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Pathogenic variants in RPE65 lead to retinal diseases, causing a vision impairment. In this work, we investigated the pathomechanism behind the frequent RPE65 variant, c.11+5G>A. Previous in silico predictions classified this change as a splice variant. Our prediction using novel software's suggested a 124-nt exon elongation containing a premature stop codon. This elongation was validated using midigenes-based approaches. Similar results were observed in patient-derived induced pluripotent stem cells (iPSC) and photoreceptor precursor cells. However, the splicing defect in all cases was detected at low levels and thereby does not fully explain the recessive condition of the resulting disease. Long-read sequencing discarded other rearrangements or variants that could explain the diseases. Subsequently, a more relevant model was employed: iPSC-derived retinal pigment epithelium (RPE) cells. In patient-derived iPSC-RPE cells, the expression of RPE65 was strongly reduced even after inhibiting a nonsense-mediated decay, contradicting the predicted splicing defect. Additional experiments demonstrated a cell-specific gene expression reduction due to the presence of the c.11+5G>A variant. This decrease also leads to the lack of the RPE65 protein, and differences in size and pigmentation between the patient and control iPSC-RPE. Altogether, our data suggest that the c.11+5G>A variant causes a cell-specific defect in the expression of RPE65 rather than the anticipated splicing defect which was predicted in silico. [ABSTRACT FROM AUTHOR]

Inherited retinal diseases (IRDs) are both genetically and clinically highly heterogeneous and have long been considered incurable. Following the successful development of a gene augmentation therapy for biallelic RPE65-associated IRD, this view has changed. As a result, many different therapeutic approaches are currently being developed, in particular a large variety of molecular therapies. These are depending on the severity of the retinal degeneration, knowledge of the pathophysiological mechanism underlying each subtype of IRD, and the therapeutic target molecule. DNA therapies include approaches such as gene augmentation therapy, genome editing and optogenetics. For some genetic subtypes of IRD, RNA therapies and compound therapies have also shown considerable therapeutic potential. In this review, we summarize the current state-of-the-art of various therapeutic approaches, including the pros and cons of each strategy, and outline the future challenges that lie ahead in the combat against IRDs. [ABSTRACT FROM AUTHOR]

Purpose: Inherited retinal diseases are a group of clinically and genetically heterogeneous disorders with approximately 270 genes involved. IMPG2 is associated with adult-onset vitelliform macular dystrophy. Here, we investigated two unrelated patients with vitelliform macular dystrophy to identify the underlying genetic cause., Methods: Whole-exome sequencing identified a putative causal complex allele consisting of c.3023-15T>A and c.3023G>A (p.(Gly1008Asp)) in IMPG2 in both individuals. To assess its effect, in vitro splice assays in HEK293T and further characterization in patient-derived photoreceptor precursor cells (PPCs) were conducted., Results: The results of the midigene splice assays in HEK293T showed that the complex allele causes a variety of splicing defects ranging from a small deletion to (multiple-)exon skipping. This finding was further validated using patient-derived PPCs that showed a significant increase of out-of-frame transcripts lacking one or multiple exons compared to control-derived PPCs. Overall, control PPCs consistently showed low levels of aberrantly spliced IMPG2 transcripts that were highly elevated in patient-derived PPCs. These differences were even more obvious upon inhibition of nonsense-mediated decay with cycloheximide., Conclusions: We report a heterozygous complex allele in IMPG2 causative for adult-onset vitelliform macular dystrophy in two unrelated individuals with mild visual loss and bilateral vitelliform lesions. The predicted causal missense mutation c.3023G>A, located in the consensus splice acceptor site, enhances the splicing effect of the upstream variant c.3023-15T>A, leading to the generation of aberrant transcripts that decrease the full-length IMPG2 levels. These results suggest a haploinsufficiency mechanism of action and highlight the complementarity of using different models to functionally assesses splicing defects.

Over the last decades, animal models have become increasingly important in therapeutic drug development and assessment. The use of these models, mainly mice and rats, allow evaluating drugs in the real-organism environment and context. However, several molecular therapeutic approaches are sequence-dependent, and therefore, the humanization of such models is required to assess the efficacy. The generation of genetically modified humanized mouse models is often an expensive and laborious process that may not always recapitulate the human molecular and/or physiological phenotype. In this chapter, we summarize basic aspects to consider before designing and generating humanized models, especially when they are aimed to test antisense-based therapies., (© 2022. The Author(s).)

Introduction: Alpha-1 antitrypsin deficiency (AATD) is a genetic condition resulting in lung and liver disease with a great clinical variability. MicroRNAs have been identified as disease modifiers; therefore miRNA deregulation could play an important role in disease heterogeneity. Members of miR-320 family are involved in regulating of multiple processes including inflammation, and have potential specific binding sites in the 3'UTR region of SERPINA1 gene. In this study we explore the involvement of miR-320c, a member of this family, in this disease., Methods: Firstly in vitro studies were carried out to demonstrate regulation of SERPINA1 gene by miR-320. Furthermore, the expression of miR-320c was analyzed in the blood of 98 individuals with different AAT serum levels by using quantitative PCR and expression was correlated to clinical parameters of the patients. Finally, HL60 cells were used to analyze induction of miR-320c in inflammatory conditions., Results: Overexpression of miR-320 members in human HepG2 cells led to inhibition of SERPINA1 expression. Analysis of miR-320c expression in patient's samples revealed significantly increased expression of miR-320c in individuals with pulmonary disease. Additionally, HL60 cells treated with the pro-inflammatory factor lipopolysaccharide (LPS) showed increase in miR-320c expression, suggesting that miR-320c responds to inflammation., Conclusion: Our findings demonstrate that miR-320c inhibits SERPINA1 expression in a hepatic cell line and its levels in blood are associated with lung disease in a cohort of patients with different AAT serum levels. These results suggest that miR-320c can play a role in AAT regulation and could be a biomarker of inflammatory processes in pulmonary diseases., (Copyright © 2020 SEPAR. Publicado por Elsevier España, S.L.U. All rights reserved.)