Biology subjects

De Angeli, P.

Publications and source records attributed to De Angeli, P..

4 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↗

bioRxiv2025

Lost in communication: How Mueller glia cells fail to maintain retinal integrity in USH1C retinal organoids

Usher syndrome type 1, caused by pathogenic variants in the USH1C gene, leads to congenital deafness and progressive retinal degeneration resulting in vision loss. While auditory deficits can be compensated by cochlea implants and hearing aids, no treatment exists to prevent retinal degeneration. Here, we generated retinal organoids from induced pluripotent stem cells of two USH1C patients to elucidate the cellular and molecular mechanisms driving ocular pathogenesis. Single-cell RNA sequencing of both healthy and USH1C retinal organoids identified differential expression of genes related to phototransduction in photoreceptors, as well as alterations in cell adhesion and canonical Wnt signaling in Muller glia cells. Analysis of intercellular communication revealed an overall reduced signaling efficiency, particularly affecting Muller glia-mediated retinal adhesion processes. Morphological characterization of organoids confirmed transcriptome changes by showing degeneration of the outer limiting membrane and loss of adherens junction architecture. Moreover, photoreceptors revealed increased susceptibility to morphological and functional changes related to phototransduction. These results demonstrate that disruption of Muller glia signaling contributes to a loss of retinal integrity, providing novel insights into USH1C pathogenesis and offering targets for therapeutic interventions.

cell biology↗

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

Mutations generated by repair of Cas9-induced double strand breaks are predictable from surrounding sequence

The exact DNA mutation produced by cellular repair of a CRISPR/Cas9-generated double strand break determines its phenotypic effect. It is known that the mutational outcomes are not random, and depend on DNA sequence at the targeted location. Here, we present a systematic study of this link. We created a high throughput assay to directly measure the edits generated by over 40,000 guide RNAs, and applied it in a range of genetic backgrounds and for alternative CRISPR/Cas9 reagents. In total, we gathered data for over 1,000,000,000 mutational outcomes in synthetic constructs, which mirror those at endogenous loci. The majority of reproducible mutations are insertions of a single base, short deletions, or long microhomology-mediated deletions. gRNAs have a cell-line dependent preference for particular outcomes, especially favouring single base insertions and microhomology-mediated deletions. We uncover sequence determinants of the produced mutations at individual loci, and use these to derive a predictor of Cas9 editing outcomes with accuracy close to the theoretical maximum. This improved understanding of sequence repair allows better design of editing experiments, and may lead to future therapeutic applications.

cell biology↗