tearing. This is an important observation for planned Phase 1/2 clinical trial to be carried out in the near term. At present, the final nonclinical safety/efficacy studies are being

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PLoS One. 2013;8:e75666. Article  CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS Dr. Karina Guziewicz participated in early phases of this study and

organized many of the images included in the manuscript figures. FUNDING Foundation Fighting Blindness Large Animal Model Translational & Research Center grant; NEI/NIH grants EY006855,

EY017549; Van Sloun Fund for Canine Genetic Research; and the Sanford and Susan Greenberg End Blindness Outstanding Achievement Prize (GDA). AUTHOR INFORMATION AUTHORS AND AFFILIATIONS *

Division of Experimental Retinal Therapies, Department of Clinical Sciences, University of Pennsylvania, School of Veterinary Medicine, Philadelphia, PA, 19104, USA Gustavo D. Aguirre & 

William A. Beltran Authors * Gustavo D. Aguirre View author publications You can also search for this author inPubMed Google Scholar * William A. Beltran View author publications You can

also search for this author inPubMed Google Scholar CONTRIBUTIONS G. Aguirre Conceptualization, Formal analysis, Investigation, Writing – original draft, Writing – review & editing,

Supervision. W. Beltran Conceptualization, Formal analysis, Investigation, Writing – review & editing, Supervision. CORRESPONDING AUTHOR Correspondence to Gustavo D. Aguirre. ETHICS

DECLARATIONS COMPETING INTERESTS The University of Pennsylvania has pending patent applications for Best Disease gene therapy and the two co-authors are among the co-inventors listed in the

patent applications; the technology has been licensed to Opus Genetics, Inc. by the University of Pennsylvania. This work was supported by the Foundation Fighting Blindness Large Animal

Model Translational & Research Center grant; NEI/NIH grants EY006855, EY017549; Van Sloun Fund for Canine Genetic Research; and the Sanford and Susan Greenberg End Blindness Outstanding

Achievement Prize (GDA). ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. RIGHTS

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Beltran, W.A. Canine Best disease as a translational model. _Eye_ 39, 412–417 (2025). https://doi.org/10.1038/s41433-024-03578-0 Download citation * Received: 08 November 2024 * Revised: 04

December 2024 * Accepted: 20 December 2024 * Published: 07 January 2025 * Issue Date: February 2025 * DOI: https://doi.org/10.1038/s41433-024-03578-0 SHARE THIS ARTICLE Anyone you share the

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