Ataxia-telangiectasia and Rad3 (ATR), a sensor of DNA damage, is associated with the regulation and control of cell division. ATR deficit is known to cause Seckel syndrome, characterized by severe proportionate short stature and microcephaly. We used a mouse model for Seckel disease to study the effect of ATR deficit on retinal development and function and we have found a new role for ATR, which is critical for the postnatal development of the photoreceptor (PR) layer in mouse retina. The structural and functional characterization of the ATR^+/s mouse retinas displayed a specific, severe and early degeneration of rod and cone cells resembling some characteristics of human retinal degenerations. A new localization of ATR in the cilia of PRs and the fact that mutant mice have shorter cilia suggests that the PR degeneration here described results from a ciliary defect.

Background: Gene therapy is a therapeutic possibility for retinitis pigmentosa (RP), in which therapeutic transgenes are currently delivered to the retina by adeno-associated viral vectors (AAVs). Although their safety and efficacy have been demonstrated in both clinical and preclinical settings, AAVs present some technical handicaps, such as limited cargo capacity and possible immunogenicity in repetitive doses. The development of alternative, non-viral delivery platforms like nanoparticles is of great interest to extend the application of gene therapy for RP. Methods: Amino-functionalized mesoporous silica-based nanoparticles (N-MSiNPs) were synthesized, physico-chemically characterized, and evaluated as gene delivery systems for human cells in vitro and for retinal cells in vivo. Transgene expression was evaluated by WB and immunofluorescence. The safety evaluation of mice subjected to subretinal injection was assessed by ophthalmological tests (electroretinogram, funduscopy, tomography, and optokinetic test). Results: N-MSiNPs delivered transgenes to human cells in vitro and to retinal cells in vivo. No adverse effects were detected for the integrity of the retinal tissue or the visual function of treated eyes. N-MSiNPs were able to deliver a therapeutic transgene candidate for RP, PRPF31, both in vitro and in vivo. Conclusions: N-MSiNPs are safe for retinal delivery and thus a potential alternative to viral vectors.

Small nuclear RNAs (snRNAs) combine with specific proteins to generate small nuclear ribonucleoproteins (snRNPs), the building blocks of the spliceosome. U4 snRNA forms a duplex with U6 and, together with U5, contributes to the tri-snRNP spliceosomal complex. Variants in RNU4-2, which encodes U4, have recently been implicated in neurodevelopmental disorders. Here we show that heterozygous inherited and de novo variants in RNU4-2 and in four RNU6 paralogs (RNU6-1, RNU6-2, RNU6-8 and RNU6-9), which encode U6, recur in individuals with nonsyndromic retinitis pigmentosa (RP), a genetic disorder causing progressive blindness. These variants cluster within the three-way junction of the U4/U6 duplex, a site that interacts with tri-snRNP splicing factors also known to cause RP (PRPF3, PRPF8, PRPF31), and seem to affect snRNP biogenesis. Based on our cohort, deleterious variants in RNU4-2 and RNU6 paralogs may explain up to ~1.4% of otherwise undiagnosed RP cases. This study highlights the contribution of noncoding RNA genes to Mendelian disease and reveals pleiotropy in RNU4-2, where distinct variants underlie neurodevelopmental disorder and retinal degeneration.

Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identified upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebrafish. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the flattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE specification network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.

Damage to the retinal pigment epithelium (RPE) in age-related macular degeneration and other diseases results in photoreceptor cell death and blindness. Replacement of RPE is therefore being explored as a therapy for several retinal diseases. To move toward a future personalized autologous transplant approach, we have prepared a biocompatible implant using RPE derived from induced pluripotent stem cells (iPSCs) reprogrammed from a healthy donor's monocytes. The correct positioning of the polarized RPE is essential to fulfill its role and protect photoreceptors from degeneration. Hence, we have used a biocompatible hydrogel matrix of fibrin and agarose that allows the surgical placement of an RPE sheet in the subretinal space, keeping its functional orientation. Our aim was to demonstrate safety and viability of the transplant in preclinical models. Pigs were used to test the feasibility of a regular vitreoretinal surgery. Our results show that this implant is suitable for subretinal transplantation allowing human RPE cells to survive and maintain their phenotype and orientation without any local or systemic adverse events. The ability to transplant the iPSC-derived RPE sheet in its natural orientation will surely increase the chance to obtain a therapeutic effect in future translational studies. Impact Statement: In the promising field of cellular therapy for retinal degenerative diseases, a new biomaterial is proposed as a scaffold to grow and surgically introduce a monolayer of retinal pigment epithelial cells into the subretinal space, keeping the orientation of the cells for a proper functional integration of the transplant. The use of induced pluripotent stem cells as the starting material for retinal pigment epithelial cells is intended to advance toward a personalized medicine approach.

Retinitis pigmentosa is a common cause of inherited blindness in adults, which in many cases is associated with an increase in the formation of reactive oxygen species (ROS) that induces DNA damage, triggering Poly-ADP-Ribose Polymerase 1 (PARP1) activation and leading to parthanatos-mediated cell death. Previous studies have shown that resveratrol (RSV) is a promising molecule that can mitigate PARP1 overactivity, but its low bioavailability is a limitation for medical use. This study examined the impact of a synthesized new acylated RSV prodrug, piceid octanoate (PIC-OCT), in the 661W cell line against H₂O₂ oxidative stress and in rd10 mice. PIC-OCT possesses a better ADME profile than RSV. In response to H₂O₂, 661W cells pretreated with PIC-OCT preserved cell viability in more than 38% of cells by significantly promoting SIRT1 nuclear translocation, preserving NAD⁺/NADH ratio, and suppressing intracellular ROS formation. These effects result from expressing antioxidant genes, maintaining mitochondrial function, reducing PARP1 nuclear expression, and preventing AIF nuclear translocation. In rd10 mice, PIC-OCT inhibited PAR-polymer formation, increased SIRT1 expression, significantly reduced TUNEL-positive cells in the retinal outer nuclear layer, preserved ERGs, and enhanced light chamber activity (all p values < 0.05). Our findings corroborate that PIC-OCT protects photoreceptors by modulating the SIRT1/PARP1 axis in models of retinal degeneration.