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Contains fulltext : 293272.pdf (Publisher’s version ) (Open Access) Leber congenital amaurosis (LCA) is a group of inherited retinal diseases characterized by early-onset, rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with ultrastructure expansion microscopy, we reveal the structural and molecular defects underlying LCA type 5 (LCA5) with nanoscale resolution. We show that LCA5-encoded lebercilin, together with retinitis pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localized at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrate that mutant mice deficient in lebercilin exhibited early axonemal defects at the bulge region and the distal OS, accompanied by reduced levels of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, adeno-associated virus-based LCA5 gene augmentation partially restored the bulge region, preserved OS axoneme structure and membrane disc formation, and resulted in photoreceptor cell survival. Our approach thus provides a next level of assessment of retinal (gene) therapy efficacy at the molecular level.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.

Contains fulltext : 293760.pdf (Publisher’s version ) (Open Access) Mutations in the lebercilin-encoding gene LCA5 cause one of the most severe forms of Leber congenital amaurosis, an early-onset retinal disease that results in severe visual impairment. Here, we report on the generation of a patient-specific cellular model to study LCA5-associated retinal disease. CRISPR-Cas9 technology was used to correct a homozygous nonsense variant in LCA5 (c.835C>T; p.Q279∗) in patient-derived induced pluripotent stem cells (iPSCs). The absence of off-target editing in gene-corrected (isogenic) control iPSCs was demonstrated by whole-genome sequencing. We differentiated the patient, gene-corrected, and unrelated control iPSCs into three-dimensional retina-like cells, so-called retinal organoids. We observed opsin and rhodopsin mislocalization to the outer nuclear layer in patient-derived but not in the gene-corrected or unrelated control organoids. We also confirmed the rescue of lebercilin expression and localization along the ciliary axoneme within the gene-corrected organoids. Here, we show the potential of combining precise single-nucleotide gene editing with the iPSC-derived retinal organoid system for the generation of a cellular model of early-onset retinal disease.

Contains fulltext : 243899.pdf (Publisher’s version ) (Open Access) Inherited retinal diseases (IRDs) cause progressive loss of light-sensitive photoreceptors in the eye and can lead to blindness. Gene-based therapies for IRDs have shown remarkable progress in the past decade, but the vast majority of forms remain untreatable. In the era of personalised medicine, induced pluripotent stem cells (iPSCs) emerge as a valuable system for cell replacement and to model IRD because they retain the specific patient genome and can differentiate into any adult cell type. Three-dimensional (3D) iPSCs-derived retina-like tissue called retinal organoid contains all major retina-specific cell types: amacrine, bipolar, horizontal, retinal ganglion cells, Müller glia, as well as rod and cone photoreceptors. Here, we describe the main applications of retinal organoids and provide a comprehensive overview of the state-of-art analysis methods that apply to this model system. Finally, we will discuss the outlook for improvements that would bring the cellular model a step closer to become an established system in research and treatment development of IRDs.

Significance The photoreceptor outer segments are primary cilia, modified for phototransduction by incorporation of stacked opsin-loaded membrane disks that are continuously regenerated. This process is disrupted in several types of inherited retinal dystrophy, but the driving force remained unclear. We show that C2orf71/PCARE (photoreceptor cilium actin regulator), associated with inherited retinal dystrophy subtype RP54, efficiently recruits the Arp2/3 complex activator WASF3 to the cilium. This activates an actin dynamics-driven expansion of the ciliary tip, resembling membrane evagination in lamellipodia formation. Colocalization of this actin dynamics module to the base of the outer segments, and absence thereof in Pcare−/− mice, suggests PCARE-regulated actin dynamics as a critical process in outer segment disk formation., The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare−/− mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Contains fulltext : 248216.pdf (Publisher’s version ) (Open Access) Leber congenital amaurosis (LCA) can be caused by mutations in more than 20 different genes. One of these, RPE65, encodes a protein essential for the visual cycle that is expressed in retinal pigment epithelium cells. In this work, we describe the generation and characterization of the human iPSC line SCTCi16-A. This hiPSC line was generated from peripheral blood mononuclear cells (PBMCs) from a patient affected with LCA caused by the homozygous c.11+5G>A variant in the RPE65 gene. Reprograming was conducted using episomal vectors containing OCT3/4, SOX2, KLF4, L-MYC, and LIN28.

Age-related macular degeneration (AMD) is a major cause of vision loss among the elderly in the Western world. Genetic variants in the complement factor H (CFH) gene are associated with AMD, but the functional consequences of many of these variants are currently unknown. In this study, we aimed to determine the effect of 64 rare and low-frequency variants in the CFH gene on systemic levels of factor H (FH) and complement activation marker C3bBbP using plasma samples of 252 carriers and 159 non-carriers. Individuals carrying a heterozygous nonsense, frameshift or missense variant in CFH presented with significantly decreased FH levels and significantly increased C3bBbP levels in plasma compared to non-carrier controls. FH and C3bBbP plasma levels were relatively stable over time in samples collected during follow-up visits. Decreased FH and increased C3bBbP concentrations were observed in carriers compared to non-carriers of CFH variants among different AMD stages, with the exception of C3bBbP levels in advanced AMD stages, which were equally high in carriers and non-carriers. In AMD families, FH levels were decreased in carriers compared to non-carriers, but C3bBbP levels did not differ. Rare variants in the CFH gene can lead to reduced FH levels or reduced FH function as measured by increased C3bBbP levels. The effects of individual variants in the CFH gene reported in this study will improve the interpretation of rare and low-frequency variants observed in AMD patients in clinical practice.

Age-related macular degeneration (AMD) is a common eye disease among the elderly in the Western world. AMD is a multifactorial disease, with a strong association with genetic variation in the complement system. One of the AMD-associated variants is the c.355G>A (p.Gly119Arg) variant in complement factor I (CFI), a central regulator of complement activation. Here, we report the generation of an iPSC line and its isogenic wildtype control derived from peripheral blood mononuclear cells of a male AMD-affected individual carrying the heterozygous variant c.355G>A (p.Gly119Arg). The line can be utilized to study the effects of this variant in disease-specific cell types.

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.

Stargardt disease is an inherited retinal disease caused by biallelic mutations in the ABCA4 gene, many of which affect ABCA4 splicing. In this study, nine antisense oligonucleotides (AONs) were designed to correct pseudoexon (PE) inclusion caused by a recurrent deep-intronic variant in ABCA4 (c.769-784C>T). First, the ability of AONs to skip the PE from the final ABCA4 mRNA transcript was assessed in two cellular models carrying the c.769-784C>T variant: a midigene assay using HEK293T cells and patient-derived fibroblasts. Based on the splicing-correcting ability of each individual AON, the three most efficacious AONs targeting independent regions of the PE were selected for a final assessment in photoreceptor precursor cells (PPCs). The final analysis in the PPC model confirmed high efficacy of AON2, -5, and -7 in promoting PE exclusion. Among the three AONs, AON2 is chosen as the lead candidate for further optimization, hereby showcasing the high potential of AONs to correct aberrant splicing events driven by deep-intronic variants.

PURPOSE: ABCA4-associated disease, a recessive retinal dystrophy, is hallmarked by a large proportion of patients with only one pathogenic ABCA4 variant, suggestive for missing heritability. METHODS: By locus-specific analysis of ABCA4, combined with extensive functional studies, we aimed to unravel the missing alleles in a cohort of 67 patients (p), with one (p = 64) or no (p = 3) identified coding pathogenic variants of ABCA4. RESULTS: We identified eight pathogenic (deep-)intronic ABCA4 splice variants, of which five are novel and six structural variants, four of which are novel, including two duplications. Together, these variants account for the missing alleles in 40.3% of patients. Furthermore, two novel variants with a putative cis-regulatory effect were identified. The common hypomorphic variant c.5603A>T p.(Asn1868Ile) was found as a candidate second allele in 43.3% of patients. Overall, we have elucidated the missing heritability in 83.6% of our cohort. In addition, we successfully rescued three deep-intronic variants using antisense oligonucleotide (AON)-mediated treatment in HEK 293-T cells and in patient-derived fibroblast cells. CONCLUSION: Noncoding pathogenic variants, novel structural variants, and a common hypomorphic allele of the ABCA4 gene explain the majority of unsolved cases with ABCA4-associated disease, rendering this retinopathy a model for missing heritability in autosomal recessive disorders. ispartof: GENETICS IN MEDICINE vol:21 issue:8 pages:1761-1771 ispartof: location:United States status: published

Nucleic acid‐based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid‐based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics., Recent advances in the delivery of nucleic acid‐based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide‐based therapeutics.

The discovery of novel intronic variants in the ABCA4 locus has contributed significantly to solving the missing heritability in Stargardt disease (STGD1). The increasing number of variants affecting pre-mRNA splicing makes ABCA4 a suitable candidate for antisense oligonucleotide (AON)-based splicing modulation therapies. In this study, AON-based splicing modulation was assessed for 15 recently described intronic variants (three near-exon and 12 deep-intronic variants). In total, 26 AONs were designed and tested in vitro using a midigene-based splice system. Overall, partial or complete splicing correction was observed for two variants causing exon elongation and all variants causing pseudoexon inclusion. Together, our results confirm the high potential of AONs for the development of future RNA therapies to correct splicing defects causing STGD1.

Contains fulltext : 237835.pdf (Publisher’s version ) (Open Access) Age-related macular degeneration (AMD) is the main cause of vision loss among the elderly in the Western world. While AMD is a multifactorial disease, the complement system was identified as one of the main pathways contributing to disease risk. The strong link between the complement system and AMD was demonstrated by genetic associations, and by elevated complement activation in local eye tissue and in the systemic circulation of AMD patients. Several complement inhibitors have been and are being explored in clinical trials, but thus far with limited success, leaving the majority of AMD patients without treatment options to date. This indicates that there is still a gap of knowledge regarding the functional implications of the complement system in AMD pathogenesis and how to bring these towards clinical translation. Many different experimental set-ups and disease models have been used to study complement activation in vivo and in vitro, and recently emerging patient-derived induced pluripotent stem cells and genome-editing techniques open new opportunities to study AMD disease mechanisms and test new therapeutic strategies in the future. In this review we provide an extensive overview of methods employed to understand the molecular processes of complement activation in AMD pathogenesis. We discuss the findings, advantages and challenges of each approach and conclude with an outlook on how recent, exciting developments can fill in current knowledge gaps and can aid in the development of effective complement-targeting therapeutic strategies in AMD.

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&prime, and 3&prime, 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 &ldquo, splicing interdependency&rdquo, 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.

Contains fulltext : 218308.pdf (Publisher’s version ) (Open Access) Inherited retinal diseases (IRDs) are a group of progressive disorders that lead to severe visual impairment or even complete blindness. IRDs display a vast heterogeneity, clinically as well as genetically, with over 250 genes identified in which mutations can cause one or more clinical subtypes of IRD. Long considered incurable diseases, intense research over the last two decades, combined with major technological advancements, have enabled the development of the first therapeutic approaches for these diseases. The approval of LuxturnaTM (voretigene neparvovec), a gene augmentation therapy vector for RPE65-associated IRD, by the US Food and Drug Administration and the European Medicines Agency, is considered a true milestone in the field, and has led to the development of similar, or different therapeutic strategies for many other subtypes of IRD. Despite these major achievements, there are still many aspects that can-and need to-be improved, including more insights into the relationship between genetic variation and cellular dysfunction, optimization of the vectors and sequences used, improving delivery methods, as well as understanding and modulating the (local) immune response. In addition, the extreme rarity of some genetic subtypes of IRDs poses an enormous challenge on the development of novel therapies, in terms of e.g., costs and regulatory affairs.[...].

Stargardt disease is a progressive retinal disorder caused by bi-allelic mutations in the ABCA4 gene that encodes the ATP-binding cassette, subfamily A, member 4 transporter protein. Over the past few years, we and others have identified several pathogenic variants that reside within the introns of ABCA4, including a recurrent variant in intron 36 (c.5196+1137G>A) of which the pathogenicity so far remained controversial. Detailed clinical characterization of this variant confirmed its pathogenic nature, and classified it as an allele of intermediate severity. Moreover, we discovered several additional ABCA4 variants clustering in intron 36. Several of these variants resulted in aberrant splicing of ABCA4, i.e., the inclusion of pseudoexons, while the splicing defects caused by the recurrent c.5196+1137G>A variant strongly increased upon differentiation of patient-derived induced pluripotent stem cells into retina-like cells. Finally, all splicing defects could be rescued by the administration of antisense oligonucleotides that were designed to specifically block the pseudoexon insertion, including rescue in 3D retinal organoids harboring the c.5196+1137G>A variant. Our data illustrate the importance of intronic variants in ABCA4 and expand the therapeutic possibilities for overcoming splicing defects in Stargardt disease., Graphical Abstract, Khan et al. report on the clinical characterization of patients harboring a recurrent deep-intronic variant in ABCA4 underlying retinal disease and demonstrate that this, and other variants close by, lead to splicing defects that can be rescued by antisense oligonucleotides.

PURPOSE. Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disease in which the retinal vasculature is affected. Patients with FEVR typically lack or have abnormal vasculature in the peripheral retina, the outcome of which can range from mild visual impairment to complete blindness. A missense mutation (p.His455Tyr) in ZNF408 was identified in an autosomal dominant FEVR family. Little, however, is known about the molecular role of ZNF408 and how its defect leads to the clinical features of FEVR. METHODS. Using CRISPR/Cas9 technology, two homozygous mutant zebrafish models with truncated znf408 were generated, as well as one heterozygous and one homozygous missense znf408 model in which the human p.His455Tyr mutation is mimicked. RESULTS. Intriguingly, all three znf408-mutant zebrafish strains demonstrated progressive retinal vascular pathology, initially characterized by a deficient hyaloid vessel development at 5 days postfertilization (dpf) leading to vascular insufficiency in the retina. The generation of stable mutant lines allowed long-term follow up studies, which showed ectopic retinal vascular hyper-sprouting at 90 dpf and extensive vascular leakage at 180 dpf. CONCLUSIONS. Together, our data demonstrate an important role for znf408 in the development and maintenance of the vascular system within the retina. Funding Agencies|Radboudumc PhD grant; Svenska Sallskapet for Medicinsk Forskning; Linkoping University; Loo och Hans Ostermans Stiftelse; Eva och Oscar Ahrens Stiftelse; Stiftelsen Sigurd och Elsa Goljes Minne; Magnus Bergvalls Stiftelse; Ogonfonden; Jeanssons Stiftelser; VetenskapsradetSwedish Research Council

Item does not contain fulltext PURPOSE OF REVIEW: Over the last years, antisense oligonucleotides (AONs) have gained attention as a therapeutic tool for the treatment of ocular diseases such as cytomegalovirus retinitis, keratitis-induced corneal neovascularization, and inherited retinal diseases (IRDs). In this review, we summarize the recent key findings, and describe the challenges and opportunities that lie ahead to translate AON-based therapies to the clinic, in particular for IRDs. RECENT FINDINGS: The efficacy of AONs to restore splice defects and cellular phenotypes associated with a common mutation in CEP290 was demonstrated in patient-derived optic cups and in a transgenic mouse model, respectively. In addition, allele-specific knockdown of a mutant RHO allele resulted in a delay of photoreceptor cell death and functional preservation of these cells in a transgenic rat model. SUMMARY: As demonstrated by several preclinical efficacy studies, AON-based therapy is moving to the clinic for the treatment of some genetic subtypes of IRD. More insights into the delivery of these molecules, the duration of the therapeutic effect, and potential off-target effects will be essential to further shape the transition to the clinic and reveal the true potential of AON-based therapy for retinal diseases.

Contains fulltext : 206339.pdf (Publisher’s version ) (Open Access) Deep-sequencing of the ABCA4 locus has revealed that ~10% of autosomal recessive Stargardt disease (STGD1) cases are caused by deep-intronic mutations. One of the most recurrent deep-intronic variants in the Belgian and Dutch STGD1 population is the c.4539+2001G>A mutation. This variant introduces a 345-nt pseudoexon to the ABCA4 mRNA transcript in a retina-specific manner. Antisense oligonucleotides (AONs) are short sequences of RNA that can modulate splicing. In this work, we designed 26 different AONs to perform a thorough screening to identify the most effective AONs to correct splicing defects associated with c.4539+2001G>A. All AONs were tested in patient-derived induced pluripotent stem cells (iPSCs) that were differentiated to photoreceptor precursor cells (PPCs). AON efficacy was assessed through RNA analysis and was based on correction efficacy, and AONs were grouped and their properties assessed. We (a) identified nine AONs with significant correction efficacies (>50%), (b) confirmed that a single nucleotide mismatch was sufficient to significantly decrease AON efficacy, and (c) found potential correlations between efficacy and some of the parameters analyzed. Overall, our results show that AON-based splicing modulation holds great potential for treating Stargardt disease caused by splicing defects in ABCA4.

Item does not contain fulltext PURPOSE: To elucidate the functional effect of the ABCA4 variant c.5461-10T-->C, one of the most frequent variants associated with Stargardt disease (STGD1). DESIGN: Case series. PARTICIPANTS: Seventeen persons with STGD1 carrying ABCA4 variants and 1 control participant. METHODS: Haplotype analysis of 4 homozygotes and 11 heterozygotes for c.5461-10T-->C and sequence analysis of the ABCA4 gene for a homozygous proband. Fibroblasts were reprogrammed from 3 persons with STGD1 into induced pluripotent stem cells, which were differentiated into photoreceptor progenitor cells (PPCs). The effect of the c.5461-10T-->C variant on RNA splicing by reverse-transcription polymerase chain reaction was analyzed using PPC mRNA. In vitro assays were performed with minigene constructs containing ABCA4 exon 39. We analyzed the natural history and ophthalmologic characteristics of 4 persons homozygous for c.5461-10T-->C. MAIN OUTCOME MEASURES: Haplotype and rare variant data for ABCA4, RNA splice defects, age at diagnosis, visual acuity, fundus appearance, visual field, electroretinography (ERG) results, fluorescein angiography results, and fundus autofluorescence findings. RESULTS: The frequent ABCA4 variant c.5461-10T-->C has a subtle effect on splicing based on prediction programs. A founder haplotype containing c.5461-10T-->C was found to span approximately 96 kb of ABCA4 and did not contain other rare sequence variants. Patient-derived PPCs showed skipping of exon 39 or exons 39 and 40 in the mRNA. HEK293T cell transduction with minigenes carrying exon 39 showed that the splice defects were the result of the c.5461-10T-->C variant. All 4 subjects carrying the c.5461-10T-->C variant in a homozygous state showed a young age of STGD1 onset, with low visual acuity at presentation and abnormal cone ERG results. All 4 demonstrated severe cone-rod dystrophy before 20 years of age and were legally blind by 25 years of age. CONCLUSIONS: The ABCA4 variant c.5461-10T-->C is located on a founder haplotype lacking other disease-causing rare sequence variants. In vitro studies revealed that it leads to mRNA exon skipping and ABCA4 protein truncation. Given the severe phenotype in persons homozygous for this variant, we conclude that this variant results in the absence of ABCA4 activity.

Contains fulltext : 190679.pdf (Publisher’s version ) (Open Access) Leber congenital amaurosis (LCA) is a rare inherited retinal disorder affecting approximately 1:50,000 people worldwide. So far, mutations in 25 genes have been associated with LCA, with CEP290 (encoding the Centrosomal protein of 290 kDa) being the most frequently mutated gene. The most recurrent LCA-causing CEP290 mutation, c.2991+1655A>G, causes the insertion of a pseudoexon into a variable proportion of CEP290 transcripts. We previously demonstrated that antisense oligonucleotides (AONs) have a high therapeutic potential for patients homozygously harbouring this mutation, although to date, it is unclear whether rescuing one single allele is enough to restore CEP290 function. Here, we assessed the AON efficacy at RNA, protein and cellular levels in samples that are compound heterozygous for this mutation, together with a protein-truncating mutation in CEP290. We demonstrate that AONs can efficiently restore splicing and increase protein levels. However, due to a high variability in ciliation among the patient-derived cell lines, the efficacy of the AONs was more difficult to assess at the cellular level. This observation points towards the importance of the severity of the second allele and possibly other genetic variants present in each individual. Overall, AONs seem to be a promising tool to treat CEP290-associated LCA, not only in homozygous but also in compound heterozygous carriers of the c.2991+1655A>G variant.

Stargardt disease is caused by variants in the ABCA4 gene, a significant part of which are noncanonical splice site (NCSS) variants. In case a gene of interest is not expressed in available somatic cells, small genomic fragments carrying potential disease-associated variants are tested for splice abnormalities using in vitro splice assays. We recently discovered that when using small minigenes lacking the proper genomic context, in vitro results do not correlate with splice defects observed in patient cells. We therefore devised a novel strategy in which a bacterial artificial chromosome was employed to generate midigenes, splice vectors of varying lengths (up to 11.7 kb) covering almost the entire ABCA4 gene. These midigenes were used to analyze the effect of all 44 reported and three novel NCSS variants on ABCA4 pre-mRNA splicing. Intriguingly, multi-exon skipping events were observed, as well as exon elongation and intron retention. The analysis of all reported NCSS variants in ABCA4 allowed us to reveal the nature of aberrant splicing events and to classify the severity of these mutations based on the residual fraction of wild-type mRNA. Our strategy to generate large overlapping splice vectors carrying multiple exons, creating a toolbox for robust and high-throughput analysis of splice variants, can be applied to all human genes.

Sequence analysis of the coding regions and splice site sequences in inherited retinal diseases is not able to uncover ∼40% of the causal variants. Whole-genome sequencing can identify most of the non-coding variants, but their interpretation is still very challenging, in particular when the relevant gene is expressed in a tissue-specific manner. Deep-intronic variants in ABCA4 have been associated with autosomal-recessive Stargardt disease (STGD1), but the exact pathogenic mechanism is unknown. By generating photoreceptor precursor cells (PPCs) from fibroblasts obtained from individuals with STGD1, we demonstrated that two neighboring deep-intronic ABCA4 variants (c.4539+2001G>A and c.4539+2028C>T) result in a retina-specific 345-nt pseudoexon insertion (predicted protein change: p.Arg1514Leufs∗36), likely due to the creation of exonic enhancers. Administration of antisense oligonucleotides (AONs) targeting the 345-nt pseudoexon can significantly rescue the splicing defect observed in PPCs of two individuals with these mutations. Intriguingly, an AON that is complementary to c.4539+2001G>A rescued the splicing defect only in PPCs derived from an individual with STGD1 with this but not the other mutation, demonstrating the high specificity of AONs. In addition, a single AON molecule rescued splicing defects associated with different neighboring mutations, thereby providing new strategies for the treatment of persons with STGD1. As many genes associated with human genetic conditions are expressed in specific tissues and pre-mRNA splicing may also rely on organ-specific factors, our approach to investigate and treat splicing variants using differentiated cells derived from individuals with STGD1 can be applied to any tissue of interest.

Leber congenital amaurosis type 10 (LCA10) is a severe inherited retinal dystrophy associated with mutations in CEP290. The deep intronic c.2991+1655A>G mutation in CEP290 is the most common mutation in LCA10 individuals and represents an ideal target for oligonucleotide therapeutics. Here, a panel of antisense oligonucleotides was designed to correct the splicing defect associated with the mutation and screened for efficacy and safety. This identified QR-110 as the best-performing molecule. QR-110 restored wild-type CEP290 mRNA and protein expression levels in CEP290 c.2991+1655A>G homozygous and compound heterozygous LCA10 primary fibroblasts. Furthermore, in homozygous three-dimensional iPSC-derived retinal organoids, QR-110 showed a dose-dependent restoration of mRNA and protein function, as measured by percentage and length of photoreceptor cilia, without off-target effects. Localization studies in wild-type mice and rabbits showed that QR-110 readily reached all retinal layers, with an estimated half-life of 58 days. It was well tolerated following intravitreal injection in monkeys. In conclusion, the pharmacodynamic, pharmacokinetic, and safety properties make QR-110 a promising candidate for treating LCA10, and clinical development is currently ongoing., Graphical Abstract

Item does not contain fulltext Antisense oligonucleotides (AONs) are small molecules able to bind to the pre-mRNA and modulate splicing. The increasing amount of intronic mutations leading to pseudoexon insertion in genes underlying inherited retinal dystrophies (IRDs) has highlighted the potential of AONs as a therapeutic tool for these disorders. Here we describe how to design and test AON molecules in vitro in order to correct pre-mRNA splicing defects involved in IRDs.

Contains fulltext : 208528.pdf (Publisher’s version ) (Open Access) 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.

Item does not contain fulltext Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991 + 1655A > G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential of naked and adeno-associated virus (AAV)-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations. 12 p.

Contains fulltext : 168161.pdf (Publisher’s version ) (Open Access) Usher syndrome (USH) is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G) was reported in 2012, leading to the insertion of a pseudoexon (PE40) into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs) to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.