William A. Beltran

Photoreceptor sensory cilia comprise a microtubule-based structural core and the phototransductive outer segment. The microtubule-based ciliary axoneme extends from the basal body through the connecting cilium into the outer segment, thereby linking the inner and outer segments. Around this central axis, specialized periciliary structures are distributed across the apical inner segment and basal outer segment. Yet how these ciliary and periciliary structures differ between rods and cones and across mammals remains incompletely defined. Using ultrastructure expansion microscopy, we compared their nanoscale molecular organization across canine, non-human primate and human retinas. Key rod–cone differences in ciliary organization were broadly conserved among all three species, whereas the scale and morphology of individual ciliary subcompartments differed between species. Periciliary structures showed even greater species-specific diversification, particularly in rods. Human photoreceptors shared several structural features with non-human primate photoreceptors but also displayed inter-individual variability in accessory inner segment-like structure. These findings refine current understanding of photoreceptor sensory cilia and associated periciliary structures in large mammals, revealing that conserved rod–cone differences co-exist with pronounced species-specific structural and molecular specializations.

To treat patients affected with bestrophinopathies caused by mutations in the BEST1 gene, a recombinant adeno-associated virus vector (OPGx-BEST1 or rAAV2/2-VMD2- BEST1 ) is being developed. The vector construct includes the human BEST1 cDNA under control of the VMD2/BEST1 promoter and is packaged in an AAV2 capsid. This study evaluated the efficacy and toxicity of OPGx-BEST1 administered by subretinal injection in dogs that were homozygous mutant or compound heterozygotes for 3 naturally occurring mutations in BEST1 . Twelve BEST1-mutant dogs were divided into 4 groups of 3 animals each, and they received in their left eye a subretinal injection of 0.15 mL of OPGx-BEST1 at 1 of 3 concentrations (9.5 × 10 ⁹ vector genome [vg]/mL; 3.0 × 10 ¹⁰ vg/mL; or 3.0 × 10 ¹¹ vg/mL) of OPGx-BEST1, resulting, respectively, in a low (1.4 × 10 ⁹ vg), high (4.5 × 10 ⁹ vg), and highest (4.5 × 10 ¹⁰ vg) dose or vehicle control. The right eyes were not injected. Subretinal injections were well tolerated and were not associated with any systemic or ocular toxicity. Electroretinography showed improved rod- and cone-mediated responses in eyes treated with OPGx-BEST1. Noninvasive retinal imaging by optical coherence tomography showed improved structural integrity with a reduction or prevention of appearance of vitelliform lesions and reversal of microdetachments in the retinal areas treated with OPGx-BEST1. These results support the use of OPGx-BEST1 in clinical studies with patients affected with bestrophinopathies and define the no-observed-adverse-effect level at 4.5 × 10 ¹⁰ vg/eye (0.15 mL, 3.0 × 10 ¹¹ vg/mL).

Inherited retinal degenerations (IRDs) are a frequent cause of vision impairment in numerous breeds of dogs. Veterinary ophthalmologists frequently suspect retinal degeneration when funduscopic examinations reveal increased tapetal reflectivity and retinal vascular attenuation; however, these clinical signs can be subtle, especially in early disease. In this study, we evaluated three deep learning architectures using fundus images to detect degenerative retinal disease in dogs. An EfficientNetV2-based convolutional neural network with a custom classification head was selected for final evaluation. Using dog-level analysis on an independent held-out test set, the model achieved 87.5% accuracy, 87.5% sensitivity, 87.5% specificity, and an area under the receiver operating characteristic curve of 0.932. Dog-level calibration analysis yielded a Brier score of 0.093. Under constrained single-image evaluation conditions, the model showed higher dog-level classification performance than the majority-vote classification of three experienced veterinary ophthalmologists, primarily by reducing false-positive classification of normal dogs as affected. Exact McNemar testing demonstrated a significant paired difference between the model and expert aggregate classifications at the dog level (p = 0.000122). Gradient-weighted Class Activation Mapping (Grad-CAM) was incorporated to provide qualitative visualization of retinal image regions associated with model predictions. These results support the potential of deep learning as an objective decision-support tool for canine fundus image interpretation, although broader multi-center and prospective validation is needed before clinical deployment.

Immunofluorescence-based mapping of photoreceptor ribbon synapse nanostructure in extended formaldehyde-fixed specimens is challenging due to diffraction-limited resolution and fixation-associated loss of antigen accessibility that restrict reliable molecular localization and structural interpretation. To circumvent these physicochemical limitations, we tested whether ultrastructure expansion microscopy (U-ExM) could overcome these barriers and enable compartment-resolved structural and molecular analysis of photoreceptor ribbon synapses in archival canine retinal tissues. Cryosections from non-fixed and formaldehyde-fixed canine retinas were immunolabeled by conventional immunohistochemistry to screen 44 antibodies targeting 29 ribbon synapse-associated proteins. The same antibody set was then evaluated in U-ExM-processed formaldehyde-fixed cryosections. U-ExM datasets were analyzed using multi-orientation visualization, line-profile measurements, and three-dimensional segmentation for ribbon morphometry in mature and developing retinas. Antibody performance differed markedly between non-fixed and formaldehyde-fixed sections, indicating fixation-dependent loss or gain of detectability across targets. U-ExM increased the fraction of compatible antibodies in formaldehyde-fixed tissue and improved effective spatial resolution within synaptic compartments. Multi-orientation visualization refined interpretation of rod spherule architecture and revealed multiple postsynaptic density 95 (PSD-95) subcellular sites of expression, including a postsynaptic pool in horizontal cell axon terminals within the invaginated compartment. U-ExM further enabled subcompartment-level visualization of active zone proteins, extended compartment-resolved mapping to the more complex cone pedicle, and supported quantitative ribbon measurements in mature retina and across postnatal development. U-ExM provides a practical framework for compartment-resolved molecular mapping and quantitative phenotyping of photoreceptor ribbon synapses in formaldehyde-fixed/frozen archival retinal tissue, enabling systematic comparisons across conditions and developmental stages in translational and comparative vision research.

Photoreceptor sensory cilia (PSCs) support outer segment formation and are associated with specialized periciliary complexes, yet their cross-species and subtype-dependent organization remains incompletely defined. Here, we used ultrastructure expansion microscopy to compare PSC architecture and associated periciliary complexes across canine, non-human primate (NHP), and human retinas. Across all three species, rods and cones shared a conserved subtype-dependent organization, with cones displaying shorter connecting cilia (CCs), longer daughter centrioles (DCs), and enlarged bulge regions relative to rods. However, the scale and elaboration of these structures differed substantially between species, with primate PSCs showing shorter CCs, more pronounced widening of the apical region, and elongated cone DCs. Periciliary complexes showed even greater diversification. Canine rods exhibited a unilateral periciliary membrane and lacked calyceal process, whereas primate rods and cones of all three species showed a circumferential periciliary membrane and robust calyceal process. Human photoreceptors were broadly NHP-like but revealed variable accessory inner segment-like structures. Together, these findings provide a comparative nanoscale framework for photoreceptor ciliary organization in large mammals and show that conserved PSC principles coexist with pronounced species- and subtype-specific specialization.

Mutations in RPGRorf15 and PDE6B cause early-onset retinitis pigmentosa, yet the molecular factors driving photoreceptor degeneration remain poorly understood. Here, we characterize the pathological accumulation of RNA:DNA hybrids (R-loops) in two canine disease models-xlpra2/RPGRorf15 and rcd1/PDE6B-to provide new insights into disease pathogenesis. Archival canine retinal tissues were used for immunohistochemistry, TUNEL, Western blotting, and PCR. Outer nuclear layer regions were isolated via laser-capture microdissection to assess gene expression changes. R-loop dynamics paralleled progression of apoptosis in both models. Most TUNEL-positive photoreceptor nuclei were also positive for R-loops and m⁶A RNA modification, suggesting potential crosstalk. Near peak apoptosis, colabeled nuclei accounted for ∼68% to 79% in xlpra2 (7-8 weeks old) and 59% to 78% in rcd1 (3-6 weeks old), although TUNEL-only nuclei were also present. At later stages (16 weeks), colabeling ranged from 76% to 100% in xlpra2 and 52% to 53% in rcd1. By 16 weeks, R-loop accumulation declined in both models, whereas γH2AX, a marker of early DNA double-strand breaks, increased. Downregulation of genes controlling R-loop metabolism was evident at 6 to 7 weeks, including CPSF6 and XRN2 (both models), as well as BRD4 and SF3B1 in xlpra2 and DHX9 in rcd1. These alterations may partly underlie the disease-associated splicing defects observed in RPGRorf15 pre-mRNA, encoded by the R-loop-prone ORF15 locus. Photoreceptor degeneration in xlpra2 and rcd1 is accompanied by overlapping molecular features, including early R-loop- and m⁶A-associated apoptosis with concurrent transcriptional alterations in R-loop-regulatory genes, followed by a progressively R-loop-independent DNA damage response.

Inherited retinal diseases (IRDs) are a diverse group of disorders that share common vision deficits ranging from early onset blindness to severe and progressive later-onset disease. We report a form of early-onset day-vision loss, cone-rod dystrophy, in the Standard poodle. Through GWAS and homozygosity mapping, a large deletion on CFA8:NC_049229.1:g.60,022,583_60,040,453del was found which removes 3’ portions of two different genes, PTPN21 and SPATA7 , presenting a challenge for assessing the actual causative gene in a multi-gene large deletion. All affected dogs were homozygous for the mutant allele, which segregated perfectly with the phenotype within the breed. The variant was absent in 1879 dogs from the Dog10K database. While the role of SPATA7 for retinal disease has been established in human patients and genetically engineered mice, the role of PTPN21 in the retina is unclear even though it is expressed in rod and cone photoreceptors. Expression of whole and truncated transcripts for both genes was detected in skin fibroblasts from controls and cases. Retinal RNA analysis of PTPN21 splicing suggests that at least one unmodified transcript is still present in mutants. Ptpn21 -/- knockout mice did not have an ocular phenotype, and IHC for rod- and cone-specific opsins detected no cone or rod abnormalities suggesting that PTPN21 loss has minimal to no contributory role towards the retinal phenotype in mutants. The variant leads to a deletion of the 3’-end of the SPATA7 transcript: XM_038545497.1:r.1,314_1,629delins[g.60,018,954–60,018,990], p.(XP_038401425.1: Asp361GlufsTer2), reducing the predicted protein from 595 to 361 AA. Ultrastructure expansion microscopy (U-ExM) enabled the detection of a distinct SPATA7 signal around the transition zone of the primary cilium in photoreceptors and fibroblasts of WT dogs, which was absent in affected dog. We posit that SPATA7 deficiency is the main cause of the condition, and propose this disease as a model for the SPATA7 -related form of cone-rod dystrophy in humans. Our work shows an example of functional refinement of a multi-gene deletion variant using a multi-technique approach.

Efficient messenger ribonucleic acid (mRNA) delivery to the retina remains challenging. This study investigated the effects of various polyethylene glycol (PEG) derivatives on the stability and uptake of cationic lipid-based mRNA lipoplexes in vitro and assessed the delivery of selected formulations to the canine retina. We present an optimized workflow for formulating mRNA lipoplexes in pure water, achieving high encapsulation efficiency. PEGylation enhanced stability of lipoplexes, particularly with PEG-DMG or hyaluronan conjugated to PEG-DPPE (HA-PEG-DPPE), maintaining size and zeta potential for 48 hours. RNA in situ hybridization (RNA-ISH) confirmed efficient internalization of PEGylated mRNA lipoplexes by cultured RAW264.7 and ARPE19 cells, though corresponding protein expression varied between cell lines. Analysis at 24 hours post-intravitreal injection of PEG-DMG- and HA-PEG-DPPE-stabilized enhanced green fluorescent protein (eGFP) mRNA lipoplexes revealed limited mRNA accumulation in inner retinal layers. In contrast, 24 hours after their subretinal administration, eGFP mRNA was detected in all retinal cell types, including photoreceptors, with accumulation comparable to endogenous rhodopsin (RHO) mRNA levels. eGFP protein expression, though, was limited to the retinal pigment epithelium (RPE). At 72 hours post-subretinal delivery, eGFP mRNA and protein persisted in the RPE. However, a marked reduction in eGFP levels was seen in other retinal layers, displaying a patchy pattern. Similarly, eGFP protein exhibited a patchy distribution across retinal layers outside the RPE. Furthermore, distinct differences in the cell types expressing the eGFP protein were observed between the two PEGylated mRNA lipoplex formulations. The data suggest that transfection efficiency in retinal cells is influenced by both intracellular processing of mRNA lipoplexes and their uptake, with the former playing a predominant role.

This study aimed to assess the applicability of the Orbit ^® Subretinal Delivery System [Orbit ^® SDS, 2nd Generation, clinical-grade device] and prototypes for subretinal injection (SRI) in dogs of different ages. Following ex vivo and in vivo testing of the clinical grade Orbit ^® SDS in 7 adult canine eyes, 8 normal dogs (13 eyes; age range: 10–72 weeks) were injected with an AAV2/5-CBA-tdTomato-WPRE vector using different Orbit ^® SDS prototype devices. Dogs underwent non-invasive fundus photography regularly post-injection (PI). Retinal imaging including optical coherence tomography (OCT) angiography (OCTA) and en face null OCT was performed in most dogs at 5- and 9-weeks PI. Following termination, eyes were examined histologically. Ex vivo and in vivo SRI could be performed in 7 adult eyes including in that of a dog with end stage retinal degeneration. SRI of AAV2/5-CBA-tdTomato-WPRE was also successful in all 13 eyes including those of juvenile animals, yet this frequently required multiple attempts and/or using custom-modified devices that were adjusted to the smaller curvature of the ocular globes. Localized tdTomato expression was found in 11/13 eyes along the cannula path in the suprachoroidal space (SCS) in the sclera and choroid. Clinical and cellular signs of inflammation were seen in 7/13 eyes. The Orbit ^® SDS device allows for controlled subretinal injections via a posterior approach in adult and juvenile dogs. There was variable vector reflux from the subretinal bleb and/or leakage from the device into the SCS. A cellular immune reaction to the AAV capsid/transgene may have contributed to retinal and choroidal inflammation.

Progressive photoreceptor loss in retinal degenerative diseases leads to irreversible vision impairment. Transplantation of human embryonic or induced pluripotent stem cell-derived photoreceptor precursor cells (PRPCs) offers potential for vision restoration. However, substantial early donor cell loss remains a major challenge. This study aims to elucidate the mechanisms underlying early PRPC loss and to evaluate host retinal responses to transplantation. PRPCs derived from human embryonic stem cells (hESC)-based retinal organoids were subretinally transplanted into both normal and degenerated canine retinas to investigate the impact of host retinal degeneration on transplant survival and integration. Single-cell RNA sequencing (scRNAseq) was performed on transplanted PRPCs 3 days post-transplantation into normal canine retinas, as well as on host retinal cells to identify molecular pathways associated with early donor cell loss. Non-invasive multimodal retinal imaging and immunohistochemical analyses were conducted to assess PRPC survival, integration, and host immune responses. Despite systemic immunosuppression, extensive early loss of human PRPCs occurred within the first week following xenotransplantation into both normal and degenerated canine retinas, suggesting that factors beyond immune activation contribute to donor cell loss. Transcriptomic analysis identified metabolic stress as a key driver of early donor cell death, characterized by dysregulation of mitochondrial function and oxidative phosphorylation pathways. Microglial infiltration into the donor cell mass was also observed in normal retinas, suggesting a response to donor cell stress and apoptosis. Beyond the initial phase of cell death, surviving donor cells integrated and persisted when transplanted into retinas with a partially preserved outer nuclear layer, whereas cell loss continued when intervention occurred at end-stage degeneration. Metabolic stress represents a critical barrier to PRPC survival following transplantation. Strategies aimed at enhancing metabolic resilience may improve transplantation outcomes. Furthermore, host retinal responses shape the transplant microenvironment, influencing donor cell survival and integration. These findings highlight the need for targeted interventions to mitigate early metabolic stress and optimize PRPC transplantation strategies for retinal degenerative diseases.