RESUMEN
Glaucoma, a leading cause of blindness, is a multifactorial condition that leads to progressive loss of retinal ganglion cells (RGCs) and vision. Therapeutic interventions based on reducing ocular hypertension are not always successful. Emerging features of glaucoma include mitochondrial dysfunction and oxidative stress. In the current study, NDI1-based gene therapy, which improves mitochondrial function and reduces reactive oxygen species, was delivered intraocularly via an adeno-associated viral vector (AAV). This AAV-NDI1 therapy protected RGCs from cell death in treated (1552.4 ± 994.0 RGCs/mm2) versus control eyes (1184.4 ± 978.4 RGCs/mm2, p < 0.05) in aged DBA/2J mice, a murine model of glaucoma. The photonegative responses (PhNRs) of RGCs were also improved in treated (6.4 ± 3.3 µV) versus control eyes (5.0 ± 3.1 µV, p < 0.05) in these mice. AAV-NDI1 also provided benefits in glaucomatous human lamina cribrosa (LC) cells by significantly increasing basal and maximal oxygen consumption rates and ATP production in these cells. Similarly, NDI1 therapy significantly protected H2O2-insulted primary porcine LC cells from oxidative stress. This study highlights the potential utility of NDI1 therapies and the benefits of improving mitochondrial function in the treatment of glaucoma.
Asunto(s)
Dependovirus , Modelos Animales de Enfermedad , Terapia Genética , Vectores Genéticos , Glaucoma , Estrés Oxidativo , Células Ganglionares de la Retina , Animales , Dependovirus/genética , Glaucoma/terapia , Glaucoma/metabolismo , Glaucoma/patología , Ratones , Terapia Genética/métodos , Células Ganglionares de la Retina/metabolismo , Células Ganglionares de la Retina/patología , Humanos , Vectores Genéticos/genética , Mitocondrias/metabolismo , Ratones Endogámicos DBA , Especies Reactivas de Oxígeno/metabolismo , PorcinosRESUMEN
Over 15% of probands in a large cohort of more than 1500 inherited retinal degeneration patients present with a clinical diagnosis of Stargardt disease (STGD1), a recessive form of macular dystrophy caused by biallelic variants in the ABCA4 gene. Participants were clinically examined and underwent either target capture sequencing of the exons and some pathogenic intronic regions of ABCA4, sequencing of the entire ABCA4 gene or whole genome sequencing. ABCA4 c.4539 + 2028C > T, p.[= ,Arg1514Leufs*36] is a pathogenic deep intronic variant that results in a retina-specific 345-nucleotide pseudoexon inclusion. Through analysis of the Irish STGD1 cohort, 25 individuals across 18 pedigrees harbour ABCA4 c.4539 + 2028C > T and another pathogenic variant. This includes, to the best of our knowledge, the only two homozygous patients identified to date. This provides important evidence of variant pathogenicity for this deep intronic variant, highlighting the value of homozygotes for variant interpretation. 15 other heterozygous incidents of this variant in patients have been reported globally, indicating significant enrichment in the Irish population. We provide detailed genetic and clinical characterization of these patients, illustrating that ABCA4 c.4539 + 2028C > T is a variant of mild to intermediate severity. These results have important implications for unresolved STGD1 patients globally with approximately 10% of the population in some western countries claiming Irish heritage. This study exemplifies that detection and characterization of founder variants is a diagnostic imperative.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Degeneración Macular , Humanos , Enfermedad de Stargardt/genética , Transportadoras de Casetes de Unión a ATP/genética , Mutación , Degeneración Macular/genética , Retina , LinajeRESUMEN
Age-related macular degeneration (AMD) is the most common cause of blindness in the aged population. However, to date there is no effective treatment for the dry form of the disease, representing 85-90% of cases. AMD is an immensely complex disease which affects, amongst others, both retinal pigment epithelium (RPE) and photoreceptor cells and leads to the progressive loss of central vision. Mitochondrial dysfunction in both RPE and photoreceptor cells is emerging as a key player in the disease. There are indications that during disease progression, the RPE is first impaired and RPE dysfunction in turn leads to subsequent photoreceptor cell degeneration; however, the exact sequence of events has not as yet been fully determined. We recently showed that AAV delivery of an optimised NADH-ubiquinone oxidoreductase (NDI1) gene, a nuclear-encoded complex 1 equivalent from S. cerevisiae, expressed from a general promoter, provided robust benefit in a variety of murine and cellular models of dry AMD; this was the first study employing a gene therapy to directly boost mitochondrial function, providing functional benefit in vivo. However, use of a restricted RPE-specific promoter to drive expression of the gene therapy enables exploration of the optimal target retinal cell type for dry AMD therapies. Furthermore, such restricted transgene expression could reduce potential off-target effects, possibly improving the safety profile of the therapy. Therefore, in the current study, we interrogate whether expression of the gene therapy from the RPE-specific promoter, Vitelliform macular dystrophy 2 (VMD2), might be sufficient to rescue dry AMD models.
Asunto(s)
Terapia Genética , Atrofia Geográfica , Proteínas de Saccharomyces cerevisiae , Anciano , Animales , Humanos , Ratones , Complejo I de Transporte de Electrón/metabolismo , Terapia Genética/métodos , Atrofia Geográfica/genética , Atrofia Geográfica/terapia , Mitocondrias/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Noncanonical splice-site mutations are an important cause of inherited diseases. Based on in vitro and stem-cell-based studies, some splice-site variants show a stronger splice defect than expected based on their predicted effects, suggesting that other sequence motifs influence the outcome. We investigated whether splice defects due to human-inherited-disease-associated variants in noncanonical splice-site sequences in ABCA4, DMD, and TMC1 could be rescued by strengthening the splice site on the other side of the exon. Noncanonical 5'- and 3'-splice-site variants were selected. Rescue variants were introduced based on an increase in predicted splice-site strength, and the effects of these variants were analyzed using in vitro splice assays in HEK293T cells. Exon skipping due to five variants in noncanonical splice sites of exons in ABCA4, DMD, and TMC1 could be partially or completely rescued by increasing the predicted strengths of the other splice site of the same exon. We named this mechanism "splicing interdependency", and it is likely based on exon recognition by splicing machinery. Awareness of this interdependency is of importance in the classification of noncanonical splice-site variants associated with disease and may open new opportunities for treatments.