Biology subjects

Sharon, D.

Publications and source records attributed to Sharon, D..

9 recordsLinked to original sources

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

RetiGene, a comprehensive gene atlas for inherited retinal diseases (IRDs)

Inherited retinal diseases (IRDs) are rare disorders, typically presenting as Mendelian traits, that result in stationary or progressive visual impairment. They are characterized by extensive genetic heterogeneity, possibly the highest among all human genetic diseases, as well as diverse inheritance patterns. Despite advances in gene discovery, limited understanding of gene function and challenges in accurately interpreting variants continue to hinder both molecular diagnosis and genetic research in IRDs. One key problem is the absence of a comprehensive and widely accepted catalogue of disease genes, which would ensure consistent genetic testing and reliable molecular diagnoses. With the rapid pace of IRD gene discovery, gene catalogues require frequent validation and updates to remain clinically and scientifically useful. To address these gaps, we developed RetiGene, an expert-curated gene atlas that integrates variant data, bulk and single-cell RNA sequencing, and functional annotations. Through the integration of diverse data sources, RetiGene supports candidate gene prioritization, functional studies, and therapeutic development in IRDs.

genetics↗

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↗

bioRxiv2023

Mouse mammary tumor virus is implicated in severity of colitis and dysbiosis in the IL-10-/- mouse model of inflammatory bowel disease

BackgroundFollowing viral infection, genetically manipulated mice lacking immunoregulatory function may develop colitis and dysbiosis in a strain specific fashion that serves as a model for inflammatory bowel disease (IBD). We found that one such model of spontaneous colitis, the interleukin (IL)-10 knockout (IL-I0-/-) model derived from the SvEv mouse, had evidence of increased mouse mammary tumor virus (MMTV) viral RNA expression compared to the SvEv wildtype. MMTV is endemic in several mouse strains as an endogenously encoded betaretrovirus that is passaged as an exogenous agent in breast milk. As MMTV requires a viral superantigen to replicate in the gut associated lymphoid tissue prior to the development of systemic infection, we evaluated whether MMTV may contribute to the development of colitis in the IL-10-/- model. ResultsViral preparations extracted from IL-10-/- weanling stomachs revealed augmented MMTV load compared to the SvEv wildtype. Illumina sequencing of the viral genome revealed that the two largest contigs shared 96.4% - 97.3% identity with the mtv-1 endogenous loci and the MMTV(HeJ) exogenous virus from the C3H mouse. The MMTV sag gene cloned from IL-10-/- spleen encoded the MTV-9 superantigen that preferentially activates T cell receptor V{beta}-12 subsets, which were expanded in the IL-10-/- versus the SvEv colon. Evidence of MMTV cellular immune responses to MMTV Gag peptides was observed in the IL-10-/- splenocytes with amplified interferon-{gamma} production versus the SvEv wildtype. To address the hypothesis that MMTV may contribute to colitis, we used HIV reverse transcriptase inhibitors, tenofovir and emtricitabine, and the HIV protease inhibitor, lopinavir boosted with ritonavir, for 12 weeks treatment versus placebo. The combination anti-retroviral therapy with known activity against MMTV was associated with reduced colonic MMTV RNA and improved histological score in IL10-/- mice, as well as diminished secretion of pro-inflammatory cytokines and modulation of the microbiome associated with colitis. ConclusionsThis study suggests that immunogenetically manipulated mice with deletion of IL-10 may have reduced capacity to contain MMTV infection in a mouse-strain specific manner, and the antiviral inflammatory responses may contribute to the complexity of IBD with the development of colitis and dysbiosis.

microbiology↗

bioRxiv2022

Huntingtin S421 phosphorylation increases kinesin and dynein engagement on early endosomes and lysosomes

Huntingtin (HTT) is a scaffolding protein that recruits motor proteins to vesicular cargoes, enabling it to regulate kinesin-1, dynein, and myosin-VI-dependent transport. To maintain the native stoichiometry of huntingtin with its interacting partners, we used CRISPR/Cas9 to induce a phosphomimetic mutation of the endogenous HTT at S421 (HTT-S421D). Using single particle tracking, optical tweezers, and immunofluorescence, we examined the effects of this mutation on the motility of early endosomes and lysosomes. In HTT-S421D cells, lysosomes exhibit longer displacements and higher processive fractions compared to wild-type (HTT-WT) cells. Kinesins and dyneins exert greater forces on early endosomes and lysosomes in cells expressing HTT-S421D. Additionally, endosomes bind to microtubules faster and are more resistant to detachment under load. The recruitment of kinesins and dyneins to microtubules is enhanced in HTT-S421D cells. In contrast, overexpression of HTT had variable effects on the processivity, displacement, and directional bias of both early endosomes and lysosomes. These data indicate that phosphorylation of the endogenous huntingtin causes early endosomes and lysosomes to move longer distances and more processively by recruiting and activating both kinesin-1 and dynein. Statement of SignificanceThe ubiquitous scaffolding protein huntingtin regulates the recruitment and activity of microtubule motors. Huntingtin phosphorylation at S421 enhances the microtubule binding and force generation of kinesin and dynein on early endosomes and lysosomes. Using optical tweezers to measure the forces exerted on endosomes in CRISPR-engineered cells, we find that a phosphomimetic huntingtin mutation (S421D) enhances both kinesin- and dynein-driven forces on early endosomes and lysosomes. The ability to modulate motor activity on a range of organelles makes huntingtin unique and suggests a significant role for huntingtin in regulating intracellular transport.

biophysics↗

bioRxiv2021

A mean-field approach for modeling the propagation of perturbations in biochemical reaction networks

Often, the time evolution of a biochemical reaction network is crucial for determining the effects of combining multiple pharmaceuticals. Here we illustrate a mathematical framework for modeling the dominant temporal behaviour of a complicated molecular pathway or biochemical reaction network in response to an arbitrary perturbation, such as resulting from the administration of a therapeutic agent. The method enables the determination of the temporal evolution of a target protein as the perturbation propagates through its regulatory network. The mathematical approach is particularly useful when the experimental data that is available for characterizing or parameterizing the regulatory network is limited or incomplete. To illustrate the method, we consider the examples of the regulatory networks for the target proteins c-Myc and Chop, which play an important role in venetoclax resistance in acute myeloid leukemia. First we show how the networks that regulate each target protein can be reduced to a mean-field model by identifying the distinct effects that groups of proteins in the regulatory network have on the target protein. Then we show how limited proteinlevel data can be used to further simplify the mean-field model to pinpoint the dominant effects of the network perturbation on the target protein. This enables a further reduction in the number of parameters in the model. The result is an ordinary differential equation model that captures the temporal evolution of the expression of a target protein when one or more proteins in its regulatory network have been perturbed. Finally, we show how the dominant effects predicted by the mathematical model agree with RNA sequencing data for the regulatory proteins comprising the molecular network, despite the model not having a priori knowledge of this data. Thus, while the approach gives a simplified model for the expression of the target protein, it allows for the interpretation of the effects of the perturbation on the regulatory network itself. This method can be easily extended to sets of target proteins to model components of a larger systems biology model, and provides an approach for partially integrating RNA sequencing data and protein expression data. Moreover, it is a general approach that can be used to study drug effects on specific protein(s) in any disease or condition.

pharmacology and toxicology↗

bioRxiv2019

Structural models of full-length JAK2 kinase

The protein JAK2 is a prototypical member of the Janus kinase family, and mediates signals from numerous cytokine receptors. The constitutively active V617F mutant of JAK2 is prevalent in many bone marrow disorders, blood cancers, and autoimmune diseases, and is an important drug target. Structures have been determined for each of the four individual domains making up JAK2, and for certain pairs of these domains, but no structure of full-length JAK2 is available, and thus the mechanisms underlying JAK2 regulation and the aberrant activity of the V617F mutant have been incompletely understood. Here we propose structural models of full-length JAK2 in both its active and inactive forms. Construction of these models was informed by long-timescale molecular dynamics simulations. Subsequent mutagenesis experiments showed that mutations at the putative interdomain interfaces modulated JAK2 activity. The models provide a structural basis for understanding JAK2 autoinhibition and activation, and suggest that the constitutive activity of the V617F mutant may arise from a dual effect of destabilizing the inactive conformation and stabilizing the active conformation.

biochemistry↗