Manifestations of systemic disease in the retina and fundus of cats and dogs.

The fundus is unique in that it is the only part of the body that allows for a noninvasive and uninterrupted view of vasculature and nervous tissue. Utilization of this can be a powerful tool in uncovering salient incidental findings which point to underlying systemic diseases, and for monitoring response to therapy. Retinal venules and arterioles allow the clinician to assess changes in vascular color, diameter, outline, and tortuosity. The retina and optic nerve may exhibit changes associated with increased or decreased thickness, inflammatory infiltrates, hemorrhages, and detachments. While some retinal manifestations of systemic disease may be nonspecific, others are pathognomonic, and may be the presenting sign for a systemic illness. The examination of the fundus is an essential part of the comprehensive physical examination. Systemic diseases which may present with retinal abnormalities include a variety of disease classifications, as represented by the DAMNIT-V acronym, for Degenerative/Developmental, Anomalous, Metabolic, Neoplastic, Nutritional, Inflammatory (Infectious/Immune-mediated/ischemic), Toxic, Traumatic and Vascular. This review details systemic illnesses or syndromes that have been reported to manifest in the fundus of companion animals and discusses key aspects in differentiating their underlying cause. Normal variations in retinal anatomy and morphology are also considered.

Metabolic changes and retinal remodeling in Heterozygous CRX mutant cats (CRXRDY/+).

CRX is a transcription factor essential for normal photoreceptor development and survival. The CRXRdy cat has a naturally occurring truncating mutation in CRX and is a large animal model for dominant Leber congenital amaurosis. This study investigated retinal remodeling that occurs as photoreceptors degenerate. CRXRdy/+ cats from 6 weeks to 10 years of age were investigated. In vivo structural changes of retinas were analyzed by fundus examination, confocal scanning laser ophthalmoscopy and spectral domain optical coherence tomography. Histologic analyses included immunohistochemistry for computational molecular phenotyping with macromolecules and small molecules. Affected cats had a cone-led photoreceptor degeneration starting in the area centralis. Initially there was preservation of inner retinal cells such as bipolar, amacrine and horizontal cells but with time migration of the deafferented neurons occurred. Early in the process of degeneration glial activation occurs ultimately resulting in formation of a glial seal. With progression the macula-equivalent area centralis developed severe atrophy including loss of retinal pigmentary epithelium. Microneuroma formation occured in advanced stages as more marked retinal remodeling occurred. This study indicates that retinal degeneration in the CrxRdy/+ cat retina follows the progressive, phased revision of retina that have been previously described for retinal remodeling. These findings suggest that therapy dependent on targeting inner retinal cells may be useful in young adults with preserved inner retinas prior to advanced stages of retinal remodeling and neuronal cell loss.

Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa.

In this study, we investigate a gene augmentation therapy candidate for the treatment of retinitis pigmentosa (RP) due to cyclic nucleotide-gated channel beta 1 (CNGB1) mutations. We use an adeno-associated virus serotype 5 with transgene under control of a novel short human rhodopsin promoter. The promoter/capsid combination drives efficient expression of a reporter gene (AAV5-RHO-eGFP) exclusively in rod photoreceptors in primate, dog, and mouse following subretinal delivery. The therapeutic vector (AAV5-RHO-CNGB1) delivered to the subretinal space of CNGB1 mutant dogs restores rod-mediated retinal function (electroretinographic responses and vision) for at least 12 months post treatment. Immunohistochemistry shows human CNGB1 is expressed in rod photoreceptors in the treated regions as well as restoration of expression and trafficking of the endogenous alpha subunit of the rod CNG channel required for normal channel formation. The treatment reverses abnormal accumulation of the second messenger, cyclic guanosine monophosphate, which occurs in rod photoreceptors of CNGB1 mutant dogs, confirming formation of a functional CNG channel. In vivo imaging shows long-term preservation of retinal structure. In conclusion, this study establishes the long-term efficacy of subretinal delivery of AAV5-RHO-CNGB1 to rescue the disease phenotype in a canine model of CNGB1-RP, confirming its suitability for future clinical development.

Alternative splicing in CEP290 mutant cats results in a milder phenotype than LCACEP290 patients.

PURPOSE: The rdAc cat has an intronic mutation in the centrosomal 290 kDa (CEP290) gene resulting in a frameshift and a premature stop codon (c.6960 + 9 T > G, p.Ile2321AlafsTer3) predicted to truncate the protein by 157 amino acids. CEP290 mutations in human patients cause a range or phenotypes including syndromic conditions and severe childhood loss of vision while the rdAc cat has a milder phenotype. We sought to further characterize the effect of rdAc mutation on CEP290 expression. METHODS: TaqMan quantitative real-time polymerase chain reaction assays were used to compare wildtype and truncated transcript levels. Relative protein abundance was analyzed by Western blot. Immunohistochemistry (IHC) was performed to detect CEP290 protein. RESULTS: CEP290 mutant cats show low-level (17.4% of wildtype cats) use of the wildtype splice site and usage of the mutant splice site. Western analysis shows retina from cats homozygous for the mutation has CEP290 protein that likely comprises a combination of both wildtype and truncated protein. IHC detects CEP290 in affected and control retina labeling the region of the interconnecting cilium. CONCLUSIONS: The comparably milder phenotype of CEP290 mutant cats is likely due to the retained production of some full-length CEP290 protein with possible functional contributions from presence of truncated protein.

Large Animal Models of Retinitis Pigmentosa in Therapy Development and Preclinical Testing.

Large animal models are valuable for developing and testing translational therapies for inherited retinal dystrophies such as retinitis pigmentosa (RP). Gene augmentation therapy has been developed utilizing such models. Adeno-associated viral (AAV) vectors have been frequently utilized and delivered by intravitreal or subretinal injection. In vivo longitudinal assessments of therapeutic outcomes are essential. These include regular ophthalmic examinations as well as detailed fundus assessments including confocal scanning laser ophthalmoscopy (cSLO) and high-resolution cross-sectional imaging of the retina by spectral domain-optical coherence tomography (SD-OCT). Retinal function assessment includes vision testing and electroretinography (ERG).

Localized alopecia and suppression of hypothalamic-pituitary-adrenal (HPA) axis in dogs following treatment with difluprednate 0.05% ophthalmic emulsion (Durezol®).

BACKGROUND: Despite the common use of topical ophthalmic corticosteroids in dogs, detailed reports on systemic and dermatologic adverse effects are limited. RESULTS: Nine purpose-bred research Beagles were treated with difluprednate 0.05% ophthalmic emulsion in one or both eyes 2-3 times daily. Some difluprednate treated dogs developed mild to severe alopecia of the periocular region, face, and distal pinna (5/9). The median duration of treatment prior to onset of dermatologic signs for difluprednate treated dogs was 550 days (453-1160 days). Diagnostic testing included complete blood count (CBC) and serum biochemistry, adrenocorticotropic hormone (ACTH) stimulation testing combined with endogenous ACTH measurement, and skin biopsy. The CBC and chemistry were within normal limits for all dogs. There were varying degrees of suppression of the hypothalamic-pituitary-adrenocortical (HPA) axis with difluprednate treatment. Dogs with the most profound alopecic changes had less pronounced HPA axis suppression compared to dogs with no integumentary changes. Skin biopsies demonstrated follicular atrophy and follicular keratosis. When topical difluprednate was reduced to unilateral therapy, the hair regrew on the untreated side of the face. In addition to the affected research dogs, a 7-year old female spayed Chihuahua that was being treated as a clinical patient with long-term difluprednate 0.05% ophthalmic emulsion developed generalized hypotrichosis on the head and body and a potbellied appearance. ACTH stimulation testing revealed suppression of the HPA axis with a mild increase in serum alkaline phosphatase (ALP) activity and a urine specific gravity of 1.016. The combination of clinical signs and laboratory abnormalities was supportive of iatrogenic hyperadrenocorticism. CONCLUSIONS: In dogs long-term use of difluprednate ophthalmic emulsion results in HPA axis suppression and in some cases iatrogenic hyperadrenocorticism. A novel pattern of localized alopecia is suspected to be related to dermal absorption and local action due to superior potency and penetration compared to other commonly utilized ophthalmic corticosteroids.

Subretinal Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in a Feline Large Animal Model.

Retinal degenerative (RD) conditions associated with photoreceptor loss such as age-related macular degeneration (AMD), retinitis pigmentosa (RP) and Leber Congenital Amaurosis (LCA) cause progressive and debilitating vision loss. There is an unmet need for therapies that can restore vision once photoreceptors have been lost. Transplantation of human pluripotent stem cell (hPSC)-derived retinal tissue (organoids) into the subretinal space of an eye with advanced RD brings retinal tissue sheets with thousands of healthy mutation-free photoreceptors and has a potential to treat most/all blinding diseases associated with photoreceptor degeneration with one approved protocol. Transplantation of fetal retinal tissue into the subretinal space of animal models and people with advanced RD has been developed successfully but cannot be used as a routine therapy due to ethical concerns and limited tissue supply. Large eye inherited retinal degeneration (IRD) animal models are valuable for developing vision restoration therapies utilizing advanced surgical approaches to transplant retinal cells/tissue into the subretinal space. The similarities in globe size, and photoreceptor distribution (e.g., presence of macula-like region area centralis) and availability of IRD models closely recapitulating human IRD would facilitate rapid translation of a promising therapy to the clinic. Presented here is a surgical technique of transplanting hPSC-derived retinal tissue into the subretinal space of a large animal model allowing assessment of this promising approach in animal models.

In Vivo Potency Testing of Subretinal rAAV5.hCNGB1 Gene Therapy in the Cngb1 Knockout Mouse Model of Retinitis Pigmentosa.

Retinitis pigmentosa type 45 (RP45) is an autosomal-recessively inherited blinding disease caused by mutations in the cyclic nucleotide-gated channel subunit beta 1 (CNGB1) gene. In this study, we developed and tested a novel gene supplementation therapy suitable for clinical translation. To this end, we designed a recombinant adeno-associated virus (rAAV) vector carrying a genome that features a novel human rhodopsin promoter (hRHO194) driving rod-specific expression of full-length human CNGB1 (rAAV5.hCNGB1). rAAV5.hCNGB1 was evaluated for efficacy in the Cngb1 knockout (Cngb1-/-) mouse model of RP45. In particular, increasing doses of rAAV5.hCNGB1 were delivered through single subretinal injection in 4-week-old Cngb1-/- mice and the treatment effect was assessed over a follow-up period of 9 months at the level of (1) retinal morphology, (2) retinal function, (3) vision-guided behavior, and (4) transgene expression. We found that subretinal treatment with rAAV5.hCNGB1 resulted in efficient expression of the human CNGB1 protein in mouse rods and was able to normalize the expression of the endogenous mouse CNGA1 subunit, which together with CNGB1 forms the native heterotetrameric cyclic guanosine monophosphate-gated cation channel in rod photoreceptors. The treatment led to a dose-dependent recovery of rod photoreceptor-driven function and preservation of retinal morphology in Cngb1-/- mice. In summary, these results demonstrate the efficacy of hCNGB1 gene supplementation therapy in the Cngb1-/- mouse model of RP45 and support the translation of this approach toward future clinical application.

Novel AAV capsids for intravitreal gene therapy of photoreceptor disorders.

Gene therapy using recombinant adeno-associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2-based peptide-display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high-level retinal transduction after intravitreal injection in mice, dogs and non-human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof-of-concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone-specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3-/- mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.

Pluripotent Stem Cell-Based Organoid Technologies for Developing Next-Generation Vision Restoration Therapies of Blindness.

Blindness, associated with death of retinal cells at the back of the eye, is caused by a number of conditions with high prevalence such as glaucoma, age-related macular degeneration, and diabetic retinopathy. In addition, a large number of orphan inherited (mostly monogenic) conditions, such as retinitis pigmentosa and Leber Congenital Amaurosis, add to the overall number of patients with blinding retinal degenerative diseases. Blindness caused by deterioration and loss of retina is so far incurable. Modern biomedical research leveraging molecular and regenerative medicine approaches had a number of groundbreaking discoveries and proof-of-principle treatments of blindness in animals. However, these methods are slow to be standardized and commercialized as therapies to benefit people losing their eyesight due to retinal degenerative conditions. In this review, we will outline major regenerative medicine approaches, which are emerging as promising for preserving or/and restoring vision. We discuss the potential of each of these approaches to reach commercialization step and be converted to treatments, which could at least ameliorate blindness caused by retinal cell death.

A CNTNAP1 Missense Variant Is Associated with Canine Laryngeal Paralysis and Polyneuropathy.

Laryngeal paralysis associated with a generalized polyneuropathy (LPPN) most commonly exists in geriatric dogs from a variety of large and giant breeds. The purpose of this study was to discover the underlying genetic and molecular mechanisms in a younger-onset form of this neurodegenerative disease seen in two closely related giant dog breeds, the Leonberger and Saint Bernard. Neuropathology of an affected dog from each breed showed variable nerve fiber loss and scattered inappropriately thin myelinated fibers. Using across-breed genome-wide association, haplotype analysis, and whole-genome sequencing, we identified a missense variant in the CNTNAP1 gene (c.2810G>A; p.Gly937Glu) in which homozygotes in both studied breeds are affected. CNTNAP1 encodes a contactin-associated protein important for organization of myelinated axons. The herein described likely pathogenic CNTNAP1 variant occurs in unrelated breeds at variable frequencies. Individual homozygous mutant LPPN-affected Labrador retrievers that were on average four years younger than dogs affected by geriatric onset laryngeal paralysis polyneuropathy could be explained by this variant. Pathologic changes in a Labrador retriever nerve biopsy from a homozygous mutant dog were similar to those of the Leonberger and Saint Bernard. The impact of this variant on health in English bulldogs and Irish terriers, two breeds with higher CNTNAP1 variant allele frequencies, remains unclear. Pathogenic variants in CNTNAP1 have previously been reported in human patients with lethal congenital contracture syndrome and hypomyelinating neuropathy, including vocal cord palsy and severe respiratory distress. This is the first report of contactin-associated LPPN in dogs characterized by a deleterious variant that most likely predates modern breed establishment.

A review of electroretinography waveforms and models and their application in the dog.

Electroretinography (ERG) is a commonly used technique to study retinal function in both clinical and research ophthalmology. ERG responses can be divided into component waveforms, analysis of which can provide insight into the health and function of different types and populations of retinal cells. In dogs, ERG has been used in the characterization of normal retinal function, as well as the diagnosis of retinal diseases and measuring effects of treatment. While many components of the recorded waveform are similar across species, dogs have several notable features that should be differentiated from the responses in humans and other animals. Additionally, modifications of standard protocols, such as changing flash frequency and stimulus color, and mathematical models of ERG waveforms have been used in studies of human retinal function but have been infrequently applied to visual electrophysiology in dogs. This review provides an overview of the origins and applications of ERG in addition to potential avenues for further characterization of responses in the dog.

Development of a protocol for maintaining viability while shipping organoid-derived retinal tissue.

Retinal organoid technology enables generation of an inexhaustible supply of three-dimensional retinal tissue from human pluripotent stem cells (hPSCs) for regenerative medicine applications. The high similarity of organoid-derived retinal tissue and transplantable human fetal retina provides an opportunity for evaluating and modeling retinal tissue replacement strategies in relevant animal models in the effort to develop a functional retinal patch to restore vision in patients with profound blindness caused by retinal degeneration. Because of the complexity of this very promising approach requiring specialized stem cell and grafting techniques, the tasks of retinal tissue derivation and transplantation are frequently split between geographically distant teams. Delivery of delicate and perishable neural tissue such as retina to the surgical sites requires a reliable shipping protocol and also controlled temperature conditions with damage-reporting mechanisms in place to prevent transplantation of tissue damaged in transit into expensive animal models. We have developed a robust overnight tissue shipping protocol providing reliable temperature control, live monitoring of the shipment conditions and physical location of the package, and damage reporting at the time of delivery. This allows for shipping of viable (transplantation-competent) hPSC-derived retinal tissue over large distances, thus enabling stem cell and surgical teams from different parts of the country to work together and maximize successful engraftment of organoid-derived retinal tissue. Although this protocol was developed for preclinical in vivo studies in animal models, it is potentially translatable for clinical transplantation in the future and will contribute to developing clinical protocols for restoring vision in patients with retinal degeneration.

Advancing Gene Therapy for PDE6A Retinitis Pigmentosa.

Mutations in the gene encoding the phosphodiesterase 6 alpha subunit (PDE6A) account for 3-4% of autosomal recessive retinitis pigmentosa (RP), and currently no treatment is available. There are four animal models for PDE6A-RP: a dog with a frameshift truncating mutation (p.Asn616ThrfsTer39) and three mouse models with missense mutations (Val685Met, Asp562Trp, and Asp670Gly) showing a range of phenotype severities. Initial proof-of-concept gene augmentation studies in the Asp670Gly mouse model and dog model used a subretinally delivered adeno-associated virus serotype 8 with a 733 tyrosine capsid mutation delivering species-specific Pde6a cDNAs. These restored some rod-mediated function and preserved retinal structure. Subsequently, a translatable vector (AAV8 with a human rhodopsin promoter and human PDE6A cDNA) was tested in the dog and the Asp670Gly mouse model. In the dog, there was restoration of rod function, a robust rod-mediated ERG, and introduction of dim-light vision. Treatment improved morphology of the photoreceptor layer, and the retina was preserved in the treated region. In the Asp670Gly mouse, therapy also preserved photoreceptors with cone survival being reflected by maintenance of cone-mediated ERG responses. These studies are an important step toward a translatable therapy for PDE6A-RP.

Transplantation of Human Embryonic Stem Cell-Derived Retinal Tissue in the Subretinal Space of the Cat Eye.

To develop biological approaches to restore vision, we developed a method of transplanting stem cell-derived retinal tissue into the subretinal space of a large-eye animal model (cat). Human embryonic stem cells (hESC) were differentiated to retinal organoids in a dish. hESC-derived retinal tissue was introduced into the subretinal space of wild-type cats following a pars plana vitrectomy. The cats were systemically immunosuppressed with either prednisolone or prednisolone plus cyclosporine A. The eyes were examined by fundoscopy and spectral-domain optical coherence tomography imaging for adverse effects due to the presence of the subretinal grafts. Immunohistochemistry was done with antibodies to retinal and human markers to delineate graft survival, differentiation, and integration into cat retina. We successfully delivered hESC-derived retinal tissue into the subretinal space of the cat eye. We observed strong infiltration of immune cells in the graft and surrounding tissue in the cats treated with prednisolone. In contrast, we showed better survival and low immune response to the graft in cats treated with prednisolone plus cyclosporine A. Immunohistochemistry with antibodies (STEM121, CALB2, DCX, and SMI-312) revealed large number of graft-derived fibers connecting the graft and the host. We also show presence of human-specific synaptophysin puncta in the cat retina. This work demonstrates feasibility of engrafting hESC-derived retinal tissue into the subretinal space of large-eye animal models. Transplanting retinal tissue in degenerating cat retina will enable rapid development of preclinical in vivo work focused on vision restoration.

Patients and animal models of CNGß1-deficient retinitis pigmentosa support gene augmentation approach.

Retinitis pigmentosa (RP) is a major cause of blindness that affects 1.5 million people worldwide. Mutations in cyclic nucleotide-gated channel ß 1 (CNGB1) cause approximately 4% of autosomal recessive RP. Gene augmentation therapy shows promise for treating inherited retinal degenerations; however, relevant animal models and biomarkers of progression in patients with RP are needed to assess therapeutic outcomes. Here, we evaluated RP patients with CNGB1 mutations for potential biomarkers of progression and compared human phenotypes with those of mouse and dog models of the disease. Additionally, we used gene augmentation therapy in a CNGß1-deficient dog model to evaluate potential translation to patients. CNGB1-deficient RP patients and mouse and dog models had a similar phenotype characterized by early loss of rod function and slow rod photoreceptor loss with a secondary decline in cone function. Advanced imaging showed promise for evaluating RP progression in human patients, and gene augmentation using adeno-associated virus vectors robustly sustained the rescue of rod function and preserved retinal structure in the dog model. Together, our results reveal an early loss of rod function in CNGB1-deficient patients and a wide window for therapeutic intervention. Moreover, the identification of potential biomarkers of outcome measures, availability of relevant animal models, and robust functional rescue from gene augmentation therapy support future work to move CNGB1-RP therapies toward clinical trials.

Gene Supplementation Rescues Rod Function and Preserves Photoreceptor and Retinal Morphology in Dogs, Leading the Way Toward Treating Human PDE6A-Retinitis Pigmentosa.

Mutations in the phosphodiesterase 6A gene (PDE6A) result in retinitis pigmentosa (RP) type 43 (RP43) and are responsible for about 4% of autosomal recessive RP. There is currently no treatment for this blinding condition. The aim of this project was to use a large-animal model to test a gene supplementation viral vector designed to be translated for use in a clinical trial for the treatment of RP43. Seven Pde6a-/- puppies were given sub-retinal injections of an adeno-associated viral vector (AAV) serotype 2/8 delivering human PDE6A cDNA under control of a short rhodopsin promoter (AAV8-PDE6A). Three puppies received ∼1 × 1011 vg in one eye and four puppies ∼5 × 1011 vg/per eye, with both eyes being injected in two animals. In vivo outcome measures included vision testing and electroretinography (ERG), as well as fundus and spectral domain-optical coherence tomography imaging. Some puppies were euthanized and their eyes processed for immunohistochemistry. All puppies had improved rod-mediated vision in the treated eye. ERGs showed improved rod-mediated responses in the higher-dose group but in only one of the lower-dose group animals. Receptor+ thickness was preserved and photoreceptor morphology improved in the treated retinal regions in all puppies. Treatment resulted in PDE6A transgene expression, accompanied by much increased levels of Pde6b, in rod outer segments in the injected retinal regions. There were several indications of improved retinal health in the PDE6A-expressing regions, including lack of abnormal cyclic guanosine monophosphate accumulation, appropriate rod opsin localization to the outer segments with a large reduction in mislocalization to other regions of the rod cell, and reduced Müller cell activation. Additionally, cone photoreceptors showed morphological improvement in the treated region, with normal-appearing inner and outer segments. AAV8-PDE6A gene supplementation therapy restored rod vision in Pde6a-/- puppies and preserved retinal morphology. These positive outcomes are an important step toward a human clinical trial to treat PDE6A-RP.

Birth of clones of the world's first cloned dog.

Animal cloning has gained popularity as a method to produce genetically identical animals or superior animals for research or industrial uses. However, the long-standing question of whether a cloned animal undergoes an accelerated aging process is yet to be answered. As a step towards answering this question, we compared longevity and health of Snuppy, the world's first cloned dog, and its somatic cell donor, Tai, a male Afghan hound. Briefly, both Snuppy and Tai were generally healthy until both developed cancer to which they succumbed at the ages of 10 and 12 years, respectively. The longevity of both the donor and the cloned dog was close to the median lifespan of Afghan hounds which is reported to be 11.9 years. Here, we report creation of 4 clones using adipose-derived mesenchymal stem cells from Snuppy as donor cells. Clinical and molecular follow-up of these reclones over their lives will provide us with a unique opportunity to study the health and longevity of cloned animals compared with their cell donors.

Gene Therapy in a Large Animal Model of PDE6A-Retinitis Pigmentosa.

Despite mutations in the rod phosphodiesterase 6-alpha (PDE6A) gene being well-recognized as a cause of human retinitis pigmentosa, no definitive treatments have been developed to treat this blinding disease. We performed a trial of retinal gene augmentation in the Pde6a mutant dog using Pde6a delivery by capsid-mutant adeno-associated virus serotype 8, previously shown to have a rapid onset of transgene expression in the canine retina. Subretinal injections were performed in 10 dogs at 29-44 days of age, and electroretinography and vision testing were performed to assess functional outcome. Retinal structure was assessed using color fundus photography, spectral domain optical coherence tomography, and histology. Immunohistochemistry was performed to examine transgene expression and expression of other retinal genes. Treatment resulted in improvement in dim light vision and evidence of rod function on electroretinographic examination. Photoreceptor layer thickness in the treated area was preserved compared with the contralateral control vector treated or uninjected eye. Improved rod and cone photoreceptor survival, rhodopsin localization, cyclic GMP levels and bipolar cell dendrite distribution was observed in treated areas. Some adverse effects including foci of retinal separation, foci of retinal degeneration and rosette formation were identified in both AAV-Pde6a and control vector injected regions. This is the first description of successful gene augmentation for Pde6a retinitis pigmentosa in a large animal model. Further studies will be necessary to optimize visual outcomes and minimize complications before translation to human studies.

CrxRdy Cat: A Large Animal Model for CRX-Associated Leber Congenital Amaurosis.

PURPOSE: Mutations in the retinal transcription factor cone-rod homeobox (CRX) gene result in severe dominant retinopathies. A large animal model, the Rdy cat, carrying a spontaneous frameshift mutation in Crx, was reported previously. The present study aimed to further understand pathogenesis in this model by thoroughly characterizing the Rdy retina. METHODS: Structural and functional changes were found in a comparison between the retinas of CrxRdy/+ kittens and those of wild-type littermates and were determined at various ages by fundus examination, electroretinography (ERG), optical coherence tomography, and histologic analyses. RNA and protein expression changes of Crx and key target genes were analyzed using quantitative reverse-transcribed PCR, Western blot analysis, and immunohistochemistry. Transcription activity of the mutant Crx was measured by a dual-luciferase transactivation assay. RESULTS: CrxRdy/+ kittens had no recordable cone ERGs. Rod responses were delayed in development and markedly reduced at young ages and lost by 20 weeks. Photoreceptor outer segment development was incomplete and was followed by progressive outer retinal thinning starting in the cone-rich area centralis. Expression of cone and rod Crx target genes was significantly down-regulated. The mutant Crx allele was overexpressed, leading to high levels of the mutant protein lacking transactivation activity. CONCLUSIONS: The CrxRdy mutation exerts a dominant negative effect on wild-type Crx by overexpressing mutant protein. These findings, consistent with those of studies in a mouse model, support a conserved pathogenic mechanism for CRX frameshift mutations. The similarities between the feline eye and the human eye with the presence of a central region of high cone density makes the CrxRdy/+ cat a valuable model for preclinical testing of therapies for dominant CRX diseases.