Preclinical Development of Antisense Oligonucleotides to Rescue Aberrant Splicing Caused by an Ultrarare ABCA4 Variant in a Child with Early-Onset Stargardt Disease.

Precision medicine is rapidly gaining recognition in the field of (ultra)rare conditions, where only a few individuals in the world are affected. Clinical trial design for a small number of patients is extremely challenging, and for this reason, the development of N-of-1 strategies is explored to accelerate customized therapy design for rare cases. A strong candidate for this approach is Stargardt disease (STGD1), an autosomal recessive macular degeneration characterized by high genetic and phenotypic heterogeneity. STGD1 is caused by pathogenic variants in ABCA4, and amongst them, several deep-intronic variants alter the pre-mRNA splicing process, generally resulting in the insertion of pseudoexons (PEs) into the final transcript. In this study, we describe a 10-year-old girl harboring the unique deep-intronic ABCA4 variant c.6817-713A>G. Clinically, she presents with typical early-onset STGD1 with a high disease symmetry between her two eyes. Molecularly, we designed antisense oligonucleotides (AONs) to block the produced PE insertion. Splicing rescue was assessed in three different in vitro models: HEK293T cells, fibroblasts, and photoreceptor precursor cells, the last two being derived from the patient. Overall, our research is intended to serve as the basis for a personalized N-of-1 AON-based treatment to stop early vision loss in this patient.

Proof-of-concept for multiple AON delivery by a single U7snRNA vector to restore splicing defects in ABCA4.

The high allelic heterogeneity in Stargardt disease (STGD1) complicates the design of intervention strategies. A significant proportion of pathogenic intronic ABCA4 variants alters the pre-mRNA splicing process. Antisense oligonucleotides (AONs) are an attractive yet mutation-specific therapeutic strategy to restore these splicing defects. In this study, we experimentally assessed the potential of a splicing modulation therapy to target multiple intronic ABCA4 variants. AONs were inserted into U7snRNA gene cassettes and tested in midigene-based splice assays. Five potent antisense sequences were selected to generate a multiple U7snRNA cassette construct, and this combination vector showed substantial rescue of all of the splicing defects. Therefore, the combination cassette was used for viral synthesis and assessment in patient-derived photoreceptor precursor cells (PPCs). Simultaneous delivery of several modified U7snRNAs through a single AAV, however, did not show substantial splicing correction, probably due to suboptimal transduction efficiency in PPCs and/or a heterogeneous viral population containing incomplete AAV genomes. Overall, these data demonstrate the potential of the U7snRNA system to rescue multiple splicing defects, but also suggest that AAV-associated challenges are still a limiting step, underscoring the need for further optimization before implementing this strategy as a potential treatment for STGD1.

Generation of three isogenic control lines from patient-derived iPSCs carrying bi-allelic ABCA4 variants underlying Stargardt disease.

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4. Employing the CRISPR/Cas9 approach, we generated isogenic control lines (RMCGENi005-A-1, RMCGENi018-A-1, RMCGENi017-A-1) for each of three induced pluripotent stem cell lines (RMCGENi005-A, RMCGENi018-A, RMCGENi017-A) derived from Stargardt patients carrying compound heterozygous ABCA4 variants. All of the generated lines showed pluripotent characteristics, no chromosomal aberrations and no indication of off-targets. The availability of these isogenic control lines will allow us to have a fair comparison between health and disease state within our studies on Stargardt disease.

Generation of iPSC lines from three Stargardt patients carrying bi-allelic ABCA4 variants.

Stargardt disease, a progressive retinal disorder, is associated with bi-allelic variants in ABCA4, a protein that is expressed in the retina. Induced pluripotent stem cell lines (RMCGENi005-A, SCTCi018-A, SCTCi017-A) were generated by lentivirus reprogramming of fibroblasts derived from Stargardt patients carrying different bi-allelic ABCA4 variants. All the generated lines showed pluripotent characteristics and no chromosomal aberrations. The availability of these lines will allow us to generate patient-derived photoreceptor precursor cells and retinal organoids to further study ABCA4 and thereby, Stargardt disease in relevant model systems carrying the patient's genetic background.

CRISPR-Cas9 correction of a nonsense mutation in LCA5 rescues lebercilin expression and localization in human retinal organoids.

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.

The Predicted Splicing Variant c.11+5G>A in RPE65 Leads to a Reduction in mRNA Expression in a Cell-Specific Manner.

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.

Detailed Phenotyping and Therapeutic Strategies for Intronic ABCA4 Variants in Stargardt Disease.

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.

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor 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.

Antisense Oligonucleotide Screening to Optimize the Rescue of the Splicing Defect Caused by the Recurrent Deep-Intronic ABCA4 Variant c.4539+2001G>A in Stargardt Disease.

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.

Splice-Modulating Oligonucleotide QR-110 Restores CEP290 mRNA and Function in Human c.2991+1655A>G LCA10 Models.

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.

An FEVR-associated mutation in ZNF408 alters the expression of genes involved in the development of vasculature.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder hallmarked by an abnormal development of retinal vasculature. A missense mutation in ZNF408 (p.H455Y) was reported to underlie autosomal dominant FEVR in a large Dutch family, and ZNF408 was shown to play a role in the development of vasculature. Nonetheless, little is known about the molecular mechanism of ZNF408-associated FEVR. To investigate this, an in vitro model of ZNF408-associated FEVR was generated by overexpressing wild-type and p.H455Y ZNF408 in human umbilical vein endothelial cells. Cells overexpressing mutant ZNF408 were unable to form a capillary-like network in an in vitro tube formation assay, thereby mimicking the clinical feature observed in patients with FEVR. Intriguingly, transcriptome analysis revealed that genes involved in the development of vasculature were deregulated by the p.H455Y mutation. Chromatin immunoprecipitation showed that p.H455Y ZNF408 has reduced DNA-binding ability, as compared to the wild-type protein. The fact that the p.H455Y mutation disrupts the expression of genes important for the development of vasculature sheds further light on the molecular mechanisms underlying ZNF408-associated FEVR.

Antisense Oligonucleotide-Based Splicing Correction in Individuals with Leber Congenital Amaurosis due to Compound Heterozygosity for the c.2991+1655A>G Mutation in CEP290.

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.

In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery.

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.

Whole-exome sequencing reveals ZNF408 as a new gene associated with autosomal recessive retinitis pigmentosa with vitreal alterations.

Retinitis pigmentosa (RP) is a group of progressive inherited retinal dystrophies that cause visual impairment as a result of photoreceptor cell death. RP is heterogeneous, both clinically and genetically making difficult to establish precise genotype-phenotype correlations. In a Spanish family with autosomal recessive RP (arRP), homozygosity mapping and whole-exome sequencing led to the identification of a homozygous mutation (c.358_359delGT; p.Ala122Leufs*2) in the ZNF408 gene. A screening performed in 217 additional unrelated families revealed another homozygous mutation (c.1621C>T; p.Arg541Cys) in an isolated RP case. ZNF408 encodes a transcription factor that harbors 10 predicted C2H2-type fingers thought to be implicated in DNA binding. To elucidate the ZNF408 role in the retina and the pathogenesis of these mutations we have performed different functional studies. By immunohistochemical analysis in healthy human retina, we identified that ZNF408 is expressed in both cone and rod photoreceptors, in a specific type of amacrine and ganglion cells, and in retinal blood vessels. ZNF408 revealed a cytoplasmic localization and a nuclear distribution in areas corresponding with the euchromatin fraction. Immunolocalization studies showed a partial mislocalization of the p.Arg541Cys mutant protein retaining part of the WT protein in the cytoplasm. Our study demonstrates that ZNF408, previously associated with Familial Exudative Vitreoretinopathy (FEVR), is a new gene causing arRP with vitreous condensations supporting the evidence that this protein plays additional functions into the human retina.

Species-dependent splice recognition of a cryptic exon resulting from a recurrent intronic CEP290 mutation that causes congenital blindness.

A mutation in intron 26 of CEP290 (c.2991+1655A>G) is the most common genetic cause of Leber congenital amaurosis (LCA), a severe type of inherited retinal degeneration. This mutation creates a cryptic splice donor site, resulting in the insertion of an aberrant exon (exon X) into ~50% of all CEP290 transcripts. A humanized mouse model with this mutation did not recapitulate the aberrant CEP290 splicing observed in LCA patients, suggesting differential recognition of cryptic splice sites between species. To further assess this phenomenon, we generated two CEP290 minigene constructs, with and without the intronic mutation, and transfected these in cell lines of various species. RT-PCR analysis revealed that exon X is well recognized by the splicing machinery in human and non-human primate cell lines. Intriguingly, this recognition decreases in cell lines derived from species such as dog and rodents, and it is completely absent in Drosophila. In addition, other cryptic splicing events corresponding to sequences in intron 26 of CEP290 were observed to varying degrees in the different cell lines. Together, these results highlight the complexity of splice site recognition among different species, and show that care is warranted when generating animal models to mimic splice site mutations in vivo.

ZNF408 is mutated in familial exudative vitreoretinopathy and is crucial for the development of zebrafish retinal vasculature.

Familial exudative vitreoretinopathy (FEVR) is a genetically heterogeneous disorder characterized by abnormal vascularization of the peripheral retina, which can result in retinal detachment and severe visual impairment. In a large Dutch FEVR family, we performed linkage analysis, exome sequencing, and segregation analysis of DNA variants. We identified putative disease-causing DNA variants in proline-alanine-rich ste20-related kinase (c.791dup; p.Ser265ValfsX64) and zinc finger protein 408 (ZNF408) (c.1363C>T; p.His455Tyr), the latter of which was also present in an additional Dutch FEVR family that subsequently appeared to share a common ancestor with the original family. Sequence analysis of ZNF408 in 132 additional individuals with FEVR revealed another potentially pathogenic missense variant, p.Ser126Asn, in a Japanese family. Immunolocalization studies in COS-1 cells transfected with constructs encoding the WT and mutant ZNF408 proteins, revealed that the WT and the p.Ser126Asn mutant protein show complete nuclear localization, whereas the p.His455Tyr mutant protein was localized almost exclusively in the cytoplasm. Moreover, in a cotransfection assay, the p.His455Tyr mutant protein retains the WT ZNF408 protein in the cytoplasm, suggesting that this mutation acts in a dominant-negative fashion. Finally, morpholino-induced knockdown of znf408 in zebrafish revealed defects in developing retinal and trunk vasculature, that could be rescued by coinjection of RNA encoding human WT ZNF408 but not p.His455Tyr mutant ZNF408. Together, our data strongly suggest that mutant ZNF408 results in abnormal retinal vasculogenesis in humans and is associated with FEVR.

Exome sequencing identifies DYNC2H1 mutations as a common cause of asphyxiating thoracic dystrophy (Jeune syndrome) without major polydactyly, renal or retinal involvement.

BACKGROUND: Jeune asphyxiating thoracic dystrophy (JATD) is a rare, often lethal, recessively inherited chondrodysplasia characterised by shortened ribs and long bones, sometimes accompanied by polydactyly, and renal, liver and retinal disease. Mutations in intraflagellar transport (IFT) genes cause JATD, including the IFT dynein-2 motor subunit gene DYNC2H1. Genetic heterogeneity and the large DYNC2H1 gene size have hindered JATD genetic diagnosis. AIMS AND METHODS: To determine the contribution to JATD we screened DYNC2H1 in 71 JATD patients JATD patients combining SNP mapping, Sanger sequencing and exome sequencing. RESULTS AND CONCLUSIONS: We detected 34 DYNC2H1 mutations in 29/71 (41%) patients from 19/57 families (33%), showing it as a major cause of JATD especially in Northern European patients. This included 13 early protein termination mutations (nonsense/frameshift, deletion, splice site) but no patients carried these in combination, suggesting the human phenotype is at least partly hypomorphic. In addition, 21 missense mutations were distributed across DYNC2H1 and these showed some clustering to functional domains, especially the ATP motor domain. DYNC2H1 patients largely lacked significant extra-skeletal involvement, demonstrating an important genotype-phenotype correlation in JATD. Significant variability exists in the course and severity of the thoracic phenotype, both between affected siblings with identical DYNC2H1 alleles and among individuals with different alleles, which suggests the DYNC2H1 phenotype might be subject to modifier alleles, non-genetic or epigenetic factors. Assessment of fibroblasts from patients showed accumulation of anterograde IFT proteins in the ciliary tips, confirming defects similar to patients with other retrograde IFT machinery mutations, which may be of undervalued potential for diagnostic purposes.