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.

Pathogenic variants in three families with distal muscle involvement.

Three families suspected of distal hereditary motor neuropathy underwent genetic screening with the aim to identify the molecular defect underlying the disease. The description of the identification reflects the shift in molecular diagnostics that was made during the last decades. Our candidate gene approach yielded a known pathogenic variant in BSCL2 (p.Asn88Ser) in one family, and via a CMT-capture, in HSPB1 (p.Arg127Trp), in addition to five other variations in Charcot-Marie-Tooth-related genes in the proband of the second family. In the third family, using whole exome sequencing, followed by linkage-by-location, a three base pair deletion in exon 33 of MYH7 (p.Glu1508del) was found, a reported pathogenic allele albeit for a myopathy. After identification of the causative molecular defect, cardiac examination was performed for patients of the third family and this demonstrated abnormalities in three out of five affected family members. Heterogeneity and expansion of clinical phenotypes beyond known characteristics requires a wider set of genes to be screened. Whole exome/genome analysis with limited prior clinical information may therefore be used to precede a detailed clinical evaluation in cases of large families, preventing screening of a too narrow set of genes, and enabling the identification of novel disease-associated genes. In our cases, the variants had been reported, and co-segregation analysis confirmed the molecular diagnosis.

Modeling ZNF408-Associated FEVR in Zebrafish Results in Abnormal Retinal Vasculature.

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.

Epithelial HMGB1 Delays Skin Wound Healing and Drives Tumor Initiation by Priming Neutrophils for NET Formation.

Regenerative responses predispose tissues to tumor formation by largely unknown mechanisms. High-mobility group box 1 (HMGB1) is a danger-associated molecular pattern contributing to inflammatory pathologies. We show that HMGB1 derived from keratinocytes, but not myeloid cells, delays cutaneous wound healing and drives tumor formation. In wounds of mice lacking HMGB1 selectively in keratinocytes, a marked reduction in neutrophil extracellular trap (NET) formation is observed. Pharmacological targeting of HMGB1 or NETs prevents skin tumorigenesis and accelerates wound regeneration. HMGB1-dependent NET formation and skin tumorigenesis is orchestrated by tumor necrosis factor (TNF) and requires RIPK1 kinase activity. NETs are present in the microenvironment of keratinocyte-derived tumors in mice and lesional and tumor skin of patients suffering from recessive dystrophic epidermolysis bullosa, a disease in which skin blistering predisposes to tumorigenesis. We conclude that tumorigenicity of the wound microenvironment depends on epithelial-derived HMGB1 regulating NET formation, thereby establishing a mechanism linking reparative inflammation to tumor initiation.

Detection and quantification of a KIF11 mosaicism in a subject presenting familial exudative vitreoretinopathy with microcephaly.

Familial exudative vitreoretinopathy (FEVR) is an inherited retinal disorder, which is primarily characterized by abnormal development of retinal vasculature. In this study, we reported a subject presenting the clinical features of FEVR as well as microcephaly. Screening of the KIF11 gene in this patient revealed a novel heterozygous protein-truncating variant (c.2717del, p.(L906*), NM_004523.3). Segregation analysis in the unaffected parents using Sanger sequencing suggested the variant to be present in a mosaic state in the unaffected mother. KIF11 exon 19 which harbors the variant was amplified from the proband and her father, as well as three different tissues of the mother, followed by amplicon-based deep sequencing. This analysis revealed that the variant is present in different tissues of the mother at various rates, i.e. in blood (16.9%), saliva (20.7%), or skin biopsy-derived fibroblast cells (6.6%). These data demonstrate the importance of deep sequencing in unaffected parents upon detection of a genetic defect in isolated cases to detect possible mosaicisms, enabling a  more reliable recurrence risk assessment and thereby improve genetic counseling.

Hypermorphic and hypomorphic AARS alleles in patients with CMT2N expand clinical and molecular heterogeneities.

Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes implicated in several dominant and recessive disease phenotypes. The canonical function of ARSs is to couple an amino acid to a cognate transfer RNA (tRNA). We identified three novel disease-associated missense mutations in the alanyl-tRNA synthetase (AARS) gene in three families with dominant axonal Charcot-Marie-Tooth (CMT) disease. Two mutations (p.Arg326Trp and p.Glu337Lys) are located near a recurrent pathologic change in AARS, p.Arg329His. The third (p.Ser627Leu) is in the editing domain of the protein in which hitherto only mutations associated with recessive encephalopathies have been described. Yeast complementation assays demonstrated that two mutations (p.Ser627Leu and p.Arg326Trp) represent loss-of-function alleles, while the third (p.Glu337Lys) represents a hypermorphic allele. Further, aminoacylation assays confirmed that the third mutation (p.Glu337Lys) increases tRNA charging velocity. To test the effect of each mutation in the context of a vertebrate nervous system, we developed a zebrafish assay. Remarkably, all three mutations caused a pathological phenotype of neural abnormalities when expressed in zebrafish, while expression of the human wild-type messenger RNA (mRNA) did not. Our data indicate that not only functional null or hypomorphic alleles, but also hypermorphic AARS alleles can cause dominantly inherited axonal CMT disease.

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.

Defects in the Cell Signaling Mediator ß-Catenin Cause the Retinal Vascular Condition FEVR.

Familial exudative vitreoretinopathy (FEVR) is an inherited blinding disorder characterized by the abnormal development of the retinal vasculature. The majority of mutations identified in FEVR are found within four genes that encode the receptor complex (FZD4, LRP5, and TSPAN12) and ligand (NDP) of a molecular pathway that controls angiogenesis, the Norrin-ß-catenin signaling pathway. However, half of all FEVR-affected case subjects do not harbor mutations in these genes, indicating that further mutated genes remain to be identified. Here we report the identification of mutations in CTNNB1, the gene encoding ß-catenin, as a cause of FEVR. We describe heterozygous mutations (c.2142_2157dup [p.His720∗] and c.2128C>T [p.Arg710Cys]) in two dominant FEVR-affected families and a de novo mutation (c.1434_1435insC [p.Glu479Argfs∗18]) in a simplex case subject. Previous studies have reported heterozygous de novo CTNNB1 mutations as a cause of syndromic intellectual disability (ID) and autism spectrum disorder, and somatic mutations are linked to many cancers. However, in this study we show that Mendelian inherited CTNNB1 mutations can cause non-syndromic FEVR and that FEVR can be a part of the syndromic ID phenotype, further establishing the role that ß-catenin signaling plays in the development of the retinal vasculature.

NANOG and CDX2 pattern distinct subtypes of human mesoderm during exit from pluripotency.

Mesoderm is induced at the primitive streak (PS) and patterns subsequently into mesodermal subtypes and organ precursors. It is unclear whether mesoderm induction generates a multipotent PS progenitor or several distinct ones with restricted subtype potentials. We induced mesoderm in human pluripotent stem cells with ACTIVIN and BMP or with GSK3-ß inhibition. Both approaches induced BRACHYURY(+) mesoderm of distinct PS-like identities, which had differing patterning potential. ACTIVIN and BMP-induced mesoderm patterned into cardiac but not somitic subtypes. Conversely, PS precursors induced by GSK3-ß inhibition did not generate lateral plate and cardiac mesoderm and favored instead somitic differentiation. The mechanism of these cell fate decisions involved mutual repression of NANOG and CDX2. Although NANOG was required for cardiac specification but blocked somitic subtypes, CDX2 was required for somitic mesoderm but blocked cardiac differentiation. In sum, rather than forming a common PS progenitor, separate induction mechanisms distinguish human mesoderm subtypes.