Photoreceptor function and structure in retinal degenerations caused by biallelic BEST1 mutations.

The only approved retinal gene therapy is for biallelic RPE65 mutations which cause a recessive retinopathy with a primary molecular defect located at the retinal pigment epithelium (RPE). For a distinct recessive RPE disease caused by biallelic BEST1 mutations, a pre-clinical proof-of-concept for gene therapy has been demonstrated in canine eyes. The current study was undertaken to consider potential outcome measures for a BEST1 clinical trial in patients demonstrating a classic autosomal recessive bestrophinopathy (ARB) phenotype. Spatial distribution of retinal structure showed a wide expanse of abnormalities including large intraretinal cysts, shallow serous retinal detachments, abnormalities of inner and outer segments, and an unusual prominence of the external limiting membrane. Surrounding the central macula extending from 7 to 30 deg eccentricity, outer nuclear layer was thicker than expected from a cone only retina and implied survival of many rod photoreceptors. Co-localized however, were large losses of rod sensitivity despite preserved cone sensitivities. The dissociation of rod function from rod structure observed, supports a large treatment potential in the paramacular region for biallelic bestrophinopathies.

Photoreceptor Function and Structure in Autosomal Dominant Vitelliform Macular Dystrophy Caused by BEST1 Mutations.

Purpose: The purpose of this study was to evaluate rod and cone function and outer retinal structure within macular lesions, and surrounding extralesional areas of patients with autosomal dominant Best vitelliform macular dystrophy caused by BEST1 mutations. Methods: Seventeen patients from seven families were examined with dark- and light-adapted chromatic perimetry and optical coherence tomography. Subsets of patients had long-term follow-up (14-22 years, n = 6) and dark-adaptation kinetics measured (n = 5). Results: Within central lesions with large serous retinal detachments, rod sensitivity was severely reduced but visual acuity and cone sensitivity were relatively retained. In surrounding extralesional areas, there was a mild but detectable widening of the subretinal space in some patients and some retinal areas. Available evidence was consistent with subretinal widening causing slower dark-adaptation kinetics. Over long-term follow-up, some eyes showed formation of de novo satellite lesions at retinal locations that years previously demonstrated subretinal widening. A subclinical abnormality consisting of a retina-wide mild thickening of the outer nuclear layer was evident in many patients and thickening increased in the subset of patients with long-term follow-up. Conclusions: Outcome measures for future clinical trials should include evaluations of rod sensitivity within central lesions and quantitative measures of outer retinal structure in normal-appearing regions surrounding the lesions.

Underdeveloped RPE Apical Domain Underlies Lesion Formation in Canine Bestrophinopathies.

Canine bestrophinopathy (cBest) is an important translational model for BEST1-associated maculopathies in man that recapitulates the broad spectrum of clinical and molecular disease aspects observed in patients. Both human and canine bestrophinopathies are characterized by focal to multifocal separations of the retina from the RPE. The lesions can be macular or extramacular, and the specific pathomechanism leading to formation of these lesions remains unclear. We used the naturally occurring canine BEST1 model to examine factors that underlie formation of vitelliform lesions and addressed the susceptibility of the macula to its primary detachment in BEST1-linked maculopathies.

BEST1 gene therapy corrects a diffuse retina-wide microdetachment modulated by light exposure.

Mutations in the BEST1 gene cause detachment of the retina and degeneration of photoreceptor (PR) cells due to a primary channelopathy in the neighboring retinal pigment epithelium (RPE) cells. The pathophysiology of the interaction between RPE and PR cells preceding the formation of retinal detachment remains not well-understood. Our studies of molecular pathology in the canine BEST1 disease model revealed retina-wide abnormalities at the RPE-PR interface associated with defects in the RPE microvillar ensheathment and a cone PR-associated insoluble interphotoreceptor matrix. In vivo imaging demonstrated a retina-wide RPE-PR microdetachment, which contracted with dark adaptation and expanded upon exposure to a moderate intensity of light. Subretinal BEST1 gene augmentation therapy using adeno-associated virus 2 reversed not only clinically detectable subretinal lesions but also the diffuse microdetachments. Immunohistochemical analyses showed correction of the structural alterations at the RPE-PR interface in areas with BEST1 transgene expression. Successful treatment effects were demonstrated in three different canine BEST1 genotypes with vector titers in the 0.1-to-5E11 vector genomes per mL range. Patients with biallelic BEST1 mutations exhibited large regions of retinal lamination defects, severe PR sensitivity loss, and slowing of the retinoid cycle. Human translation of canine BEST1 gene therapy success in reversal of macro- and microdetachments through restoration of cytoarchitecture at the RPE-PR interface has promise to result in improved visual function and prevent disease progression in patients affected with bestrophinopathies.

Bestrophin 1 and retinal disease.

Mutations in the gene BEST1 are causally associated with as many as five clinically distinct retinal degenerative diseases, which are collectively referred to as the "bestrophinopathies". These five associated diseases are: Best vitelliform macular dystrophy, autosomal recessive bestrophinopathy, adult-onset vitelliform macular dystrophy, autosomal dominant vitreoretinochoroidopathy, and retinitis pigmentosa. The most common of these is Best vitelliform macular dystrophy. Bestrophin 1 (Best1), the protein encoded by the gene BEST1, has been the subject of a great deal of research since it was first identified nearly two decades ago. Today we know that Best1 functions as both a pentameric anion channel and a regulator of intracellular Ca2+ signaling. Best1 is an integral membrane protein which, within the eye, is uniquely expressed in the retinal pigment epithelium where it predominantly localizes to the basolateral plasma membrane. Within the brain, Best1 expression has been documented in both glial cells and astrocytes where it functions in both tonic GABA release and glutamate transport. The crystal structure of Best1 has revealed critical information about how Best1 functions as an ion channel and how Ca2+ regulates that function. Studies using animal models have led to critical insights into the physiological roles of Best1 and advances in stem cell technology have allowed for the development of patient-derived, "disease in a dish" models. In this article we review our knowledge of Best1 and discuss prospects for near-term clinical trials to test therapies for the bestrophinopathies, a currently incurable and untreatable set of diseases.

Bestrophinopathy: An RPE-photoreceptor interface disease.

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment.

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.

Pharmacological Modulation of Photoreceptor Outer Segment Degradation in a Human iPS Cell Model of Inherited Macular Degeneration.

Degradation of photoreceptor outer segments (POS) by retinal pigment epithelium (RPE) is essential for vision, and studies have implicated altered POS processing in the pathogenesis of some retinal degenerative diseases. Consistent with this concept, a recently established hiPSC-RPE model of inherited macular degeneration, Best disease (BD), displayed reduced rates of POS breakdown. Herein we utilized this model to determine (i) if disturbances in protein degradation pathways are associated with delayed POS digestion and (ii) whether such defect(s) can be pharmacologically targeted. We found that BD hiPSC-RPE cultures possessed increased protein oxidation, decreased free-ubiquitin levels, and altered rates of exosome secretion, consistent with altered POS processing. Application of valproic acid (VPA) with or without rapamycin increased rates of POS degradation in our model, whereas application of bafilomycin-A1 decreased such rates. Importantly, the negative effect of bafilomycin-A1 could be fully reversed by VPA. The utility of hiPSC-RPE for VPA testing was further evident following examination of its efficacy and metabolism in a complementary canine disease model. Our findings suggest that disturbances in protein degradation pathways contribute to the POS processing defect observed in BD hiPSC-RPE, which can be manipulated pharmacologically. These results have therapeutic implications for BD and perhaps other maculopathies.

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.

Altered miRNA expression in canine retinas during normal development and in models of retinal degeneration.

BACKGROUND: Although more than 246 loci/genes are associated with inherited retinal diseases, the mechanistic events that link genetic mutations to photoreceptor cell death are poorly understood. miRNAs play a relevant role during retinal development and disease. Thus, as a first step in characterizing miRNA involvement during disease expression and progression, we examined miRNAs expression changes in normal retinal development and in four canine models of retinal degenerative disease. RESULTS: The initial microarray analysis showed that 50 miRNAs were differentially expressed (DE) early (3 vs. 7 wks) in normal retina development, while only 2 were DE between 7 and 16 wks, when the dog retina is fully mature. miRNA expression profiles were similar between dogs affected with xlpra2, an early-onset retinal disease caused by a microdeletion in RPGRORF15, and normal dogs early in development (3 wks) and at the peak of photoreceptor death (7 wks), when only 2 miRNAs were DE. However, the expression varied much more markedly during the chronic cell death stage at 16 wks (118 up-/55 down-regulated miRNAs). Functional analyses indicated that these DE miRNAs are associated with an increased inflammatory response, as well as cell death/survival. qRT-PCR of selected apoptosis-related miRNAs ("apoptomirs") confirmed the microarray results in xlpra2, and extended the analysis to the early-onset retinal diseases rcd1 (PDE6B-mutation) and erd (STK38L-mutation), as well as the slowly progressing prcd (PRCD-mutation). The results showed up-regulation of anti-apoptotic (miR-9, -19a, -20, -21, -29b, -146a, -155, -221) and down-regulation of pro-apoptotic (miR-122, -129) apoptomirs in the early-onset diseases and, with few exceptions, also in the prcd-mutants. CONCLUSIONS: Our results suggest that apoptomirs might be expressed by diseased retinas in an attempt to counteract the degenerative process. The pattern of expression in diseased retinas mirrored the morphology and cell death kinetics previously described for these diseases. This study suggests that common miRNA regulatory mechanisms may be involved in retinal degeneration processes and provides attractive opportunities for the development of novel miRNA-based therapies to delay the progression of the degenerative process.

Recombinant AAV-mediated BEST1 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.

Canine multifocal retinopathy in the Australian Shepherd: a case report.

A 1-year-old Australian Shepherd (AS) was presented for a routine hereditary eye examination. During the examination multiple raised, brown to orange lesions were noted in the fundus, which could not be attributed to a known retinal disease in this breed. As they clinically most closely resembled canine multifocal retinopathy (cmr) and no indication of an acquired condition was found, genetic tests for BEST1 gene mutations were performed. These showed the dog to be homozygous for the cmr1 (C73T/R25X) gene defect. Furthermore, ultrasound (US), electroretinography (ERG), and optical coherence tomography were performed, confirming changes typical for cmr. Subsequently, the AS pedigree members were genetically and clinically tested, demonstrating autosomal recessive inheritance with no clinical symptoms in carrier animals, as was previously described for cmr. To our knowledge, this is the first reported case of canine multifocal retinopathy in the AS breed. Further investigations are under way.

Molecular consequences of BEST1 gene mutations in canine multifocal retinopathy predict functional implications for human bestrophinopathies.

PURPOSE: Bestrophin-1 gene (BEST1) mutations are responsible for a broad spectrum of human retinal phenotypes, jointly called bestrophinopathies. Canine multifocal retinopathy (cmr), caused by mutations in the dog gene ortholog, shares numerous phenotypic features with human BEST1-associated disorders. The purpose of this study was the assessment of molecular consequences and pathogenic outcomes of the cmr1 (C(73)T/R(25)X) premature termination and the cmr2 (G(482)A/G(161)D) missense mutation of the canine model compared with the C(87)G/Y(29)X mutation observed in human patients. METHODS: Dogs carrying the BEST1 mutation were introduced into a breeding colony and used to produce either carrier or affected offspring. Eyes were collected immediately after euthanatization at the disease-relevant ages and were harvested for expression studies. In parallel, an in vitro cell culture model system was developed and compared with in vivo RESULTS: The results demonstrate that cmr1 and human C(87)G-mutated transcripts bypass the nonsense-mediated mRNA decay machinery, suggesting the AUG proximity effect as an underlying transcriptional mechanism. The truncated protein, however, is not detectable in either species. The in vitro model accurately recapitulates transcriptional and translational expression events observed in vivo and, thus, implies loss of bestrophin-1 function in cmr1-dogs and Y(29)X-affected patients. Immunofluorescence microscopy of cmr2 mutant showed mislocalization of the protein. CONCLUSIONS: Molecular evaluation of cmr mutations in vivo and in vitro constitutes the next step toward elucidating genotype-phenotype interactions concerning human bestrophinopathies and emphasizes the importance of the canine models for studying the complexity of the BEST1 disease mechanism.

Assessment of canine BEST1 variations identifies new mutations and establishes an independent bestrophinopathy model (cmr3).

PURPOSE: Mutations in bestrophin 1 (BEST1) are associated with a group of retinal disorders known as bestrophinopathies in man and canine multifocal retinopathies (cmr) in the dog. To date, the dog is the only large animal model suitable for the complex characterization and in-depth studies of Best-related disorders. In the first report of cmr, the disease was described in a group of mastiff-related breeds (cmr1) and the Coton de Tulear (cmr2). Additional breeds, e.g., the Lapponian herder (LH) and others, subsequently were recognized with similar phenotypes, but linked loci are unknown. Analysis of the BEST1 gene aimed to identify mutations in these additional populations and extend our understanding of genotype-phenotype associations. METHODS: Animals were subjected to routine eye exams, phenotypically characterized, and samples were collected for molecular studies. Known BEST1 mutations were assessed, and the canine BEST1 coding exons were amplified and sequenced in selected individuals that exhibited a cmr compatible phenotype but that did not carry known mutations. Resulting sequence changes were genotyped in several different breeds and evaluated in the context of the phenotype. RESULTS: Seven novel coding variants were identified in exon 10 of cBEST1. Two linked mutations were associated with cmr exclusive to the LH breed (cmr3). Two individuals of Jämthund and Norfolk terrier breeds were heterozygous for two conservative changes, but these were unlikely to have disease-causing potential. Another three substitutions were found in the Bernese mountain dog that were predicted to have a deleterious effect on protein function. Previously reported mutations were excluded from segregation in these populations, but cmr1 was confirmed in another mastiff-related breed, the Italian cane corso. CONCLUSIONS: A third independent canine model for human bestrophinopathies has been established in the LH breed. While exhibiting a phenotype comparable to cmr1 and cmr2, the novel cmr3 mutation is predicted to be based on a distinctly different molecular mechanism. So far cmr2 and cmr3 are exclusive to a single dog breed each. In contrast, cmr1 is found in multiple related breeds. Additional sequence alterations identified in exon 10 of cBEST1 in other breeds exhibit potential disease-causing features. The inherent genetic and phenotypic variation observed with retinal disorders in canines is complicated further by cmr3 being one of four distinct genetic retinal traits found to segregate in LH. Thus, a combination of phenotypic, molecular, and population analysis is required to establish a strong phenotype-genotype association. These results indicate that cmr has a larger impact on the general dog population than was initially suspected. The complexity of these models further confirms the similarity to human bestrophinopathies. Moreover, analyses of multiple canine models will provide additional insight into the molecular basis underlying diseases caused by mutations in BEST1.

Bestrophin gene mutations cause canine multifocal retinopathy: a novel animal model for best disease.

PURPOSE: Canine multifocal retinopathy (cmr) is an autosomal recessive disorder of multiple dog breeds. The disease shares a number of clinical and pathologic similarities with Best macular dystrophy (BMD), and cmr is proposed as a new large animal model for Best disease. METHODS: cmr was characterized by ophthalmoscopy and histopathology and compared with BMD-affected patients. BEST1 (alias VMD2), the bestrophin gene causally associated with BMD, was evaluated in the dog. Canine ortholog cDNA sequence was cloned and verified using RPE/choroid 5'- and 3'-RACE. Expression of the canine gene transcripts and protein was analyzed by Northern and Western blotting and immunocytochemistry. All exons and the flanking splice junctions were screened by direct sequencing. RESULTS: The clinical phenotype and pathology of cmr closely resemble lesions of BMD. Canine VMD2 spans 13.7 kb of genomic DNA on CFA18 and shows a high level of conservation among eukaryotes. The transcript is predominantly expressed in RPE/choroid and encodes bestrophin, a 580-amino acid protein of 66 kDa. Immunocytochemistry of normal canine retina demonstrated specific localization of protein to the RPE basolateral plasma membranes. Two disease-specific sequence alterations were identified in the canine VMD2 gene: a C(73)T stop mutation in cmr1 and a G(482)A missense mutation in cmr2. CONCLUSIONS: The authors propose these two spontaneous mutations in the canine VMD2 gene, which cause cmr, as the first naturally occurring animal model of BMD. Further development of the cmr models will permit elucidation of the complex molecular mechanism of these retinopathies and the development of potential therapies.