Efficient correction of ABCA4 variants by CRISPR-Cas9 in hiPSCs derived from Stargardt disease patients.

Inherited retinal dystrophies comprise a broad group of genetic eye diseases without effective treatment. Among them, Stargardt disease is the second most prevalent pathology. This pathology triggers progressive retinal degeneration and vision loss in children and adults. In recent years, the evolution of several genome editing technologies, such as the CRISPR-Cas9 system, has revolutionized disease modeling and personalized medicine. Human induced pluripotent stem cells also provide a valuable tool for in vitro disease studies and therapeutic applications. Here, we show precise correction of two ABCA4 pathogenic variants in human induced pluripotent stem cells from two unrelated patients affected with Stargardt disease. Gene editing was achieved with no detectable off-target genomic alterations, demonstrating efficient ABCA4 gene correction without deleterious effects. These results will contribute to the development of emerging gene and cell therapies for inherited retinal dystrophies.

Expanding the retinal phenotype of RP1: from retinitis pigmentosa to a novel and singular macular dystrophy.

PURPOSE: This study aimed to identify the underlying genetic cause(s) of inherited retinal dystrophy (IRD) in 12 families of Kuwaiti origin affected by macular dystrophy and four Spanish patients affected by retinitis pigmentosa (RP). METHODS: Clinical diagnoses were based on standard ophthalmic evaluations (best-corrected visual acuity, retinography, fundus autofluorescence imaging, optical coherence tomography, electroretinography and visual field tests). Panel-based whole exome sequencing was used to simultaneously analyse 224 IRD genes in one affected member of each family. The putative causative variants were confirmed by Sanger sequencing and cosegregation analyses. Haplotype analysis was performed using single nucleotide polymorphisms. RESULTS: A homozygous missense mutation c.606C>A (p.Asp202Glu) in RP1 was found to be the molecular cause of IRD in all 12 families from Kuwait. These patients exhibited comparable symptoms, including progressive decline in visual acuity since adolescence. Fundus autofluorescence images revealed bilateral macular retinal pigment epithelium disturbances, with neither perimacular flecks nor peripheral alterations. A shared haplotype spanning at least 1.1 Mb was identified in all families, suggesting a founder effect. Furthermore, RP1 variants involving nonsense and/or frameshifting mutations (three of them novel) were identified in three Spanish autosomal-recessive RP families and one dominant RP pedigree. CONCLUSION: This study describes, for the first time, a macular dystrophy phenotype caused by an RP1 mutation; establishing a new genotype-phenotype correlation in this gene, expanding its mutation spectrum and further highlighting the clinical heterogeneity associated with IRD.

Characterization of the cone-rod dystrophy retinal phenotype caused by novel homozygous DRAM2 mutations.

Cone-rod dystrophies (CRD) are a group of Inherited Retinal Dystrophies (IRD) characterized by the primary involvement of cone photoreceptors, resulting in the degeneration of the central retina, or macula. Although there are more than 55 CRD genes, a considerable percentage of cases remain unsolved. In this context, the present study aimed to describe and characterize the phenoptype and the genetic cause of 3 CRD families from a cohort of IRD cases. Clinical evaluation in each patient was supported by a complete ophthalmological examination, including visual acuity measurement, fundus retinography, fundus autofluorescence imaging, optical coherence tomography and full-field electroretinography. Molecular diagnoses were performed by whole exome sequencing analyzing a group of 279 IRD genes, and cosegregation of the identified pathogenic variants was confirmed by Sanger sequencing. Three novel homozygous mutations in the autophagy gene DRAM2 were identified as the molecular cause of disease in the three families: c.518-1G>A, c.628_629insAG and c.693+2T>A. Clinical data revealed that the 3 patients presented a shared CRD phenotype with adult-onset macular involvement and later peripheral degeneration, although the age of onset, evolution and severity were variable. In order to characterize the transcription effects of these variants, mRNA expression studies were performed. The results showed alterations in the DRAM2 transcription, including alternative splicing forms and lower levels of mRNA, which correlated with the phenotypic variability observed between patients. For instance, frameshift mutations were related to a less severe phenotype, with circumscribed mid-peripheral involvement, and lower levels of mRNA, suggesting an activation of the nonsense-mediated decay (NMD) pathway; while a more severe and widespread retinal degeneration was associated to the inframe alternative splicing variant reported, possibly due to a malfunctioning or toxicity of the resulting protein. Following these findings, DRAM2 expression was assessed in several human tissues by semi-quantitative RT-PCR and two isoforms were detected ubiquitously, yet with a singular tissue-specific pattern in retina and brain. Altogether, although the unique retinal phenotype described did not correlate with the ubiquitous expression, the retinal-specific expression and the essential role of autophagy in the photoreceptor survival could be key arguments to explain this particular DRAM2 phenotype.