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.

[Canine inherited retinal degeneration as model to study disease mechanisms and therapy for ciliopathies].

Humans have a highly developed retina and obtain approximately 80% of their external information from vision. Photoreceptor cells, which are located in the outermost layer of the neuroretina and recognize light signals, are highly specialized sensory cilia that share structural and functional features with primary cilia. Genetic disorders of the retina or photoreceptor cells are termed inherited retinal diseases (IRDs) and are caused by variants in one of more than 280 genes identified to date. Among the genes responsible for IRDs, many are shared with those responsible for ciliopathies. In studies of inherited diseases, mouse models are commonly used due to their advantages in breeding, handling, and relative feasibility in creating pathological models. On the other hand, structural, functional, and genetic differences in the retina between mice and humans can be a barrier in IRD research. To overcome the limitations of mouse models, larger vertebrate models of IRDs can be a useful research subject. In particular, canines have retinas that are structurally and functionally similar and eyes that are anatomically comparable to those of humans. In addition, due to their unique veterinary clinical surveillance and genetic background, naturally occurring canine IRDs are more likely to be identified than in other large animals. To date, pathogenic mutations related to canine IRDs have been identified in more than 30 genes, contributing to the understanding of pathogeneses and to the development of new therapies. This review provides an overview of the roles of the canine IRD models in ciliopathy research.

Combination HSCT and intravenous AAV-mediated gene therapy in a canine model proves pivotal for translation of Krabbe disease therapy.

Hematopoietic stem cell transplantation (HSCT) is the only approved treatment for presymptomatic infantile globoid cell leukodystrophy (GLD [Krabbe disease]). However, correction of disease is not complete, and outcomes remain poor. Herein we evaluated HSCT, intravenous (IV) adeno-associated virus rh10 vector (AAVrh10) gene therapy, and combination HSCT + IV AAVrh10 in the canine model of GLD. While HSCT alone resulted in no increase in survival as compared with untreated GLD dogs (∼16 weeks of age), combination HSCT + IV AAVrh10 at a dose of 4E13 genome copies (gc)/kg resulted in delayed disease progression and increased survival beyond 1 year of age. A 5-fold increase in AAVrh10 dose to 2E14 gc/kg, in combination with HSCT, normalized neurological dysfunction up to 2 years of age. IV AAVrh10 alone resulted in an average survival to 41.2 weeks of age. In the peripheral nervous system, IV AAVrh10 alone or in addition to HSCT normalized nerve conduction velocity, improved ultrastructure, and normalized GALC enzyme activity and psychosine concentration. In the central nervous system, only combination therapy at the highest dose was able to restore galactosylceramidase activity and psychosine concentrations to within the normal range. These data have now guided clinical translation of systemic AAV gene therapy as an addition to HSCT (NCT04693598, NCT05739643).

Recent advances in the understanding of cilia mechanisms and their applications as therapeutic targets.

The primary cilium is a single immotile microtubule-based organelle that protrudes into the extracellular space. Malformations and dysfunctions of the cilia have been associated with various forms of syndromic and non-syndromic diseases, termed ciliopathies. The primary cilium is therefore gaining attention due to its potential as a therapeutic target. In this review, we examine ciliary receptors, ciliogenesis, and ciliary trafficking as possible therapeutic targets. We first discuss the mechanisms of selective distribution, signal transduction, and physiological roles of ciliary receptors. Next, pathways that regulate ciliogenesis, specifically the Aurora A kinase, mammalian target of rapamycin, and ubiquitin-proteasome pathways are examined as therapeutic targets to regulate ciliogenesis. Then, in the photoreceptors, the mechanism of ciliary trafficking which takes place at the transition zone involving the ciliary membrane proteins is reviewed. Finally, some of the current therapeutic advancements highlighting the role of large animal models of photoreceptor ciliopathy are discussed.

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.

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.

Monocular retinopathy of prematurity-like retinal vasculopathy in a dog.

PURPOSE: To describe a case of monocular retinopathy of prematurity (ROP)-like vasculopathy without oxygen supplementation in the dog. METHODS: Fundus photographs (RetCam), spectral-domain optical coherence tomography (sdOCT), confocal scanning laser ophthalmoscopy (cSLO), and fluorescein angiography (FA), as well as postmortem histology and immunohistochemistry (Collagen IV and anti-vWF antibodies), were carried out to characterize the vascular abnormalities. RESULTS: Ophthalmic examination showed peripheral and mid-temporal avascular areas in the tapetal region, neovascularization and abnormally dilated and tortuous retinal vessels in the left eye. sdOCT demonstrated not only cross-sectional views of preretinal fibrovascular proliferation but also extensive proliferation extraretinally into the vitreous. FA emphasized demarcation of vascular and avascular zones with neovascular tufts "popcorns." Histology and immunohistochemistry confirmed presence of abnormally dilated vessels and the intravitreal blood vessels. CONCLUSIONS: ROP is a disease of abnormally developed retinal vascularization associated with oxygen supplementation therapy, potentially causing blindness in premature infants. Although the mechanism of ROP-like vasculopathy in our case is unclear, it is important to appreciate that the abnormal vascular pattern seen in ROP in premature infants can occur in canines without oxygen administration.

CCDC66 frameshift variant associated with a new form of early-onset progressive retinal atrophy in Portuguese Water Dogs.

Aberrant photoreceptor function or morphogenesis leads to blinding retinal degenerative diseases, the majority of which have a genetic aetiology. A variant in PRCD previously identified in Portuguese Water Dogs (PWDs) underlies prcd (progressive rod-cone degeneration), an autosomal recessive progressive retinal atrophy (PRA) with a late onset at 3-6 years of age or older. Herein, we have identified a new form of early-onset PRA (EOPRA) in the same breed. Pedigree analysis suggested an autosomal recessive inheritance. Four PWD full-siblings affected with EOPRA diagnosed at 2-3 years of age were genotyped (173,661 SNPs) along with 2 unaffected siblings, 2 unaffected parents, and 15 unrelated control PWDs. GWAS, linkage analysis and homozygosity mapping defined a 26-Mb candidate region in canine chromosome 20. Whole-genome sequencing in one affected dog and its obligatory carrier parents identified a 1 bp insertion (CFA20:g.33,717,704_33,717,705insT (CanFam3.1); c.2262_c.2263insA) in CCDC66 predicted to cause a frameshift and truncation (p.Val747SerfsTer8). Screening of an extended PWD population confirmed perfect co-segregation of this genetic variant with the disease. Western blot analysis of COS-1 cells transfected with recombinant mutant CCDC66 expression constructs showed the mutant transcript translated into a truncated protein. Furthermore, in vitro studies suggest that the mutant CCDC66 is mislocalized to the nucleus relative to wild type CCDC66. CCDC66 variants have been associated with inherited retinal degenerations (RDs) including canine and murine ciliopathies. As genetic variants affecting the primary cilium can cause ciliopathies in which RD may be either the sole clinical manifestation or part of a syndrome, our findings further support a role for CCDC66 in retinal function and viability, potentially through its ciliary function.

Krabbe disease successfully treated via monotherapy of intrathecal gene therapy.

Globoid cell leukodystrophy (GLD; Krabbe disease) is a progressive, incurable neurodegenerative disease caused by deficient activity of the hydrolytic enzyme galactosylceramidase (GALC). The ensuing cytotoxic accumulation of psychosine results in diffuse central and peripheral nervous system (CNS, PNS) demyelination. Presymptomatic hematopoietic stem cell transplantation (HSCT) is the only treatment for infantile-onset GLD; however, clinical outcomes of HSCT recipients often remain poor, and procedure-related morbidity is high. There are no effective therapies for symptomatic patients. Herein, we demonstrate in the naturally occurring canine model of GLD that presymptomatic monotherapy with intrathecal AAV9 encoding canine GALC administered into the cisterna magna increased GALC enzyme activity, normalized psychosine concentration, improved myelination, and attenuated inflammation in both the CNS and PNS. Moreover, AAV-mediated therapy successfully prevented clinical neurological dysfunction, allowing treated dogs to live beyond 2.5 years of age, more than 7 times longer than untreated dogs. Furthermore, we found that a 5-fold lower dose resulted in an attenuated form of disease, indicating that sufficient dosing is critical. Finally, postsymptomatic therapy with high-dose AAV9 also significantly extended lifespan, signifying a treatment option for patients for whom HSCT is not applicable. If translatable to patients, these findings would improve the outcomes of patients treated either pre- or postsymptomatically.

Intrastromal Gene Therapy Prevents and Reverses Advanced Corneal Clouding in a Canine Model of Mucopolysaccharidosis I.

Mucopolysaccharidosis type I (MPS I) is an autosomal recessive lysosomal storage disease characterized by severe phenotypes, including corneal clouding. MPS I is caused by mutations in alpha-l-iduronidase (IDUA), a ubiquitous enzyme that catalyzes the hydrolysis of glycosaminoglycans. Currently, no treatment exists to address MPS I corneal clouding other than corneal transplantation, which is complicated by a high risk for rejection. Investigation of an adeno-associated virus (AAV) IDUA gene addition strategy targeting the corneal stroma addresses this deficiency. In MPS I canines with early or advanced corneal disease, a single intrastromal AAV8G9-IDUA injection was well tolerated at all administered doses. The eyes with advanced disease demonstrated resolution of corneal clouding as early as 1 week post-injection, followed by sustained corneal transparency until the experimental endpoint of 25 weeks. AAV8G9-IDUA injection in the MPS I canine eye with early corneal disease prevented the development of advanced corneal changes while restoring clarity. Biodistribution studies demonstrated vector genomes in ocular compartments other than the cornea and in some systemic organs; however, a capsid antibody response was detected in only the highest dosed subject. Collectively, the results suggest that intrastromal AAV8G9-IDUA therapy prevents and reverses visual impairment associated with MPS I corneal clouding.

Genome-wide association study and whole-genome sequencing identify a deletion in LRIT3 associated with canine congenital stationary night blindness.

Congenital stationary night blindness (CSNB), in the complete form, is caused by dysfunctions in ON-bipolar cells (ON-BCs) which are secondary neurons of the retina. We describe the first disease causative variant associated with CSNB in the dog. A genome-wide association study using 12 cases and 11 controls from a research colony determined a 4.6 Mb locus on canine chromosome 32. Subsequent whole-genome sequencing identified a 1 bp deletion in LRIT3 segregating with CSNB. The canine mutant LRIT3 gives rise to a truncated protein with unaltered subcellular expression in vitro. Genetic variants in LRIT3 have been associated with CSNB in patients although there is limited evidence regarding its apparently critical function in the mGluR6 pathway in ON-BCs. We determine that in the canine CSNB retina, the mutant LRIT3 is correctly localized to the region correlating with the ON-BC dendritic tips, albeit with reduced immunolabelling. The LRIT3-CSNB canine model has direct translational potential enabling studies to help understand the CSNB pathogenesis as well as to develop new therapies targeting the secondary neurons of the retina.

Author Correction: Variabilities in retinal function and structure in a canine model of cone-rod dystrophy associated with RPGRIP1 support multigenic etiology.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

Mapping of Canine Models of Inherited Retinal Diseases.

The gene/mutation discovery approaches for inherited retinal diseases (RDs) in the dog model have seen considerable development over the past 25 years. Initial attempts were focused on candidate genes, followed by genome-wide approaches including linkage analysis and DNA-chip-based genome-wide association study. Combined, there are as many as 32 mutations in 27 genes that have been associated with canine retinal diseases to date. More recently, next-generation sequencing has become one of the key methods of choice. With increasing knowledge of the molecular basis of RDs and follow-up surveys in different subpopulations, the conventional understanding of RDs as simple Mendelian traits is being challenged. Modifiers and involvement of multiple genes that alter the disease expression are complicating the prediction of the disease course. In this chapter, advances in the gene/mutation discovery approaches for canine RDs are reviewed, and a multigenic form of canine RD is discussed using a form of canine cone-rod dystrophy as an example.

Variabilities in retinal function and structure in a canine model of cone-rod dystrophy associated with RPGRIP1 support multigenic etiology.

Defects in the cilia gene RPGRIP1 cause Leber congenital amaurosis and cone-rod dystrophy in humans. A form of canine cone-rod dystrophy (cord1) was originally associated with a homozygous insertion in RPGRIP1 (RPGRIP1 ins/ins) as the primary disease locus while a homozygous deletion in MAP9 (MAP9 del/del) was later identified as a modifier associated with the early onset form. However, we find further variability in cone electroretinograms (ERGs) ranging from normal to absent in an extended RPGRIP1 ins/ins canine colony, irrespective of the MAP9 genotype. Ophthalmoscopically, cone ERGabsent RPGRIP1 ins/ins eyes show discolouration of the tapetal fundus with varying onset and disease progression, while sd-OCT reveals atrophic changes. Despite marked changes in cone ERG and retinal morphology, photopic vision-guided behaviour is comparable between normal and cone ERGabsent RPGRIP1 ins/ins littermates. Cone morphology of the dogs lacking cone ERG are truncated with shortened outer and inner segments. Immunohistochemically, cone ERGabsent RPGRIP1 ins/ins retinas have extensive L/M-opsin mislocalization, lack CNGB3 labelling in the L/M-cones, and lack GC1 in all cones. Our results indicate that cord1 is a multigenic disease in which mutations in neither RPGRIP1 nor MAP9 alone lead to visual deficits, and additional gene(s) contribute to cone-specific functional and morphologic defects.

Canine genome assembly correction facilitates identification of a MAP9 deletion as a potential age of onset modifier for RPGRIP1-associated canine retinal degeneration.

Retinal degeneration (RD) in the Miniature Long Haired Dachshund (MLHD) is a cone-rod dystrophy resulting in eventual blindness in affected individuals. In a previous study, a 44-nucleotide insertion (ins44) in exon 2 of RPGRIP1 was associated with RD. However, results on an extended population of MLHD revealed a variable RD onset age for ins44 homozygous dogs. Further investigations using a genome-wide association study comparing early onset and late onset RD cases identified an age of onset modifying locus for RD, approximately 30 Mb upstream of RPGRIP1 on chr15. In this investigation, target enriched sequencing identified a MAP9 deletion spanning approximately 22 kb associated with early RD onset. Identification of the deletion required correction to the CanFam3.1 genome build as canine MAP9 is part of a historic tandem duplication, resulting in incomplete assembly of this genome region. The deletion breakpoints were identified in MAP9 intron 10 and in a downstream partial MAP9 pseudogene. The fusion of these two genes, which we have called MAP9 EORD (microtubule-associated protein, early onset retinal degeneration), is in frame and is expressed at the RNA level, with the 3' region containing several predicted deleterious variants. We speculate that MAP9 associates with α-tubulin in the basal body of the cilium. RPGRIP1 is also known to locate to the cilium, where it is closely associated with RPGR. RPGRIP1 mutations also cause redistribution of α-tubulin away from the ciliary region in photoreceptors. Hence, a MAP9 partial deficit is a particularly attractive candidate to synergise with a partial RPGRIP1 deficit to cause a more serious disease.

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.