Human retinal organoids from induced pluripotent stem cells (hiPSCs) can be used to confirm the localization of proteins in retinal cell types and to test transduction and expression patterns of gene therapy vectors. Here, we compared the onset of CRB protein expression in human fetal retina with human iPSC-derived retinal organoids. We show that CRB2 protein precedes the expression of CRB1 in the developing human retina. Our data suggest the presence of CRB1 and CRB2 in human photoreceptors and Müller glial cells. Thus the fetal CRB complex formation is replicated in hiPSC-derived retina. CRB1 patient iPSC retinal organoids showed disruptions at the outer limiting membrane as found in Crb1 mutant mice. Furthermore, AAV serotype 5 (AAV5) is potent in infecting human Müller glial cells and photoreceptors in hiPSC-derived retinas and retinal explants. Our data suggest that human photoreceptors can be efficiently transduced by AAVs in the presence of photoreceptor segments.

Based on best-corrected visual acuity survival probabilities, the intervention window for gene therapy for RPGR-associated retinal dystrophies is relatively broad in patients with RP. RPGR-ORF15 mutations were associated with COD/CORD and with a more severe phenotype in RP. High myopia is a risk factor for faster best-corrected visual acuity decline.

Purpose: To describe the phenotype, long-term clinical course, clinical variability, and genotype of patients with CRB1-associated retinal dystrophies.Design: Retrospective cohort study. Participants: Fifty-five patients with CRB1-associated retinal dystrophies from 16 families. Methods: A medical record review of 55 patients for age at onset, medical history, initial symptoms, bestcorrected visual acuity, ophthalmoscopy, fundus photography, full-field electroretinography (ffERG), Goldmann visual fields (VFs), and spectral-domain optical coherence tomography.Main Outcome Measures: Age at onset, visual acuity survival time, visual acuity decline rate, and electroretinography and imaging findings.Results: A retinitis pigmentosa (RP) phenotype was present in 50 patients, 34 of whom were from a Dutch genetic isolate (GI), and 5 patients had a Leber congenital amaurosis phenotype. The mean follow-up time was 15.4 years (range, 0e55.5 years). For the RP patients, the median age at symptom onset was 4.0 years. In the RP group, median ages for reaching low vision, severe visual impairment, and blindness were 18, 32, and 44 years, respectively, with a visual acuity decline rate of 0.03 logarithm of the minimum angle of resolution per year. The presence of a truncating mutation did not alter the annual decline rate significantly (P ¼ 0.75). Asymmetry in visual acuity was found in 31% of patients. The annual VF decline rate was 5% in patients from the genetic isolate, which was significantly faster than in non-GI patients (P < 0.05). Full-field electroretinography responses were extinguished in 50% of patients, were pathologically attenuated without a documented rod or cone predominance in 30% of patients, and showed a rodecone dysfunction pattern in 20% of RP patients. Cystoid fluid collections in the macula were found in 50% of RP patients.Conclusions: Mutations in the CRB1 gene are associated with a spectrum of progressive retinal degeneration. Visual acuity survival analyses indicate that the optimal intervention window for subretinal gene therapy is within the first 2 to 3 decades of life. Ophthalmology 2017;124:884-895 ª 2017 by the American Academy of Ophthalmology Supplemental material available at www.aaojournal.org.Mutations in the CRB1 gene are associated with a wide variety of severe retinal dystrophies with variable phenotypes, including panretinal dystrophies such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), and coneerod dystrophy, as well as central phenotypes such as isolated macular dystrophy and foveal retinoschisis. Retinitis pigmentosa is a clinically and genetically heterogeneous disorder. Mutations in the CRB1 gene have been associated with RP12, a distinct form of RP characterized by preservation of para-arteriolar retinal pigment epithelium, progressive visual field (VF) loss starting from the first decade of life, and early macular involvement. 4 Other common features are hyperopia and optic disc drusen, previously described in a Dutch genetic isolate (GI). 5Classic forms of earl...

PURPOSE. The purpose of this study was to investigate the phenotype and long-term clinical course of female carriers of RPGR mutations.METHODS. This was a retrospective cohort study of 125 heterozygous RPGR mutation carriers from 49 families.RESULTS. Eighty-three heterozygotes were from retinitis pigmentosa (RP) pedigrees, 37 were from cone-/cone-rod dystrophy (COD/CORD) pedigrees, and 5 heterozygotes were from pedigrees with mixed RP/CORD or unknown diagnosis. Mutations were located in exon 1-14 and in ORF15 in 42 of 125 (34%) and 83 of 125 (66%) subjects, respectively. The mean age at the first examination was 34.4 years (range, 2.1 to 86.0 years). The median follow-up time in heterozygotes with longitudinal data (n ¼ 62) was 12.2 years (range, 1.1 to 52.2 years). Retinal pigmentary changes were present in 73 (58%) individuals. Visual symptoms were reported in 51 (40%) cases. Subjects with both symptoms and pigmentary fundus changes were older than the other heterozygotes (P ¼ 0.01) and had thinner foveal outer retinas (P ¼ 0.006). Complete expression of the RP or CORD phenotype was observed in 29 (23%) heterozygotes, although usually in milder forms than in affected male relatives. Best-corrected visual acuity (BCVA) was <20/40 and <20/400 in at least one eye in 45 of 116 (39%) and 11 of 116 (9%) heterozygotes, respectively. Myopia was observed in 74 of 101 (73%) subjects and was associated with lower BCVA (P ¼ 0.006). Increasing age was associated with lower BCVA (P ¼ 0.002) and decreasing visual field size (P ¼ 0.012; I4e isopter).CONCLUSIONS. RPGR mutations lead to a phenotypic spectrum in female carriers, with myopia as a significantly aggravating factor. Complete disease expression is observed in some individuals, who may benefit from future (gene) therapeutic options.

AimTo investigate the natural history in a Belgian cohort of CRB1-associated retinal dystrophies.MethodsAn in-depth retrospective study focusing on visual function and retinal structure.ResultsForty patients from 35 families were included (ages: 2.5–80.1 years). In patients with a follow-up of >1 year (63%), the mean follow-up time was 12.0 years (range: 2.3–29.2 years). Based on the patient history, symptoms and/or electroretinography, 22 patients (55%) were diagnosed with retinitis pigmentosa (RP), 15 (38%) with Leber congenital amaurosis (LCA) and 3 (8%) with macular dystrophy (MD), the latter being associated with the p.(Ile167_Gly169del) mutation (in compound heterozygosity). MD later developed into a rod-cone dystrophy in one patient. Blindness at initial presentation was seen in the first decade of life in LCA, and in the fifth decade of life in RP. Eventually, 28 patients (70%) reached visual acuity-based blindness (<0.05). Visual field-based blindness (<10°) was documented in 17/25 patients (68%). Five patients (13%) developed Coats-like exudative vasculopathy. Intermediate/posterior uveitis was found in three patients (8%). Cystoid maculopathy was common in RP (9/21; 43%) and MD (3/3; 100%). Macular involvement, varying from retinal pigment epithelium alterations to complete outer retinal atrophy, was observed in all patients.ConclusionBi-allelic CRB1 mutations result in a range of progressive retinal disorders, most of which are generalised, with characteristically early macular involvement. Visual function and retinal structure analysis indicates a window for potential intervention with gene therapy before the fourth decade of life in RP and the first decade in LCA.