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

ABSTRACT: 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.

SUBMITTER: Siles L

PROVIDER: S-EPMC10034418 | biostudies-literature | 2023 Jun

REPOSITORIES: biostudies-literature

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

Siles Laura L Ruiz-Nogales Sheila S Navinés-Ferrer Arnau A Méndez-Vendrell Pilar P Pomares Esther E

Molecular therapy. Nucleic acids 20230303

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 ...[more]

PMID: 36969552

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Project description:PurposeThe aim of this study was to analyze and report pathogenic variants in the ABCA4 gene in Brazilian patients with a clinical diagnosis of Stargardt disease.MethodsThis retrospective study evaluated variants in the ABCA4 gene in Brazilian patients with Stargardt disease. The patients' visual acuity and age of symptom onset were obtained from previous medical records. The patients were classified according to the autofluorescence patterns.ResultsFifty patients aged between 10 and 65 years from 44 families were included in the study. Among these cases, the mean age of symptom onset was 14 years (range, 5-40 years). ABCA4 gene sequencing was conclusive in 40 patients (80%), negative in two patients (4%), and inconclusive in eight patients (16%). Four families carried homozygous pathogenic variants. Segregation analysis results were available for 23 families. One novel variant was found: p.Ala2084Pro. The most frequent pathogenic variant in this group was p.Arg602Trp (12/100 alleles). Based on the phenotypic characteristics assessed with fundus autofluorescence imaging, 12 patients were classified as having type I phenotype, 16 as having type II, and 18 patients as having type III. The cases classified as type III phenotype included patients who were homozygous for the p.Asn96Asp and p.Arg2030* variants. One patient with a type I phenotype carried the homozygous intronic variant c.3862+1G>A.ConclusionsNext-generation sequencing was effective for the molecular diagnosis of genetic diseases and specifically allowed a conclusive diagnosis in 80% (40/50) of the patients. As the ABCA4 gene does not show a preferential region for pathogenic variants, the diagnosis of Stargardt disease depends on broader analysis of the gene. The most common pathogenic variants in the ABCA4 gene described in the literature were also found in these Brazilian patients. Although some genotype-phenotype correlations were found, more studies regarding the progression of Stargardt disease will help increase our understanding of the pathogenicity of these gene variants.

Project description:Over 1,500 variants in the ABCA4 locus cause phenotypes ranging from severe, early-onset retinal degeneration to very late-onset maculopathies. The resulting ABCA4/Stargardt disease is the most prevalent Mendelian eye disorder, although its underlying clinical heterogeneity, including penetrance of many alleles, are not well-understood. We hypothesized that a share of this complexity is explained by trans-modifiers, i.e., variants in unlinked loci, which are currently unknown. We sought to identify these by performing exome sequencing in a large cohort for a rare disease of 622 cases and compared variation in seven genes known to clinically phenocopy ABCA4 disease to cohorts of ethnically matched controls. We identified a significant enrichment of variants in 2 out of the 7 genes. Moderately rare, likely functional, variants, at the minor allele frequency (MAF) <0.005 and CADD>25, were enriched in ROM1, where 1.3% of 622 patients harbored a ROM1 variant compared to 0.3% of 10,865 controls (p = 2.41E04; OR 3.81 95% CI [1.77; 8.22]). More importantly, analysis of common variants (MAF>0.1) identified a frequent haplotype in PRPH2, tagged by the p.Asp338 variant with MAF = 0.21 in the matched general population that was significantly increased in the patient cohort, MAF 0.25, p = 0.0014. Significant differences were also observed between ABCA4 disease subgroups. In the late-onset subgroup, defined by the hypomorphic p.Asn1868Ile variant and including c.4253+43G>A, the allele frequency for the PRPH2 p.Asp338 variant was 0.15 vs 0.27 in the remaining cohort, p = 0.00057. Known functional data allowed suggesting a mechanism by which the PRPH2 haplotype influences the ABCA4 disease penetrance. These associations were replicated in an independent cohort of 408 patients. The association was highly statistically significant in the combined cohorts of 1,030 cases, p = 4.00E-05 for all patients and p = 0.00014 for the hypomorph subgroup, suggesting a substantial trans-modifying role in ABCA4 disease for both rare and common variants in two unlinked loci.

Dataset Information

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

Publications

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

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