Significance Canine 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.

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 noninvasive 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.

AAV gene therapies aimed at curing inherited retinal diseases to date have typically focused on photoreceptors and retinal pigmented epithelia within the relatively accessible outer retina. However, therapeutic targeting in diseases such as congenital stationary night blindness (CSNB) that involve defects in ON-bipolar cells (ON-BCs) within the mid-retina has been challenged by the relative inaccessibility of the target cell in intact retinas, the limited transduction efficiency of these cells by existing AAV serotypes, poor availability of established ON-BC-specific promoters, and absence of appropriate patient-relevant large animal models. Here, we demonstrate safe and effective ON-BC targeting by AAV gene therapy in a recently characterized naturally-occurring canine model of CSNB, LRIT3-CSNB. To effectively target ON-BCs, new AAV capsid variants with ON-BC tropism and ON-BC specific modified GRM6 promoters were adopted to ensure cell-specific transgene expression. Notably, subretinal injection of one vector, AAVK9#4-shGRM6-cLRIT3-WPRE, significantly recovered rod-derived b-wave in all treated eyes (6/6) of adult dogs injected at 1-3 years of age. The robust therapeutic effect was evident 7 weeks post-injection and was sustained for at least 1 year in all treated eyes. Scotopic vision was significantly improved in treated eyes based on visually-guided obstacle course navigation. Restoration of LRIT3 signals was confirmed by immunohistochemistry. Thus, we report on the first ON-BC functional rescue in a large animal model using a novel AAV capsid variant and modified promoter construct optimized for ON-BC specificity, thereby establishing both proof-of-concept and a novel translational platform for treatment of CSNB in patients with defects in photoreceptor-to-bipolar signaling.

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