Biology subjects

Wissinger, B.

Publications and source records attributed to Wissinger, B..

3 recordsLinked to original sources

bioRxiv2026

A mutation-agnostic and allele-specific ASO strategy demonstrates potent functional rescue and retinal preservation in RHO-linked retinitis pigmentosa

Autosomal dominant retinitis pigmentosa (adRP) caused by RHO mutations is a leading form of inherited retinal degeneration. Extensive allelic heterogeneity of RHO pathogenic variants limits the translational applicability of mutation-specific gene therapies. To address this, we developed SNARE (SNP-guided Silencing of Aberrant RHO Expression), a mutation-independent, allele-specific antisense oligonucleotide (ASO) strategy. SNARE selectively suppresses mutant RHO transcripts by targeting the common, benign c.-26A/G single-nucleotide polymorphism (SNP) as an allelic discriminator. Candidate gapmer ASOs were screened in engineered reporter lines and validated in patient-derived retinal organoids, identifying RHOligo-A as the lead c.-26A-targeting candidate. In vitro, RHOligo-A achieved robust, preferential knockdown of the target allele, improving RHO localization in retinal organoids, and demonstrated a favorable safety profile with minimal transcriptomic off-target effects and no detectable immunostimulatory activity. Subsequent validation in a novel, humanized RHOP347L/WT mouse model, achieved sustained c.-26A-linked allele-selective suppression, retinal structure preservation, and significantly restored visual function, upon a single intravitreal administration. These findings establish RHOligo-A and SNARE as a scalable, mutation-independent therapeutic platform with strong translational potential and substantial clinical reach for RHO-associated adRP.

genetics↗

bioRxiv2024

EDSpliCE, a CRISPR-Cas9 gene editing platform to rescue splicing, effectively corrects inherited retinal dystrophy-associated splicing defects

BackgroundCorrect splicing of transcripts is essential to ensure the production of functional gene products in eukaryotic cells. Missplicing of transcripts has been identified as the underlying molecular mechanisms behind various disease-causing variants in a wide range of inherited genetic conditions. Achieving therapeutic splicing correction is possible through antisense oligonucleotide and CRISPR/Cas9 strategies. However, while antisense oligonucleotides offer effective modulation, they do not enable for permanent correction. On the other hand, current CRISPR/Cas9 approaches often rely on dual-gRNA-inducing deletion of larger pieces of DNA, containing the site(s) responsible for the splicing defect, particularly the elimination of pseudoexons, raising concerns about potential chromosomal instability. ResultsThe novel gene editing strategy, Enhanced-Deletion Splicing Correction Editing (EDSpliCE), just uses single gRNAs to effectively correct aberrant splicing caused by pseudoexon sequence inclusion into the mature mRNA. By employing Cas9 fused to a human exonuclease (TREX2), EDSpliCE achieves targeted enhanced deletions of sequences involved in pseudoexon recognition, thereby restoring correct splicing of the pre-mRNA. By addressing two isolated (ABCA4:c.5197-557G>T and USH2A:c.7595-2144A>G) and two clustered (ABCA4:c.5196+1013A>G and ABCA4:c.5196+1056A>G) pathogenic deep-intronic variants, we demonstrated effective splicing rescue in minigene assay employing distinct single gRNAs. Further validation in patient-derived fibroblasts for the common USH2A:c.7595-2144A>G variant confirmed consistent and high splicing correction. Additionally, the characterization of achieved gene editing affirmed the generation of enhanced deletions by EDSpliCE, revealed high directionality of editing events for all the single gRNAs tested in patient-derived fibroblasts and did not show higher off-target editing potential on selected loci. ConclusionsThe successful implementation of the EDSpliCE platform for splicing correction and modulation offers a promising and versatile gene editing approach to address splicing defects, potentially providing a safer option to existing gene editing strategies.

genomics↗

bioRxiv2018

Molecular and clinical analysis of 27 German patients with Leber congenital amaurosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies (IRD) and the most frequent cause of inherited blindness in children. The phenotypic overlap with other early-onset and severe IRDs as well as difficulties associated with the ophthalmic examination of infants can complicate the clinical diagnosis. To date, 25 genes have been implicated in the pathogenesis of LCA. The disorder is usually inherited in an autosomal recessive fashion, although rare dominant cases have been reported. We report the mutation spectra and frequency of genes in 27 German index patients initially diagnosed with LCA. A total of 108 LCA- and other genes implicated in IRD were analysed using a cost-effective targeted next-generation sequencing procedure based on molecular inversion probes (MIPs). Sequencing and variant filtering led to the identification of putative pathogenic variants in 25 cases, thereby leading to a detection rate of 93%. The mutation spectrum comprises 34 different alleles, 17 of which are novel. In line with previous studies, the genetic results led to a revision of the initial clinical diagnosis in a substantial proportion of cases, demonstrating the importance of genetic testing in IRD. In addition, our detection rate of 93% shows that MIPs are a cost-efficient and sensitive tool for targeted next-generation sequencing in IRD.

genetics↗