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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.

Inherited retinal dystrophies (IRDs) are genetic diseases affecting 1 in every 3000 individuals worldwide. Nowadays, more than 250 genes have been associated with different forms of IRD. In the last decade, it has been shown that gene therapy is a promising approach to correct the genetic defects underlying IRD. In fact, voretigene neparvovec-rzyl (Luxturna™), the first commercialized gene therapy drug to treat RPE65-associated Leber congenital amaurosis, has opened new venues. However, IRDs are highly heterogeneous at genetic level making the design of novel strategies complicated. Unfortunately, the size of several frequently mutated genes is not suitable for the approved conventional therapeutic viral vectors; therefore, there is an urgent need for the development of alternatives, such as those targeting the pre-mRNA. In this mini-review, the potential of RNA-based strategies for IRDs is discussed.

This study aimed to evaluate the stability of ‘Big Top’ nectarine fruit quality (fruit weight, fruit mineral elements and fruit phytochemical composition such as soluble solids content, titratable acidity, individual sugars, individual organic acids, total ascorbic acid content, total phenolics content, and antioxidant capacity) when grafted on 20 Prunus rootstocks over two consecutive seasons. For most of the evaluated traits, rootstock was the main source of variability, whereas for Mg, malic and citric acids, and glucose most of the variability was observed among years. Similarly, the interaction year × rootstock was not significant for most traits (14 out of 21), hence highlighting that most rootstocks responded in a similar manner to changes in the weather conditions. Thus said, some important micro- and macro-nutrients such as Ca, Mg, Fe, Zn, together with taste- (fructose, glucose and sucrose) or health-related (antioxidant capacity) compounds showed a differential influence of the rootstock depending on the year conditions and thereby suggested that climatic conditions can be a limiting factor in the choice of rootstocks for a given nectarine cultivar. Overall, the results from this study indicated that the cherry-plum hybrid Krimsk-1 and the peach-plum hybrid PS rootstocks are the most suitable rootstocks for ‘Big Top’ under the conditions investigated herein. Both rootstocks induced high values on sugar profile, ascorbic acid, antioxidant activity, and TPC of ‘Big Top’ nectarine being relatively stable regardless of the weather conditions. Finally, the rootstocks IRTA-1 and Rootpac-20 also induced good fruit quality and phytochemical properties to ‘Big Top’ fruit. info:eu-repo/semantics/acceptedVersion

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&gt, 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&gt, 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 (&gt, 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.

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.

The objective of this work was to evaluate the agronomic performance (vigor, yield, yield efficiency, number of root suckers), fruit quality (fruit weight, fruit size, flesh firmness, soluble solids content, and titratable acidity), leaf and fruit mineral nutrition (macro and micro elements), leaf chlorophyll concentration and iron chlorosis susceptibility of ‘Big Top’ nectarine cultivar grafted on 20 Prunus rootstocks and grown in loamy and calcareous soil under the hot climate conditions of the Ebro river basin (Spain). After the 10 years of the study (at 11th leaf), statistical analysis showed significant differences among rootstocks for most of the traits evaluated. Based on vigor and cumulative yield, ‘Big Top’ trees from Padac 04-03 rootstock were found to be the most vigorous and productive, followed by Castore, GF-677, Ishtara®, PS and Rootpac® 70. However, the most efficient rootstocks were Controller 5, Adesoto® 101, Rootpac® 40, Krymsk® 1, Ishtara®, Penta, IRTA-1, Polluce, and Padac-150. ‘Big Top’ fruits from Rootpac® 40 had the highest fruit weight and fruit size (>70 mm), with good soluble solids content and titratable acidity, but less firmness than the other ‘Big Top’ fruits. After 3 months with no application of chelate, chlorosis symptoms were visible in most of the trees, with those from Krymsk® 1 and PS showing the highest susceptibility. In contrast, AD-105, Adesoto® 101, Cadaman®, GF-677, Padac-150, Rootpac® 40 and Tetra were the least susceptible rootstocks. Controller 5, IRTA-1, Padac-04.03 and Penta had moderate susceptibility. In conclusion, Rootpac® 40, Ishtara®, IRTA-1 and Padac-150 may represent a good compromise between canopy size control, yield, yield efficiency, fruit size, and susceptibility to iron chlorosis. info:eu-repo/semantics/acceptedVersion

Inherited retinal dystrophies (IRDs) are genetic diseases affecting 1 in every 3000 individuals worldwide. Nowadays, more than 250 genes have been associated with different forms of IRD. In the last decade, it has been shown that gene therapy is a promising approach to correct the genetic defects underlying IRD. In fact, voretigene neparvovec-rzyl (Luxturna™), the first commercialized gene therapy drug to treat RPE65-associated Leber congenital amaurosis, has opened new venues. However, IRDs are highly heterogeneous at genetic level making the design of novel strategies complicated. Unfortunately, the size of several frequently mutated genes is not suitable for the approved conventional therapeutic viral vectors; therefore, there is an urgent need for the development of alternatives, such as those targeting the pre-mRNA. In this mini-review, the potential of RNA-based strategies for IRDs is discussed.