Gene Editing for CEP290-Associated Retinal Degeneration.

BACKGROUND: CEP290-associated inherited retinal degeneration causes severe early-onset vision loss due to pathogenic variants in CEP290. EDIT-101 is a clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) gene-editing complex designed to treat inherited retinal degeneration caused by a specific damaging variant in intron 26 of CEP290 (IVS26 variant). METHODS: We performed a phase 1-2, open-label, single-ascending-dose study in which persons 3 years of age or older with CEP290-associated inherited retinal degeneration caused by a homozygous or compound heterozygous IVS26 variant received a subretinal injection of EDIT-101 in the worse (study) eye. The primary outcome was safety, which included adverse events and dose-limiting toxic effects. Key secondary efficacy outcomes were the change from baseline in the best corrected visual acuity, the retinal sensitivity detected with the use of full-field stimulus testing (FST), the score on the Ora-Visual Navigation Challenge mobility test, and the vision-related quality-of-life score on the National Eye Institute Visual Function Questionnaire-25 (in adults) or the Children's Visual Function Questionnaire (in children). RESULTS: EDIT-101 was injected in 12 adults 17 to 63 years of age (median, 37 years) at a low dose (in 2 participants), an intermediate dose (in 5), or a high dose (in 5) and in 2 children 9 and 14 years of age at the intermediate dose. At baseline, the median best corrected visual acuity in the study eye was 2.4 log10 of the minimum angle of resolution (range, 3.9 to 0.6). No serious adverse events related to the treatment or procedure and no dose-limiting toxic effects were recorded. Six participants had a meaningful improvement from baseline in cone-mediated vision as assessed with the use of FST, of whom 5 had improvement in at least one other key secondary outcome. Nine participants (64%) had a meaningful improvement from baseline in the best corrected visual acuity, the sensitivity to red light as measured with FST, or the score on the mobility test. Six participants had a meaningful improvement from baseline in the vision-related quality-of-life score. CONCLUSIONS: The safety profile and improvements in photoreceptor function after EDIT-101 treatment in this small phase 1-2 study support further research of in vivo CRISPR-Cas9 gene editing to treat inherited retinal degenerations due to the IVS26 variant of CEP290 and other genetic causes. (Funded by Editas Medicine and others; BRILLIANCE ClinicalTrials.gov number, NCT03872479.).

FOVEAL PHENOTYPES IN CHOROIDEREMIA ON ADAPTIVE OPTICS SCANNING LIGHT OPHTHALMOSCOPY.

PURPOSE: Choroideremia is an X-linked inherited retinal degeneration involving the choriocapillaris, retinal pigment epithelium, and photoreceptors. Adaptive optics scanning light ophthalmoscopy allows visualization of retinal structure at the level of individual cells and is well poised to provide insight into the pathophysiologic mechanisms underpinning the retinal degeneration in choroideremia. METHODS: Foveal adaptive optics scanning light ophthalmoscopy images of 102 eyes of 54 individuals with choroideremia were analyzed. Measures were compared with those from standard clinical imaging. Visual acuity was also measured and compared with quantitative foveal metrics. RESULTS: The 3 distinct phenotypes observed were: relatively normal (5 eyes, 4 individuals), spiderweb (9 eyes, 7 individuals), and salt and pepper (87 eyes, 47 individuals). Peak cone density (86 eyes of 51 individuals) was significantly lower in choroideremia than in healthy retinas (P < 0.0001, range: 29,382-157,717 cones/mm2). Peak cone density was significantly related to extent of retained ellipsoid zone on en face optical coherence tomography (r2 = 0.47, P = 0.0009) and inversely related to visual acuity (r2 = 0.20, P = 0.001). CONCLUSION: Distinct phenotypes can be observed on adaptive optics scanning light ophthalmoscopy imaging in choroideremia that cannot always be discerned on standard clinical imaging. Quantitative measures on adaptive optics imaging are related to the structural and functional severity of disease.

Ocular Biomarkers of Riboflavin Transporter Deficiency.

BACKGROUND: To describe the clinical presentation with a focus on ocular manifestations and response to riboflavin supplementation of 3 patients with riboflavin transporter deficiency (RTD) caused by mutations in SLC52A2 ( SLC52A2- RTD). METHODS: This is a retrospective review of records of 3 children (aged 18, n = 2 and age = 8, n = 1) with SLC52A2- RTD. Patients underwent comprehensive ophthalmic evaluations including color vision testing, pattern visual-evoked potentials (pVEPs, 1 patient) and spectral domain optical coherence tomography (SD-OCT) imaging. Patients received riboflavin supplements from the time of the molecular diagnosis of RTD. RESULTS: Two unrelated 18-year-old patients with SLC52A2- RTD had a symptomatic onset with sensorineural hearing loss and auditory neuropathy/dys-synchrony since age 3 and 11, respectively. On examination 7 years after symptomatic onset, they showed subnormal visual acuities (20/30 and 20/60, both eyes, respectively), preserved color vision, and a thin but measurable retinal ganglion cell layer (GCL) and nerve fiber (RNFL). The inner and outer nuclear layers were normal. The asymptomatic SLC52A2- positive brother of one of these patients started riboflavin supplementation right after the molecular diagnosis and had normal vision and SD-OCTs 7 years later. Onset of riboflavin supplementation in one of the 2 symptomatic cases resulted in acute improvement of the pattern visual-evoked potential and vision. CONCLUSIONS: Retinal ganglion cells and their axons are uniquely susceptible to RTD compared with other highly energy-dependent retinal neurons, such as photoreceptors, raising the possibility for alternative mechanisms of disease or protection. Riboflavin supplementation results in acute functional improvement of vision and long-term preservation of GCL and RNFL if initiated early.

Adeno-Associated Virus Serotype 2-hCHM Subretinal Delivery to the Macula in Choroideremia: Two-Year Interim Results of an Ongoing Phase I/II Gene Therapy Trial.

PURPOSE: To assess the safety of the subretinal delivery of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human choroideremia (CHM)-encoding cDNA in CHM. DESIGN: Prospective, open-label, nonrandomized, dose-escalation, phase I/II clinical trial. PARTICIPANTS: Fifteen CHM patients (ages 20-57 years at dosing). METHODS: Patients received uniocular subfoveal injections of low-dose (up to 5 × 1010 vector genome [vg] per eye, n = 5) or high-dose (up to 1 × 1011 vg per eye, n = 10) of a recombinant adeno-associated virus serotype 2 (AAV2) vector carrying a human CHM-encoding cDNA (AAV2-hCHM). Patients were evaluated preoperatively and postoperatively for 2 years with ophthalmic examinations, multimodal retinal imaging, and psychophysical testing. MAIN OUTCOME MEASURES: Visual acuity, perimetry (10-2 protocol), spectral-domain OCT (SD-OCT), and short-wavelength fundus autofluorescence (SW-FAF). RESULTS: We detected no vector-related or systemic toxicities. Visual acuity returned to within 15 letters of baseline in all but 2 patients (1 developed acute foveal thinning, and 1 developed a macular hole); the rest showed no gross changes in foveal structure at 2 years. There were no significant differences between intervention and control eyes in mean light-adapted sensitivity by perimetry or in the lateral extent of retinal pigment epithelium relative preservation by SD-OCT and SW-FAF. Microperimetry showed nonsignificant (< 3 standard deviations of the intervisit variability) gains in sensitivity in some locations and participants in the intervention eye. There were no obvious dose-dependent relationships. CONCLUSIONS: Visual acuity was within 15 letters of baseline after the subfoveal AAV2-hCHM injections in 13 of 15 patients. Acute foveal thinning with unchanged perifoveal function in 1 patient and macular hole in 1 patient suggest foveal vulnerability to the subretinal injections. Longer observation intervals will help establish the significance of the minor differences in sensitivities and rate of disease progression observed between intervention and control eyes.

Clinical Perspective: Treating RPE65-Associated Retinal Dystrophy.

Until recently, there was no approved treatment for a retinal degenerative disease. Subretinal injection of a recombinant adeno-associated virus (AAV) delivering the normal copy of the human RPE65 cDNA led to reversal of blindness first in animal models and then in humans. This led to the first US Food and Drug Administration (FDA)-approved gene therapy product for a genetic disease, voretigene neparvovec-rzyl (Luxturna). Luxturna was then approved by the European Medicines Association and is now available in the US through Spark Therapeutics and worldwide through Novartis. Not only has treatment with Luxturna changed the lives of people previously destined to live a life of blindness, but it has fueled interest in developing additional gene therapy reagents targeting numerous other genetic forms of inherited retinal disease. This review describes many of the considerations for administration of Luxturna and describes how lessons from experience with Luxturna could lead to additional gene-based treatments of blindness.

Variants in myelin regulatory factor (MYRF) cause autosomal dominant and syndromic nanophthalmos in humans and retinal degeneration in mice.

Nanophthalmos is a rare, potentially devastating eye condition characterized by small eyes with relatively normal anatomy, a high hyperopic refractive error, and frequent association with angle closure glaucoma and vision loss. The condition constitutes the extreme of hyperopia or farsightedness, a common refractive error that is associated with strabismus and amblyopia in children. NNO1 was the first mapped nanophthalmos locus. We used combined pooled exome sequencing and strong linkage data in the large family used to map this locus to identify a canonical splice site alteration upstream of the last exon of the gene encoding myelin regulatory factor (MYRF c.3376-1G>A), a membrane bound transcription factor that undergoes autoproteolytic cleavage for nuclear localization. This variant produced a stable RNA transcript, leading to a frameshift mutation p.Gly1126Valfs*31 in the C-terminus of the protein. In addition, we identified an early truncating MYRF frameshift mutation, c.769dupC (p.S264QfsX74), in a patient with extreme axial hyperopia and syndromic features. Myrf conditional knockout mice (CKO) developed depigmentation of the retinal pigment epithelium (RPE) and retinal degeneration supporting a role of this gene in retinal and RPE development. Furthermore, we demonstrated the reduced expression of Tmem98, another known nanophthalmos gene, in Myrf CKO mice, and the physical interaction of MYRF with TMEM98. Our study establishes MYRF as a nanophthalmos gene and uncovers a new pathway for eye growth and development.

Compound Heterozygous Mutations in ZNF408 in a Patient with a Late Onset Pigmentary Retinopathy and Relatively Preserved Central Retina.

PURPOSE: To describe in detail the phenotype of a patient with compound heterozygous mutations in ZNF408 and an adult-onset pigmentary retinopathy rather than familial exudative vitreoretinopathy as expected with heterozygous mutations in this gene. METHODS: A 70-year-old male presented with a pigmentary retinopathy, which prompted a genetic evaluation that revealed two variants in trans in the ZNF408 gene. He underwent an ophthalmic examination, kinetic fields, electroretinography (ERG), spectral-domain optical coherence tomography (SD-OCT), fundus autofluorescence, wide-angle fluorescein angiography and near-infrared imaging. RESULTS: Visual acuity was 20/20 for both eyes. Fundus examination showed epiretinal membrane, vascular attenuation and peripheral bone spicule pigmentation in both eyes. Fluorescein angiography showed no vascular anomalies in both eyes. Fundus autofluorescence showed a preserved island of fundus autofluorescence centrally. Visual field by kinetic perimetry (V-4e stimulus) showed generalized constriction to 40 degrees of eccentricity and by an I-4e target showed generalized constriction to 10 degrees of eccentricity. ERG showed detectable but reduced cone-mediated responses. SD-OCT demonstrated preserved outer nuclear layer thickness centrally, which decreased with eccentricity. Static perimetry showed substantial rod and cone sensitivities centrally that declined with eccentricity. A next-generation sequencing panel revealed bi-allelic variants (p.Arg567Ter; c.1699C > T and p.Leu566His; c.1697 T > A) in the ZNF408 gene. CONCLUSIONS: ZNF408-associated retinal dystrophies can present with predominantly retinal findings and should be considered in the differential diagnosis of retinitis pigmentosa. Our study revealed a novel variant p.L566H, which to our knowledge has not previously been reported.

Unicoronal Craniosynostosis: Is There a Lateral Difference in Retinal Morphology?

BACKGROUND: Craniosynostosis is the premature fusion of cranial sutures in pediatric patients, which may lead to elevated intracranial pressure due to cerebro-cephalic disproportion between a growing brain and constricted skull. It is unknown whether this increased pressure is distributed equally throughout the cranial vault, or whether certain areas of the brain experience greater pressure at these regions of premature osseous fusion. METHODS: Optical coherence tomography (OCT) is a noninvasive modality for detecting elevated intracranial pressure. Optical coherence tomography was utilized to measure the peripapillary retinal nerve fiber layer (RNFL) thickness in patients undergoing surgical correction of craniosynostosis. Retinal nerve fiber layer in the eye ipsilateral to the unicoronal suture fusion was compared to the RNFL in the eye contralateral to the unicoronal suture fusion. RESULTS: During the study interval, 21 patients met inclusion criteria. Median age at operative intervention was 8.0 months, and 28.6% patients presented with left-sided unicoronal craniosynostosis, whereas 71.4% of patients presented with right-sided unicoronal craniosynostosis. Rather than universal increase on the affected side of coronal suture fusion, retinal nerve fiber layer thickness parameters showed a rotation phenomenon, such that the patterns of elevation had a 45° circumferential rotation in the direction of intorsion. CONCLUSIONS: The explanation for these results remains elusive, but they likely indicate either intracranial changes transmitted differentially to the peripapillary retina, or differing retinal morphology, between the ipsilateral and contralateral eyes in unicoronal craniosynostosis.

Structural correlates of atypical visual and motor cortical oscillations in pediatric-onset multiple sclerosis.

We have previously demonstrated that pediatric-onset multiple sclerosis (POMS) negatively impacts the visual pathway as well as motor processing speed. Relationships between MS-related diffuse structural damage of gray and white matter (WM) tissue and cortical responses to visual and motor stimuli remain poorly understood. We used magnetoencephalography in 14 POMS patients and 15 age- and sex-matched healthy controls to assess visual gamma (30-80 Hz), motor gamma (60-90 Hz), and motor beta (15-30 Hz) cortical oscillatory responses to a visual-motor task. Then, 3T MRI was used to: (a) calculate fractional anisotropy (FA) of the posterior visual and corticospinal motor WM pathways and (b) quantify volume and thickness of the cuneus and primary motor cortex. Visual gamma band power was reduced in POMS and was associated with reduced FA of the optic radiations but not with loss of cuneus volume or thickness. Activity in the primary motor cortex, as measured by postmovement beta rebound amplitude associated with peak latency, was decreased in POMS, although this reduction was not predicted by structural metrics. Our findings implicate loss of WM integrity as a contributor to reduced electrical responses in the visual cortex in POMS. Future work in larger cohorts will inform on the cognitive implications of this finding in terms of visual processing function and will determine whether the progressive loss of brain volume known to occur in POMS ultimately contributes to both progressive dysfunction in such tasks as well as progressive reduction in cortical electrical responses in the visual cortex.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

FREQUENT SUBCLINICAL MACULAR CHANGES IN COMBINED BRAF/MEK INHIBITION WITH HIGH-DOSE HYDROXYCHLOROQUINE AS TREATMENT FOR ADVANCED METASTATIC BRAF MUTANT MELANOMA: Preliminary Results From a Phase I/II Clinical Treatment Trial.

PURPOSE: To assess the potential ocular toxicity of a combined BRAF inhibition (BRAFi) + MEK inhibition (MEKi) + hydroxychloroquine (HCQ) regime used to treat metastatic BRAF mutant melanoma. METHODS: Patients with stage IV metastatic melanoma and BRAF V600E mutations (n = 11, 31-68 years of age) were included. Treatment was with oral dabrafenib, 150 mg bid, trametinib, 2 mg/day, and HCQ, 400 mg to 600 mg bid. An ophthalmic examination, spectral domain optical coherence tomography, near-infrared and short-wavelength fundus autofluorescence, and static perimetry were performed at baseline, 1 month, and q/6 months after treatment. RESULTS: There were no clinically significant ocular events; there was no ocular inflammation. The only medication-related change was a separation of the photoreceptor outer segment tip from the apical retinal pigment epithelium that could be traced from the fovea to the perifoveal retina noted in 9/11 (82%) of the patients. There were no changes in retinal pigment epithelium melanization or lipofuscin content by near-infrared fundus autofluorescence and short-wavelength fundus autofluorescence, respectively. There were no inner retinal or outer nuclear layer changes. Visual acuities and sensitivities were unchanged. CONCLUSION: BRAFi (trametinib) + MEKi (dabrafenib) + HCQ causes very frequent, subclinical separation of the photoreceptor outer segment from the apical retinal pigment epithelium without inner retinal changes or signs of inflammation. The changes suggest interference with the maintenance of the outer retinal barrier and/or phagocytic/pump functions of the retinal pigment epithelium by effective MEK inhibition.

Natural History of the Central Structural Abnormalities in Choroideremia: A Prospective Cross-Sectional Study.

PURPOSE: To describe in detail the central retinal structure of a large group of patients with choroideremia (CHM). DESIGN: A prospective, cross-sectional, descriptive study. PARTICIPANTS: Patients (n = 97, age 6-71 years) with CHM and subjects with normal vision (n = 44; ages 10-50 years) were included. METHODS: Subjects were examined with spectral-domain optical coherence tomography (SD OCT) and near-infrared reflectance imaging. Visual acuity (VA) was measured during their encounter or obtained from recent ophthalmic examinations. Visual thresholds were measured in a subset of patients (n = 24) with automated static perimetry within the central regions (±15°) examined with SD OCT. MAIN OUTCOME MEASURES: Visual acuity and visual thresholds; total nuclear layer, inner nuclear layer (INL), and outer nuclear layer (ONL) thicknesses; and horizontal extent of the ONL and the photoreceptor outer segment (POS) interdigitation zone (IZ). RESULTS: Earliest abnormalities in regions with normally appearing retinal pigment epithelium (RPE) were the loss of the POS and ellipsoid zone associated with rod dysfunction. Transition zones (TZs) from relatively preserved retina to severe ONL thinning and inner retinal thickening moved centripetally with age. Most patients (88%) retained VAs better than 20/40 until their fifth decade of life. The VA decline coincided with migration of the TZ near the foveal center. There were outer retinal tubulations in degenerated, nonatrophic retina in the majority (69%) of patients. In general, RPE abnormalities paralleled photoreceptor degeneration, although there were regions with detectable but abnormally thin ONL co-localizing with severe RPE depigmentation and choroidal thinning. CONCLUSIONS: Abnormalities of the POS and rod dysfunction are the earliest central abnormalities observed in CHM. Foveal function is relatively preserved until the fifth decade of life. Migration of the TZs to the foveal center with foveal thinning and structural disorganization heralded central VA loss. The relationships established may help outline the eligibility criteria and outcome measures for clinical trials for CHM.

Obstructive sleep apnea and central serous chorioretinopathy.

PURPOSE: The purpose of this study was to determine if there is an association between obstructive sleep apnea (OSA) and central serous chorioretinopathy (CSCR). METHODS: Patients with CSCR without a history of steroid use or secondary retinal disease were matched based on age/gender/body mass index with control patients and administered the Berlin Questionnaire to assess for OSA risk. Patients were scored "OSA+" if they were at "high risk" on the Berlin Questionnaire or reported a previous OSA diagnosis. Rates of OSA+ were compared between the 2 groups, odds ratio and its 95% confidence interval was calculated using exact conditional logistic regression. RESULTS: Forty-eight qualifying patients with CSCR were identified. There were no statistically significant differences between the CSCR and control groups by age (mean = 55 years), gender (79% male), body mass index (mean = 28.2), history of diabetes, or hypertension. Within the CSCR group, 22 patients (45.8%) were OSA+ versus 21 control patients (43.8%) (difference = 2.1%; 95% confidence interval, -18.2% to 22.2%; exact odds ratio = 1.08, 95% confidence interval, 0.47-2.49; P = 1.00). CONCLUSION: When compared with matched controls, patients with CSCR did not have statistically significant higher rates of OSA risk or previous diagnosis. This finding contrasts with previous work showing a strong association between the diseases. The divergence is likely due to our matching controls for body mass index, a significant risk factor for OSA.

Gene therapy rescues photoreceptor blindness in dogs and paves the way for treating human X-linked retinitis pigmentosa.

Hereditary retinal blindness is caused by mutations in genes expressed in photoreceptors or retinal pigment epithelium. Gene therapy in mouse and dog models of a primary retinal pigment epithelium disease has already been translated to human clinical trials with encouraging results. Treatment for common primary photoreceptor blindness, however, has not yet moved from proof of concept to the clinic. We evaluated gene augmentation therapy in two blinding canine photoreceptor diseases that model the common X-linked form of retinitis pigmentosa caused by mutations in the retinitis pigmentosa GTPase regulator (RPGR) gene, which encodes a photoreceptor ciliary protein, and provide evidence that the therapy is effective. After subretinal injections of adeno-associated virus-2/5-vectored human RPGR with human IRBP or GRK1 promoters, in vivo imaging showed preserved photoreceptor nuclei and inner/outer segments that were limited to treated areas. Both rod and cone photoreceptor function were greater in treated (three of four) than in control eyes. Histopathology indicated normal photoreceptor structure and reversal of opsin mislocalization in treated areas expressing human RPGR protein in rods and cones. Postreceptoral remodeling was also corrected: there was reversal of bipolar cell dendrite retraction evident with bipolar cell markers and preservation of outer plexiform layer thickness. Efficacy of gene therapy in these large animal models of X-linked retinitis pigmentosa provides a path for translation to human treatment.

Stimulus type and duration affect magnitude and evolution of flicker-induced hyperemia measured by laser speckle flowgraphy at the optic disc and peripapillary vessels.

Neurovascular coupling is a vital mechanism employed by the cerebrovascular system, including the eye, to regulate blood flow in periods of neuronal activation. This study aims to investigate if laser speckle flowgraphy (LSFG) can detect coupling response elicited by flickering light stimuli and how variations in stimulus type and duration can affect the magnitude and evolution of blood flow in the optic nerve head (ONH) and peripapillary vessels. Healthy adults were exposed to two types of 10-Hz flicker stimuli: a photopic negative response-like stimulus (PhNR-S) or a visual evoked potential-like stimulus (VEP-S)-each presented in separate 10- and 60-s epochs. Both PhNR-S and VEP-S significantly increased ONH blood flow (p < 0.001) immediately after flicker cessation, with a trend of 60-s stimuli (PhNR-S = 11.6%; VEP-S = 10.4%) producing a larger response than 10-s stimuli (PhNR-S = 7.5%; VEP-S = 6.2%). Moreover, exposure to 60-s stimuli elicited a significantly prolonged ONH hyperemic response, especially with PhNR-S. Lastly, stimulation with either 60-s stimuli elicited a robust increase in blood flow within the peripapillary arterioles (p < 0.01) and venules (p < 0.01) as well. Flicker stimulation with common visual electrophysiology stimuli (PhNR-S and VEP-S) induced a demonstrable increase in ONH and peripapillary vessel blood flow, which varied with flicker duration. Our results validate that LSFG is a robust method to quantify flicker-induced hyperemic responses and to study neurovascular coupling in humans.

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.

Cone photoreceptors are the main targets for gene therapy of NPHP5 (IQCB1) or NPHP6 (CEP290) blindness: generation of an all-cone Nphp6 hypomorph mouse that mimics the human retinal ciliopathy.

Leber congenital amaurosis (LCA), a severe autosomal recessive childhood blindness, is caused by mutations in at least 15 genes. The most common molecular form is a ciliopathy due to NPHP6 (CEP290) mutations and subjects have profound loss of vision. A similarly severe phenotype occurs in the related ciliopathy NPHP5 (IQCB1)-LCA. Recent success of retinal gene therapy in one form of LCA prompted the question whether we know enough about human NPHP5 and NPHP6 disease to plan such treatment. We determined that there was early-onset rapid degeneration of rod photoreceptors in young subjects with these ciliopathies. Rod outer segment (OS) lamination, when detectable, was disorganized. Retinal pigment epithelium lipofuscin accumulation indicated that rods had existed in the past in most subjects. In contrast to early rod losses, the all-cone human fovea in NPHP5- and NPHP6-LCA of all ages retained cone nuclei, albeit with abnormal inner segments and OS. The rd16 mouse, carrying a hypomorphic Nphp6 allele, was a good model of the rod-dominant human extra-foveal retina. Rd16 mice showed normal genesis of photoreceptors, including the formation of cilia, followed by abnormal elaboration of OS and rapid degeneration. To produce a model of the all-cone human fovea in NPHP6-LCA, we generated rd16;Nrl-/- double-mutant mice. They showed substantially retained cone photoreceptors with disproportionate cone function loss, such as in the human disease. NPHP5- and NPHP6-LCA across a wide age spectrum are thus excellent candidates for cone-directed gene augmentation therapy, and the rd16;Nrl-/- mouse is an appropriate model for pre-clinical proof-of-concept studies.