Biology subjects

Banin, E.

Publications and source records attributed to Banin, E..

8 recordsLinked to original sources

bioRxiv2026

Wavelet Transform improves time-frequency characterization of the Electroretinographic Oscillatory Potentials as compared to Short-Time Fourier Transform across experimental and clinical models

Purpose: To compare continuous wavelet transform (CWT) and short-time Fourier transform (STFT) analyses of the oscillatory potentials (OPs) of the dark-adapted full-field electroretinogram (FFERG). Methods: FFERG recordings from Royal College of Surgeons (RCS) and healthy Brown Norway (BN) rats, wild-type and Atoh7 enhancer-knockout (KO) mice, and patients with diabetic retinopathy and healthy controls were analyzed with STFT and with CWT employing the real Morlet wavelet, after 60 Hz high-pass and sharp 60 Hz notch filtering. Time- and frequency-localized OP features were quantified and the methods compared on detection of transient OP signals. Both were additionally validated using synthetic OP-like bursts of known frequency embedded in each cohort's recorded noise. Results: CWT revealed distinct time-frequency patterns and disease-specific OP alterations across all rat, mouse, and human cohorts, with improved temporal localization and frequency resolution, whereas STFT merged transient features and was more susceptible to noise. CWT-derived dominant-frequency differences reached significance in all three models; the corresponding STFT difference reached significance only in rats (separate tests; methods not directly compared). In synthetic bursts, both methods recovered injected frequencies to within 1-2.5 Hz, but only CWT resolved two bursts separated by 15 ms. Conclusions: CWT employing the real Morlet wavelet provides a robust, physiologically relevant evaluation of OPs, with improved temporal localization and reduced spectral dispersion relative to STFT in clinical and experimental settings. Translational Relevance: CWT analysis may improve the characterization of inner retinal dysfunction by preserving transient OP time-frequency structure, as illustrated in preclinical rodent models and diabetic retinopathy patients.

physiology↗

bioRxiv2026

Transcript-Specific Site-Directed RNA Editing Reveals Principles for Splice-aware guide RNA Design

Site-directed RNA editing (SDRE) utilizing the adenosine deaminase acting on RNA (ADAR) enzymes is commonly facilitated by guide RNAs (gRNAs) optimized to enhance on-target editing and minimize bystander effects. However, the impact of gRNA binding and ADAR-mediated SDRE on canonical pre-mRNA splicing remains poorly understood. Here, we developed an in vitro transcript-specific editing strategy that enables selective targeting and direct comparison of SDRE in pre-mRNA and mature mRNA. Using splice-relevant variants associated with inherited retinal diseases, we investigated the effects of SDRE on exonic, near-canonical intronic, and deep intronic splice variants. We identified gRNA-induced splice perturbation at exonic and intronic targets and observed that higher editing levels could be associated with increased splice disruption. Conversely, SDRE of two exonic splice variants and a deep intronic variant resulted in increased production of correctly spliced transcripts, demonstrating the potential of SDRE for splice modulation. Finally, by dissecting the effects of ADAR expression and gRNA design on editing and splicing outcomes, we established a system for identifying design principles that reduce splice interference and enhance the generation of correctly spliced, edited transcripts. These findings highlight the importance of considering transcript context and splicing consequences in the development of SDRE-based therapeutic strategies. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741163v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@55e0cforg.highwire.dtl.DTLVardef@1a5c29org.highwire.dtl.DTLVardef@98aec7org.highwire.dtl.DTLVardef@1fab73b_HPS_FORMAT_FIGEXP M_FIG Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiy C_FIG

molecular biology↗

bioRxiv2026

Single anticodon-edited tRNA therapy targeting highly prevalent Arg>Ter premature termination codons causing inherited retinal diseases

Nonsense variants cause 18% of inherited retinal diseases (IRDs), yet current therapies require variant-specific development, leaving most patients untreated. Here, we combined a large-scale genetic analysis literature survey of >37,500 IRD patients with anticodon-edited (ACE)-tRNA engineering to create a single, gene-agnostic therapy targeting Arg>Ter nonsense variants which are the most prevalent subclass (35%) of premature stop codons (PTCs). We developed an optimized ACE-tRNA (V3) that achieved up to 86% readthrough across 13 clinically relevant variants, restored native PRCD localization in the arRP-causing p.R22* mutant, and demonstrated activity in photoreceptor-like cells. To enable translation, we established an AAV2/7m8 production platform (1*10{superscript 1}{superscript 2}-1*10{superscript 1}3 GC/mL) and defined 1*10 GC/eye as the safe dose in mice. This patient genetics-guided strategy positions ACE-tRNA_V3 as a promising candidate for preclinical development, offering a precision medicine approach that targets the most common nonsense variant class with a single therapeutic molecule.

genetics↗

bioRxiv2025

Smarcc1 is essential for the patterning of the optic stalk and differentiation of the optic nerve head astrocytes

The optic nerve is essential for vision, and its development depends on the transient neuroectodermal optic stalk, which forms alongside the optic cup through coordinated morphogenesis and differentiation into optic nerve astrocytes that provide lifelong support for retinal ganglion cell (RGC) axons. Here, we delineate key steps in astrocyte formation from the optic stalk and uncover stage- and lineage-specific roles of Smarcc1 (Baf155) and Smarcc2 (Baf170), scaffolding subunits of SWI/SNF chromatin remodeling complexes. Both factors are co-expressed in early retinal pigment epithelium (RPE) and optic stalk progenitors, with Smarcc2 persisting in differentiated derivatives, including RPE and optic nerve astrocytes. Conditional deletion using Dct-Cre revealed reciprocal compensation in pigmented lineages, whereas Smarcc1 loss uniquely caused optic nerve head (ONH) morphogenetic failure, characterized by glial lamina collapse, RGC degeneration, and progressive visual decline. Spatial transcriptomics and functional assays demonstrate that Smarcc1 drives the transition of dorsal optic stalk progenitors from a pigmented, RPE-like state to astrocyte progenitors by repressing pigment gene programs and enabling Pax2/Sox2 activity. After specification, Smarcc1 is required for glial lamina assembly, essential for ONH integrity and RGC survival. These findings establish a mechanism in which Smarcc1-mediated chromatin remodeling couples astrocyte progenitor specification and differentiation with local morphogenesis, ensuring long-term retinal function.

developmental biology↗

bioRxiv2024

Multigenerational Proteolytic Inactivation of Restriction Upon Subtle Genomic Hypomethylation

Restriction-modification (R-M) systems, present in most bacterial genomes, protect against phage infection by detecting and degrading invading foreign DNA. However, like many prokaryotic anti-phage systems, R-M systems pose a significant risk of auto-immunity, exacerbated by the presence of hundreds to thousands of potential cleavage sites in the bacterial genome. In Pseudomonas aeruginosa, restriction inactivation upon growth at high temperatures was previously described, however, which system is being inactivated, the underlying mechanism, as well as the timing of recovery, remain unknown. Here, we report that P. aeruginosa Type I methyltransferase (HsdMS) and restriction endonuclease (HsdR) components are degraded by two Lon-like proteases when replicating above 41 {degrees}C, which induces partial genome hypomethylation and simultaneously prevents self-targeting, respectively. Interestingly, upon return to 37 {degrees}C, methyltransferase activity returns gradually, with restriction activity not fully recovering for over 60 bacterial generations, representing the longest bacterial memory to our knowledge. Forced expression of HsdR over the first 45 generations is toxic, demonstrating the fitness benefit of HsdR inactivation. Our findings demonstrate that type I R-M is tightly regulated post-translationally with a remarkable memory effect to ensure genomic stability and emphasize the importance of mitigating auto-toxicity for bacterial defense systems.

microbiology↗

bioRxiv2024

The prevalent NR2E3 c.932G>A mutation induces aberrant splicing that can be rescued using splice-shifting antisense oligonucleotides

Mutations in NR2E3 have been implicated in several progressive retinal disease phenotypes such as enhanced S-cone syndrome, Goldmann-Favre syndrome and retinitis pigmentosa. One of the most frequent mutations in NR2E3 is c.932G>A (p.R311Q), where pathogenicity is thought to stem from the resulting amino acid substitution. However, multiple studies that evaluated the effect of this substitution on the protein, did not elucidate the molecular basis underlying the pathogenicity. Primed by bioinformatic analyses, we hypothesized and experimentally validated that the NR2E3 c.932G>A mutation leads to aberrant splicing which results in a short, non-functional protein isoform. Using cell models expressing WT and mutant constructs of the full NR2E3 sequence (including exonic and intronic regions), we observed that the mutated transcript exhibits a high level (75%) of aberrant splicing through gain of a novel splice acceptor site within exon 6. This mis-splicing results in the in-frame loss of 186 base pairs that code for a portion of the protein ligand binding domain. We further designed and evaluated splice-shifting antisense oligonucleotides (ASOs), that circumvented the aberrant splicing. The best performing ASO successfully restored 70% of the total NR2E3 full-length isoform levels and demonstrated rescue of nuclear localization and rhodopsin transcriptional activation. This study demonstrates the importance of understanding splicing consequences of pathogenic mutations, allowing the design and development of ASO-based therapies. Our findings set the stage for the potential treatment of NR2E3-related retinal degeneration caused by the c.932G>A mutation using splice-shifting ASOs.

genomics↗

bioRxiv2023

Fine-tuning FAM161A gene augmentation therapy to restore retinal function

In 15 years, inherited retinal diseases have seen gene therapy as a springboard to hope. Many preclinical investigations focused on vectors with maximal gene expression capabilities. But despite an efficient gene transfer, little physiological improvement was noted for certain ciliopathies. FAM161A is an essential protein for the structure of photoreceptor connecting cilium (CC). In the absence of FAM161A, cilia disorganize resulting in outersegment collapses and vision impairment. Within the human retina, FAM161A produces two isoforms: the long with exon 4, and the short, lacking it. To restore CC in Fam161a-deficient mice, we compared AAV vectors with different promoter activities, doses, and human isoforms injected subretinally in 14-days Fam161atm1b/tm1b mice, shortly after the onset of cilium disorganization. All vectors improved cell survival, but only combining both isoforms using the weak FCBR1-F0.4 promoter allowed precise FAM161A expression in the CC and enhanced retinal function. Our study on FAM161A gene replacement for RP28, a rod-cone-related disease, underscores the critical need for precise therapeutic gene regulation, appropriate vector dosing and delivery of both isoforms. Fine tuning of therapeutic gene expression, tailored to disease traits, is crucial for restoring retinal function. This precision is pivotal for secure gene therapy involving structural proteins like FAM161A.

neuroscience↗

bioRxiv2019

Resolving the dark matter of ABCA4 for 1,054 Stargardt disease probands through integrated genomics and transcriptomics

Missing heritability in human diseases represents a major challenge. Although whole-genome sequencing enables the analysis of coding and non-coding sequences, substantial costs and data storage requirements hamper its large-scale use to (re)sequence genes in genetically unsolved cases. The ABCA4 gene implicated in Stargardt disease (STGD1) has been studied extensively for 22 years, but thousands of cases remained unsolved. Therefore, single molecule molecular inversion probes were designed that enabled an automated and cost-effective sequence analysis of the complete 128-kb ABCA4 gene. Analysis of 1,054 unsolved STGD and STGD-like probands resulted in bi-allelic variations in 448 probands. Twenty-seven different causal deep-intronic variants were identified in 117 alleles. Based on in vitro splice assays, the 13 novel causal deep-intronic variants were found to result in pseudo-exon (PE) insertions (n=10) or exon elongations (n=3). Intriguingly, intron 13 variants c.1938-621G>A and c.1938-514G>A resulted in dual PE insertions consisting of the same upstream, but different downstream PEs. The intron 44 variant c.6148-84A>T resulted in two PE insertions that were accompanied by flanking exon deletions. Structural variant analysis revealed 11 distinct deletions, two of which contained small inverted segments. Uniparental isodisomy of chromosome 1 was identified in one proband. Integrated complete gene sequencing combined with transcript analysis, identified pathogenic deep-intronic and structural variants in 26% of bi-allelic cases not solved previously by sequencing of coding regions. This strategy serves as a model study that can be applied to other inherited diseases in which only one or a few genes are involved in the majority of cases.

genetics↗