A naturally occurring canine model of syndromic congenital microphthalmia.

In humans, the prevalence of congenital microphthalmia is estimated to be 0.2-3.0 for every 10,000 individuals, with nonocular involvement reported in ∼80% of cases. Inherited eye diseases have been widely and descriptively characterized in dogs, and canine models of ocular diseases have played an essential role in unraveling the pathophysiology and development of new therapies. A naturally occurring canine model of a syndromic disorder characterized by microphthalmia was discovered in the Portuguese water dog. As nonocular findings included tooth enamel malformations, stunted growth, anemia, and thrombocytopenia, we hence termed this disorder Canine Congenital Microphthalmos with Hematopoietic Defects. Genome-wide association study and homozygosity mapping detected a 2 Mb candidate region on canine chromosome 4. Whole-genome sequencing and mapping against the Canfam4 reference revealed a Short interspersed element insertion in exon 2 of the DNAJC1 gene (g.74,274,883ins[T70]TGCTGCTTGGATT). Subsequent real-time PCR-based mass genotyping of a larger Portuguese water dog population found that the homozygous mutant genotype was perfectly associated with the Canine Congenital Microphthalmos with Hematopoietic Defects phenotype. Biallelic variants in DNAJC21 are mostly found to be associated with bone marrow failure syndrome type 3, with a phenotype that has a certain degree of overlap with Fanconi anemia, dyskeratosis congenita, Shwachman-Diamond syndrome, Diamond-Blackfan anemia, and reports of individuals showing thrombocytopenia, microdontia, and microphthalmia. We, therefore, propose Canine Congenital Microphthalmos with Hematopoietic Defects as a naturally occurring model for DNAJC21-associated syndromes.

Retinal response to light exposure in BEST1-mutant dogs evaluated with ultra-high resolution OCT.

Mutations in BEST1 cause an autosomal recessive disease in dogs where the earliest changes localize to the photoreceptor-RPE interface and show a retina-wide micro-detachment that is modulated by light exposure. The purpose of this study was to define the spatial and temporal details of the outer retina and its response to light with ultra-high resolution OCT across a range of ages and with different BEST1 mutations. Three retinal regions were selected in each eye: near the fovea-like area, near the optic nerve, both in the tapetal area, and inferior to the optic nerve in the non-tapetal area. The OS+ slab thickness was defined between the peak near the junction of inner and outer segments (IS/OS) and the transition between basal RPE, Bruch membrane, choriocapillaris and proximal tapetum (RPE/T). In wildtype (WT) dogs, two tapetal regions showed additional hyperscattering OCT peaks within the OS+ slab likely representing cone and rod outer segment tips (COST and ROST). The inferior non-tapetal region of WT dogs had only one of these peaks, likely ROST. In dogs with BEST1 mutations, all three locations showed a single peak, likely suggesting optical silence of COST. Light-dependent expansion of the micro-detachment by about 10 um was detectable in both tapetal and non-tapetal retina across all ages and BEST1 mutations.

Frameshift Variant in AMPD2 in Cirneco dell'Etna Dogs with Retinopathy and Tremors.

While the manifestations of many inherited retinal disorders are limited to loss of vision, others are part of a syndrome that affects multiple tissues, particularly the nervous system. Most syndromic retinal disorders are thought to be recessively inherited. Two dogs out of a litter of Cirneco dell' Etna dogs, both males, showed signs of retinal degeneration, along with tremors and signs described as either atypical seizures or paroxysmal dyskinesias, while the other two male littermates were normal. We named this oculo-neurological syndrome CONS (Cirneco oculo-neurological syndrome), and undertook homozygosity mapping and whole-genome sequencing to determine its potential genetic etiology. Notably, we detected a 1-bp deletion in chromosome 6 that was predicted to cause a frameshift and premature stop codon within the canine AMPD2 gene, which encodes adenosine monophosphate deaminase, an enzyme that converts adenosine 5'-monophosphate (AMP) to inosine 5'-monophosphate (IMP). Genotyping of the available Cirneco population suggested perfect segregation between cases and controls for the variant. Moreover, this variant was absent in canine genomic databases comprised of thousands of unaffected dogs. The AMPD2 genetic variant we identified in dogs presents with retinal manifestations, adding to the spectrum of neurological manifestations associated with AMPD2 variants in humans.

Consensus guidelines for nomenclature of companion animal inherited retinal disorders.

Companion animals, namely dogs, cats, and horses, can be affected with many forms of hereditary retinal disease. The number of such diseases characterized in the last decade has increased substantially, and nomenclature is nonstandardized, heterogenous, and confusing. We provide in this viewpoint article consensus guidelines for naming of companion animal hereditary retinal diseases, either prospectively or retrospectively. These consensus guidelines have been developed with the purpose of standardizing nomenclature. We provide examples for the iterative nomenclature process and a comprehensive File S1 on proposed renaming of previously described diseases.

Cone-Driven, Geniculocortical Responses in Canine Models of Outer Retinal Disease.

Purpose: Canine models of inherited retinal degeneration are used for proof of concept of emerging gene and cell-based therapies that aim to produce functional restoration of cone-mediated vision. We examined functional magnetic resonance imaging (MRI) measures of the postretinal response to cone-directed stimulation in wild-type (WT) dogs, and in three different retinal disease models. Methods: Temporal spectral modulation of a uniform field of light around a photopic background was used to target the canine L/M (hereafter "L") and S cones and rods. Stimuli were designed to separately target the postreceptoral luminance (L+S) and chrominance (L-S) pathways, the rods, and all photoreceptors jointly (light flux). These stimuli were presented to WT, and mutant PDE6B-RCD1, RPGR-XLPRA2, and NPHP5-CRD2 dogs during pupillometry and functional MRI (fMRI). Results: Pupil responses in WT dogs to light flux, L+S, and rod-directed stimuli were consistent with responses being driven by cone signals alone. For WT animals, both luminance and chromatic (L-S) stimuli evoked fMRI responses in the lateral geniculate nucleus or visual cortex; RCD1 animals with predominant rod loss had similar responses. Responses to cone-directed stimulation were reduced in XLPRA2 and absent in CRD2. NPHP5 gene augmentation restored the cortical response to luminance stimulation in a CRD2 animal. Conclusions: Cone-directed stimulation during fMRI can be used to measure the integrity of luminance and chrominance responses in the dog visual system. The NPHP5-CRD2 model is appealing for studies of recovered cone function. Translational Relevance: fMRI assessment of cone-driven cortical response provides a tool to translate cell/gene therapies for vision restoration.

Molecular characterization of MAP9 in the photoreceptor sensory cilia as a modifier in canine RPGRIP1-associated cone-rod dystrophy.

Photoreceptors possess a highly specialized primary cilium containing expanded ciliary membrane discs called the outer segment. The photoreceptor cilium is essential for the maintenance of the outer segment, and pathogenic variants in more than 50 cilia-related genes have been identified as causing non-syndromic inherited retinal diseases in patients. The retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1) is a structural protein localized to the photoreceptor cilium and biallelic RPGRIP1 variants have been associated with non-syndromic human inherited retinal diseases. In a canine cone-rod dystrophy model, a naturally occurring 44-bp exonic insertion in RPGRIP1 (RPGRIP1ins44/ins44) is the primary disease locus while an additional homozygous variant in MAP9 (microtubule associated protein 9) (MAP9aff/aff) acts as a modifier associated with early disease onset. MAP9 was originally identified as a microtubule-binding protein stabilizing microtubule structure during both mitosis and interphase in human cell lines. However, the roles of MAP9 in primary cilia, including photoreceptor neurosensory cilia, have not been well understood. Hence, we characterized the pathogenic phenotypes associated with homozygous MAP9 variant, and investigated the molecular function of MAP9 in primary cilia using the RPGRIP1-associated oligogenic canine cone-rod dystrophy model as well as cultured cells. Both functionally and structurally, the RPGRIP1ins44/ins44 MAP9aff/aff retina exhibited progressive cone photoreceptor degeneration starting earlier than the retina affected by RPGRIP1ins44/ins44 alone. Based on immunostaining of canine retinal sections and cultured cells, we found that MAP9 is prominently localized in the basal body of primary cilia and played an important role in maintaining the structure of ciliary microtubule axoneme. These findings suggest that the affected MAP9, together with mutant RPGRIP1, is deprived of critical roles in cilia organization and maintenance resulting in altered cilia structure and function giving rise to early onset and accelerated disease progression in the RPGRIP1ins44/ins44 MAP9aff/aff double homozygote cone-rod dystrophy canine model.

Extended functional rescue following AAV gene therapy in a canine model of LRIT3-congenital stationary night blindness.

Congenital stationary night blindness (CSNB) is a group of inherited retinal diseases in which either rod-to-ON-bipolar cell (ON-BC) signaling, or rod function is affected leading to impaired vision under low light conditions. One type of CSNB is associated with defects in genes (NYX, GRM6, TRPM1, GPR179, and LRIT3) involved in the mGluR6 signaling cascade at the ON-BC dendritic tips. We have previously characterized a canine model of LRIT3-CSNB and demonstrated short-term safety and efficacy of an ON-BC targeting AAV-LRIT3 (AAVK9#4-shGRM6-cLRIT3-WPRE) gene therapy. Herein, we demonstrate long-term functional recovery and molecular restoration following subretinal injection of the ON-BC targeting AAV-LRIT3 vector in all eight treated eyes for up to 32 months. Following subretinal administration of the therapeutic vector, expression of the LRIT3 transgene, as well as restoration of mGluR6 signaling cascade member TRPM1, were confirmed in the outer plexiform layer (OPL) of the treated area. However, further investigation of the transgene LRIT3 transcript expression by RNA in situ hybridization (RNA-ISH) revealed off-target expression in non-BCs including the photoreceptors, inner nuclear, and ganglion cell layers, despite the use of a mutant AAVK9#4 capsid and an improved mGluR6 promoter designed to specifically transduce and promote expression in ON-BCs. While the long-term therapeutic potential of AAVK9#4-shGRM6-cLRIT3-WPRE is promising, we highlight the necessity for further optimization of AAV-LRIT3 therapy in the canine CSNB model prior to its clinical application.

Natural disease history of a canine model of oligogenic RPGRIP1-cone-rod dystrophy establishes variable effects of previously and newly mapped modifier loci.

Canine RPGRIP1-cone-rod dystrophy (CRD), a model for human inherited retinal diseases (IRDs), was originally identified as autosomal recessive early-onset blindness. However, later studies revealed extensive phenotypic variability among RPGRIP1 mutants. This led to the identification of a homozygous MAP9 variant as a modifier associated with early-onset disease. Based on further phenotypic variation affecting cone photoreceptor function, we report mapping of L3 as an additional modifier locus, within a 4.1-Mb locus on canine chromosome 30. We establish the natural disease history of RPGRIP1-CRD based on up to 9-year long-term functional and structural retinal data from 58 dogs including 44 RPGRIP1 mutants grouped according to the modifier status. RPGRIP1 mutants affected by both MAP9 and L3 modifiers exhibited the most severe phenotypes with rapid disease progression. MAP9 alone was found to act as an overall accelerator of rod and cone diseases, while L3 had a cone-specific effect. Ultrastructural analysis of photoreceptors revealed varying degrees of rod and cone damage, while the connecting cilia appeared structurally preserved in all groups. We conclude that RPGRIP1-CRD is an oligogenic disease with at least three loci contributing to the pathogenesis. While the RPGRIP1 variant is required for developing the disease, MAP9 and L3 modifiers exacerbate the phenotype, individually and cumulatively. Oligogenic canine RPGRIP1-CRD illustrates the impact of multiple genetic modifiers on disease phenotype and thus has the potential to reveal new targets for broad-spectrum therapies for oligogenic or polygenic forms of human IRDs.

Identification of circular RNAs hosted by the RPGR ORF15 genomic locus.

Mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRorf15) cause X-linked retinitis pigmentosa, a severe and early onset inherited retinal degeneration. The underlying pathogenic mechanisms and variability in disease severity remain to be fully elucidated. The present study examines structural features of the ORF15 exonic region to provide new insights into the disease pathogenesis. Using canine and human RNA samples, we identified several novel RPGR ORF15-like linear RNA transcripts containing cryptic introns (exitrons) within the annotated exon ORF15. Furthermore, using outward-facing primers designed inside exitrons in the ORF15 exonic region, we found many of previously unidentified circular RNAs (circRNAs) that formed via back fusion of linear parts of the RPGRorf15 pre-mRNAs. These circRNAs (resistant to RNAse R treatment) were found in all studied cells and tissues. Notably, some circRNAs were present in cytoplasmic and polysomal RNA fractions. Although certain RPGR circRNAs may be cell type specific, we found some of the same circRNAs expressed in different cell types, suggesting similarities in their biogenesis and functions. Sequence analysis of RPGR circRNAs revealed several remarkable features, including identification of N6-methyladenosine (m6A) consensus sequence motifs and high prevalence of predictive microRNA binding sites pointing to the functional roles of these circRNAs. Our findings also illustrate the presence of non-canonical RPGR circRNA biogenesis pathways independent of the known back splicing mechanism. The obtained data on novel RPGR circRNAs further underline structural complexity of the RPGR ORF15 region and provide a potential molecular basis for the disease phenotypic heterogeneity.

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.

Cone-driven, geniculo-cortical responses in canine models of outer retinal disease.

Purpose: Canine models of inherited retinal degeneration are used for proof-of-concept of emerging gene and cell-based therapies that aim to produce functional restoration of cone-mediated vision. We examined functional MRI measures of the post-retinal response to cone-directed stimulation in wild type (WT) dogs, and in three different retinal disease models. Methods: Temporal spectral modulation of a uniform field of light around a photopic background was used to target the canine L/M (hereafter "L") and S cones and rods. Stimuli were designed to separately target the post-receptoral luminance (L+S) and chrominance (L-S) pathways, the rods, and all photoreceptors jointly (light flux). These stimuli were presented to WT, and mutant PDE6B-RCD1, RPGR-XLPRA2, and NPHP5-CRD2 dogs during pupillometry and fMRI. Results: Pupil responses in WT dogs to light flux, L+S, and rod-directed stimuli were consistent with responses being driven by cone signals alone. For WT animals, both luminance and chromatic (L-S) stimuli evoked fMRI responses in the lateral geniculate nucleus (LGN) or visual cortex; RCD1 animals with predominant rod loss had similar responses. Responses to cone-directed stimulation were reduced in XLPRA2 and absent in CRD2. NPHP5 gene augmentation restored the cortical response to luminance stimulation in a CRD2 animal. Conclusions: Cone-directed stimulation during fMRI can be used to measure the integrity of luminance and chrominance responses in the dog visual system. The NPHP5-CRD2 model is appealing for studies of recovered cone function. Translational Relevance: fMRI assessment of cone driven cortical response provides a tool to translate cell/gene therapies for vision restoration.

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.

Photochemical Restoration of Light Sensitivity in the Degenerated Canine Retina.

Photopharmacological compounds such as azobenzene-based photoswitches have been shown to control the conductivity of ionic channels in a light-dependent manner and are considered a potential strategy to restore vision in patients with end-stage photoreceptor degeneration. Here, we report the effects of DENAQ, a second-generation azobenzene-based photoswitch on retinal ganglion cells (RGC) in canine retinas using multi-electrode array (MEA) recordings (from nine degenerated and six WT retinas). DENAQ treatment conferred increased light sensitivity to RGCs in degenerated canine retinas. RGC light responses were observed in degenerated retinas following ex vivo application of 1 mM DENAQ (n = 6) or after in vivo DENAQ injection (n = 3, 150 µL, 3-10 mM) using 455 nm light at intensities as low as 0.2 mW/cm2. The number of light-sensitive cells and the per cell response amplitude increased with light intensity up to the maximum tested intensity of 85 mW/cm2. Application of DENAQ to degenerated retinas with partially preserved cone function caused appearance of DENAQ-driven responses both in cone-driven and previously non-responsive RGCs, and disappearance of cone-driven responses. Repeated stimulation slowed activation and accelerated recovery of the DENAQ-driven responses. The latter is likely responsible for the delayed appearance of a response to 4 Hz flicker stimulation. Limited aqueous solubility of DENAQ results in focal drug aggregates associated with ocular toxicity. While this limits the therapeutic potential of DENAQ, more potent third-generation photoswitches may be more promising, especially when delivered in a slow-release formulation that prevents drug aggregation.

Retinal Vascular Plexuses Are Unequally Affected in Canine Inherited Retinal Degenerations.

Purpose: To characterize the progression of vascular changes that occur in each retinal plexus, in three canine models of inherited retinal degeneration. Methods: In this retrospective cohort study, we examined the retinal imaging records of 44 dogs from a research colony that had undergone optical coherence tomography angiography (OCTA) imaging. Animals enrolled included crd2/NPHP5 and xlpra2/RPGR mutant dogs imaged at different stages of photoreceptor loss, as well as RHOT4R/+ dogs after acute light-induced rod degeneration. Also included were normal controls imaged at similar ages. OCT angiograms of the superficial vascular plexus combined with the intermediate capillary plexus (SVP + ICP), and the deep capillary plexus (DCP) were analyzed using the AngioTool software to calculate vessel density and other vascular parameters. Results: A reduction in vessel density was seen over time in both the SVP + ICP and DCP in all mutant dogs but was more pronounced in the DCP. Scans were subclassified based on outer nuclear layer (ONL) thinning compared to age-matched normal controls. When ONL loss was 0% to 50%, vessel density in the DCP was significantly lower than in age-matched controls. In all cases, when ONL loss exceeded 87.5%, vessel density in the SVP + ICP was significantly reduced as well. In the acute light-induced rod degeneration model, the vascular regression changes were observed mainly in the DCP. Conclusions: Vessel density reduction in dogs undergoing retinal degeneration is first detected by OCTA in the DCP, and only at later stages in the SVP + ICP.

Systemic immunosuppression promotes survival and integration of subretinally implanted human ESC-derived photoreceptor precursors in dogs.

Regenerative therapies aimed at replacing photoreceptors are a promising approach for the treatment of otherwise incurable causes of blindness. However, such therapies still face significant hurdles, including the need to improve subretinal delivery and long-term survival rate of transplanted cells, and promote sufficient integration into the host retina. Here, we successfully delivered in vitro-derived human photoreceptor precursor cells (PRPCs; also known as immature photoreceptors) to the subretinal space of seven normal and three rcd1/PDE6B mutant dogs with advanced inherited retinal degeneration. Notably, while these xenografts were rejected in dogs that were not immunosuppressed, transplants in most dogs receiving systemic immunosuppression survived up to 3-5 months postinjection. Moreover, differentiation of donor PRPCs into photoreceptors with synaptic pedicle-like structures that established contact with second-order neurons was enhanced in rcd1/PDE6B mutant dogs. Together, our findings set the stage for evaluating functional vision restoration following photoreceptor replacement in canine models of inherited retinal degeneration.

Targeting ON-bipolar cells by AAV gene therapy stably reverses LRIT3-congenital stationary night blindness.

SignificanceCanine models of inherited retinal diseases have helped advance adeno-associated virus (AAV)-based gene therapies targeting specific cells in the outer retina for treating blinding diseases in patients. However, therapeutic targeting of diseases such as congenital stationary night blindness (CSNB) that exhibit defects in ON-bipolar cells (ON-BCs) of the midretina remains underdeveloped. Using a leucine-rich repeat, immunoglobulin-like and transmembrane domain 3 (LRIT3) mutant canine model of CSNB exhibiting ON-BC dysfunction, we tested the ability of cell-specific AAV capsids and promotors to specifically target ON-BCs for gene delivery. Subretinal injection of one vector demonstrated safety and efficacy with robust and stable rescue of electroretinography signals and night vision up to 1 y, paving the way for clinical trials in patients.

Novel insights into chorioretinal and juxtapapillary colobomas by optical coherence tomography.

PURPOSE: The purpose was to describe the in vivo microanatomy of typical and atypical chorioretinal and juxtapapillary colobomas in the dog. METHODS: Three cross-breed dogs were found to be affected with colobomas. Two of the cases were NEHJ1 homozygous and Collie Eye Anomaly (CEA) affected and had the typical optic nerve head colobomas seen with the disease. The third case had an unexpected atypical coloboma. In vivo retinal photography and non-invasive retinal imaging by confocal scanning laser ophthalmoscope (cSLO) and optical coherence tomography (OCT) were done, and the eye affected with the atypical coloboma was collected and processed for histopathological evaluation. RESULTS: The majority of the defining features within the CEA defects were similar, with the extent of change to the choroid being of note. Similar to the first two cases, the atypical coloboma demonstrated absent normal retina, RPE, and choroid within the coloboma. Prominent intercalary membranes and vitreal strands attached to the depth of the coloboma were also apparent in all affected eyes. However, unlike the CEA-associated colobomas, the atypical coloboma possessed normal choroid surrounding the lesion and the depth of the lesion was apparent throughout. CONCLUSIONS: Advanced retinal imaging enables the appreciation of microanatomical changes that occur in the living eye. The ability of OCT to enhance visualization of abnormal retinal structures and detect subtle neurosensory retinal defects has allowed for the in vivo characterization of features observed in typical and atypical colobomas, as well as the appreciation of some of the resulting structural changes not visible by ophthalmoscopy alone.

Retinal structural and microvascular abnormalities in retinal dysplasia imaged by OCT and OCT angiography.

OBJECTIVE: To describe the in vivo structural characteristics of multifocal and geographic retinal dysplasia visualized with advanced retinal imaging including confocal scanning laser ophthalmoscopy (cSLO), optical coherence tomography (OCT), en face OCT, and the novel vascular imaging technique OCT angiography (OCTA). DOGS STUDIED AND PROCEDURES: Two dogs were diagnosed with unilateral multifocal or geographic retinal dysplasia and underwent advanced retinal imaging under general anesthesia at the Retinal Disease Studies Facility of the University of Pennsylvania. RESULTS: In both cases, the morphological pattern of the lesions was similar including outer retinal folds that invaginated and formed tubular retinal rosettes, surrounding a central inner retinal thickening (multifocal) or plaque (geographic). The two dogs had multiple vascular anomalies in the lesions such as increased tortuosity, abnormal change of vessel diameter including aneurysms and capillary network disruption. We also identified increased autofluorescence by AF cSLO with short wavelength light source (488 nm and barrier filter at 500 nm), and several areas of photoreceptor loss associated with the lesions. CONCLUSION: The use of OCTA allowed the identification of microvascular abnormalities associated with multifocal and geographic retinal dysplasia in two dogs. To our knowledge, this is the first report where the dye-free OCTA technique is used to study vascular lesions in canine retinas.