Biology subjects

Collin, R. W. J.

Publications and source records attributed to Collin, R. W. J..

6 recordsLinked to original sources

bioRxiv2026

Genome sequencing reveals novel pathogenic deep-intronic PCDH15 variants, amenable to antisense oligonucleotide-based splice correction

Despite substantial advances in diagnostic testing, 10-15% of Usher syndrome patients remain without a genetic diagnosis, having significant implications for genetic counseling and potential future therapeutic interventions. In this study, genome sequencing data from probands clinically presenting with Usher syndrome were analyzed. Two novel deep-intronic variants were identified in PCDH15, c.3983+3635A>G and c.3123-1728A>G, in two independent patients. Both deep-intronic variants were classified as likely pathogenic and predicted to alter PCDH15 pre-mRNA splicing. Using a minigene splice assay and iPSC-derived photoreceptor precursor cells from patients, we confirmed that both variants lead to the inclusion of a pseudoexon in the PCDH15 transcript introducing a stop codon and subsequent premature termination of protein translation. We designed and evaluated antisense oligonucleotides (ASOs) with the purpose of redirecting aberrant pre-mRNA splicing caused by both deep-intronic variants. For both variants, designed ASOs were successful in restoring normal splicing patterns, highlighting their potential as a future therapeutic intervention strategy to halt the progression of retinitis pigmentosa caused by these novel variants. Overall, these findings contribute to the understanding of Usher syndrome caused by deep-intronic pathogenic variants in PCDH15 and describe for the first time the use of an ASO-mediated splice correction strategy for individuals diagnosed with these variants.

genetics↗

bioRxiv2026

The Lipidome of iPSC-Derived Retinal Organoids and RPE Partially Resembles that of the Human Retina

New approach methodologies (NAMs), including induced pluripotent stem cell (iPSC)-derived retinal organoids (ROs) and retinal pigment epithelium (iRPE), are increasingly applied to study retinal disease mechanisms and therapeutic strategies. However, these models often remain relatively immature. Given the high lipid content and complex metabolism of the retina, it is unclear to what extent iPSC-derived systems recapitulate the human retinal lipidome. Here, we compared the lipidomic profiles of ROs and iRPE, collected at several differentiation stages, with those of post-mortem adult human macular, non-macular and RPE plus choroid (pmRPE). The lipidome of iRPE differed markedly from pmRPE, whereas prolonged differentiation of ROs resulted in a lipidomic profile increasingly resembling that of the post-mortem retina. Moreover, ROs showed similarities to both macular and non-macular lipidome. These findings show that iPSC-derived models can become valuable NAMs to study lipid-related retinal disorders and provide a framework to optimize differentiation protocols.

molecular biology↗

bioRxiv2026

Usherin in the pineal gland: altered sleep in zebrafish models of Usher syndrome type 2a

Usher syndrome type 2A (USH2a), the most common form of hereditary deaf-blindness, is frequently accompanied by fatigue and poor sleep quality. As these sleep problems occur independently of visual decline, it is hypothesized that the USH2A-encoded protein usherin regulates sleep and circadian rhythmicity via an extra-retinal mechanism. Ush2a knockout zebrafish models were utilized to investigate this hypothesis. Immunohistochemical analysis demonstrated usherin localisation in pineal gland photoreceptor cells in wild-type larvae, alongside the USH2 complex proteins Adgrv1 and Whrna. Cross-species transcriptomic and proteomic analyses confirmed USH2A expression in all mammalian pineal gland tissues studied. Circadian clock gene expression was measured over 24 h and showed preserved oscillatory patterns in wild-type and mutant zebrafish. Ex vivo superfusion of pineal glands revealed sustained circadian melatonin release with comparable phase and period to controls, although potential differences in absolute melatonin levels could not be excluded. Despite intact clock gene expression and melatonin release in ush2a mutants, behavioural classification over 24-h recordings revealed altered sleep-wake behaviour: ush2a mutants displayed elevated daytime sleep and significantly prolonged and more variable sleep latency. The dissociation between intact molecular rhythms and abnormal sleep behaviour likely implicates that usherin plays a role in sleep-wake regulation independent of the circadian pacemaker and melatonin synthesis. These findings suggest a novel role of usherin in the pineal gland and establish a mechanistic link between usherin dysfunction and sleep disturbances, providing a biological basis for the fatigue and sleep problems reported in USH2a patients.

animal behavior and cognition↗

bioRxiv2025

Long-term daily light exposure boosts photoreceptor maturation in retinal organoids

Induced pluripotent stem cell-derived retinal organoids (ROs) have become promising personalized models to study inherited retinal diseases (IRDs) and develop new innovative therapies. Although ROs mimic key retinal features and show some light responsiveness, their differentiation and maturation remain limited and lengthy. We hypothesize that standard dark culture conditions limit the expression of genes and proteins related to retinal function, delaying differentiation and complicating disease modeling. Therefore, we investigated whether daily light exposure could promote photoreceptor maturation. ROs were exposed to six hours of daily light starting from day 70 in vitro. Light conditioning led to enhanced photoreceptor maturation, specifically an increase in rod photoreceptors, a late-born cell type, without signs of increased stress or cell death. Our findings suggest that daily light exposure enhances RO differentiation, opening new avenues to investigate molecular and cellular phenotypes in IRDs and accelerate therapy development in more relevant functional models.

cell biology↗

bioRxiv2023

Probing the sub-cellular mechanisms of LCA5-Leber Congenital Amaurosis and associated gene therapy with expansion microscopy

Leber Congenital Amaurosis (LCA) is a group of Inherited Retinal Diseases (IRDs) characterized by the early onset and rapid loss of photoreceptor cells. Despite the discovery of a growing number of genes associated with this disease, the molecular mechanisms of photoreceptor cell degeneration of most LCA subtypes remain poorly understood. Here, using retina-specific affinity proteomics combined with Ultrastructure Expansion Microscopy (U-ExM), we revealed the structural and molecular defects underlying LCA type 5 (LCA5) with unprecedented resolution. We showed that LCA5-encoded lebercilin, together with Retinitis Pigmentosa 1 protein (RP1) and the intraflagellar transport (IFT) proteins IFT81 and IFT88, localize at the bulge region of the photoreceptor outer segment (OS), a region crucial for OS membrane disc formation. Next, we demonstrated that mutant mice deficient for lebercilin exhibit early axonemal defects at the bulge region and the distal OS, accompanied by reduced level of RP1 and IFT proteins, affecting membrane disc formation and presumably leading to photoreceptor death. Finally, we probed the LCA5 gene augmentation therapy strategy using U-ExM by monitoring its subcellular outcome. We found that, expression of LCA5 partially restores the bulge region, preserves OS axoneme structure and membrane disc formation, as well as photoreceptor survival.

molecular biology↗

bioRxiv2022

Antisense oligonucleotide therapy for the common Stargardt disease type 1-causing variant in ABCA4

The c.5461-10T>C p.[Thr1821Aspfs*6,Thr1821Valfs*13] variant has been identified as the most common severe Stargardt disease type 1 (STGD1)-associated variant in ABCA4. STGD1 is the most recurrent hereditary form of maculopathy and so far, no treatment is available for STGD1. In STGD1 patients homozygous for this variant, the onset of the disease typically is in childhood and patients are legally blind by early adulthood. The variant leads to exon skipping and generates out-of-frame ABCA4 transcripts that prevent the translation of functional ABCA4 protein. We applied antisense oligonucleotides (AONs) to restore the wild-type RNA splicing in ABCA4 c.5461-10T>C. The effect of AONs was investigated in vitro using an ABCA4 midigene model and 3D human retinal organoids (ROs) homozygous for the ABCA4 c.5461-10T>C variant. The mRNA in untreated ROs contained only disease-associated isoforms, whereas the organoids treated with the lead AON sequence showed 53% splicing correction and restoration of ABCA4 protein. Collectively, these data identified the lead candidate QR-1011 as a potent splice-correcting AON to be further developed as therapeutic intervention for patients harboring the severe ABCA4 c.5461-10T>C variant.

molecular biology↗