NAR Molecular Medicine

BackgroundLMNA-related congenital muscular dystrophy (L-CMD) is a rare, life-threatening genetic disorder caused by point mutations in the LMNA gene, for which no effective treatment currently exists. It is characterized by early-onset muscle weakness, dropped-head syndrome, hypotonia, cardiac complications, and restrictive lung disease, frequently leading to premature death. The LMNA c.745C>T (p.R249W) mutation is the most prevalent amongst L-CMD patients. Given its monogenic nature, L-CMD represents a compelling candidate for gene therapy approaches. ResultsIn this study, we investigated the therapeutic potential of adenine base editing (ABE) to correct the pathogenic LMNA c.745C>T (p.R249W) mutation in human myoblasts. We evaluated multiple ABE variants and single-guide RNAs (sgRNAs), identifying optimal combinations that achieved efficient and specific correction of the mutant allele. However, we found that editing can also introduce an adjacent bystander mutation, c.743T>C (p.L248P). To determine the functional consequences of base editing, we established clonal cell lines reverted to wild type or harboring the p.L248P variant. Whereas wild-type edited cells showed a clear correction for all the studied parameters that were abnormal in R249W myoblasts, we found that L248P cells show nuclear abnormalities resembling those of R249W mutant cells, and their cellular function is partially compromised. These results demonstrate that ABE can effectively target the LMNA c.745C>T mutation but also reveal the significant impact of bystander edits on cellular physiology. ConclusionsOur findings provide proof-of-concept for the application of base editing as a therapeutic strategy for L-CMD, while underscoring the necessity of precise editing technologies to ensure both efficacy and safety in future clinical translation.

Effective and safe strategies to restore gene expression in haploinsufficiency disorders or to augment protective protein signaling have long been attractive but challenging therapeutic goals. Here, we demonstrate the potential of antisense oligonucleotides (ASOs) to upregulate gene targets through mRNA stabilization. The 3 untranslated regions (UTRs) of mRNAs regulate stability via activating and repressive elements. One prominent destabilizing cis-element found in ~30% of transcripts is the Pumilio RNA-binding protein recognition element (PRE), which can be targeted to enable gene-specific protein upregulation. A scan for PREs highlights hundreds of disease-relevant transcripts that may be targetable via ASO-mediated gene-specific blocking of Pumilio binding. We show that masking specific PREs with ASOs produces sustained protein upregulation through mRNA stabilization using multiple reporter and endogenous gene targets. Using PRE-masking ASOs, we neutralized PRE-mediated destabilization of TANK-binding kinase-1 (TBK1) mRNA and restored TBK1 protein expression in participant-derived fibroblasts with TBK1 haploinsufficiency, causal for amyotrophic lateral sclerosis. Additionally, by targeting PRE sites in the myoprotective gene follistatin (FST), we achieved effective FST mRNA upregulation in tested mouse tissues. These findings suggest that ASO strategies that neutralize destabilizing 3UTR sequences like PREs may provide a powerful approach to upregulate haploinsufficiency-related genes or enhance protective protein levels for therapeutic benefit.

Duchenne muscular dystrophy is a lethal neuromuscular disorder caused by the absence of dystrophin, for which no curative treatment is available. RNA-based approaches have shown promising results; however, their evaluation is hindered by the lack of robust and rapid delivery methods for differentiated human muscle cells, which represent the most physiologically relevant in vitro models for assessing therapeutic strategies. Here, we establish a versatile lipid nanoparticle platform enabling efficient delivery of diverse RNA therapeutics across a range of human muscle models, including myotubes, induced pluripotent stem cell-derived myotubes, myoblasts, cardiomyocytes, and 3D engineered skeletal muscle tissues. Remarkably, a single commercially available lipid nanoparticle formulation supports delivery of cargos spanning more than 300-fold in size, from short antisense oligonucleotides ([~]20 nt) to complex CRISPR-based editors (up to [~]6.7 kb), including Cas9 nucleases, adenine base editors, and CRISPRa systems. This enables efficient gene correction and transcriptional modulation, resulting in dystrophin restoration or compensatory utrophin upregulation in relevant Duchenne muscular dystrophy models. Together, our results establish a single lipid nanoparticle formulation as a versatile platform for RNA delivery in human muscle systems and provide a practical framework for the rapid preclinical assessment of emerging therapies for Duchenne muscular dystrophy and other neuromuscular disorders. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=92 SRC="FIGDIR/small/741396v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@a18d71org.highwire.dtl.DTLVardef@4d0faforg.highwire.dtl.DTLVardef@140b078org.highwire.dtl.DTLVardef@1a235_HPS_FORMAT_FIGEXP M_FIG C_FIG Created in BioRender. Leclerc, D. (2026) https://BioRender.com/90h0fme

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a complex multisystem disorder. Its hallmark feature, post-exertional malaise (PEM), is a delayed and prolonged worsening of symptoms following even minor exertion. As PEM commonly includes muscle pain, weakness, exercise intolerance and impaired recovery, skeletal muscle dysfunction may contribute to its pathophysiology, but the underlying molecular processes remain poorly understood. We generated induced pluripotent stem cell-derived skeletal muscle myotubes from individuals with ME/CFS (n = 6) and healthy controls (n = 7) to investigate disease-associated transcriptomic alterations and identify potential therapeutic candidates. Myogenic progenitor cells from both groups differentiated into elongated, multinucleated myotubes expressing titin and myosin heavy chain, while qPCR profiling of myogenic differentiation markers further supported successful differentiation. Gene-level analysis identified three differentially expressed genes at an FDR-adjusted p < 0.05: ERVH48-1, CDX4 and SLC2A14. Isoform-switching analysis identified 16 genes with significant differential isoform usage, including CACNA1H, MIEF2, SMAD2 and CHD4. Gene set enrichment analysis identified broader pathway-level alterations involving RNA processing, protein synthesis, proteostasis, mitochondrial quality control, metabolism and muscle-related processes. Library of Integrated Network-based Cellular Signatures Phase 2 (LINCS2) analysis further prioritised 14 compounds associated with mitochondrial metabolism, glucose and lipid regulation, PPAR signalling, calcium and excitatory signalling, neuroimmune activity and cellular stress responses. Overall, ME/CFS iPSC-derived myotubes showed modest gene-level differences alongside broader isoform- and pathway-level alterations, supporting their use for investigating skeletal muscle biology and prioritising candidates for functional validation.

Myotonic dystrophy type 1 (DM1) is caused by a CTG expansion in the 3' untranslated region of the DMPK gene that results in the expression of expanded CUG RNA which trap splicing factors resulting in widespread alternative splicing dysregulation. A panel of these splicing events, or splicing index (SI), is currently used to measure splicing dysregulation in DM1 patients. Despite significant interest in splicing, little is known about its progression over time in DM1. The objective of this study was to evaluate the changes in SI over 3 years in a well-phenotyped cohort of DM1 participants and identify factors contributing to these changes. RNA was extracted from vastus lateralis (VL) biopsies of 20 DM1 participants taken 3 years apart and the SI score was measured using targeted RNA sequencing. Baseline SI scores correlated with percent predicted muscle strength of the ankle dorsiflexors, knee extensors and hand grip. On average, SI scores worsened over 3 years, but the progression varied markedly among participants. Adult/juvenile phenotypes and lower baseline SI scores were among the factors associated with higher progression of SI scores over time. These findings emphasize the importance of integrating clinical phenotype and baseline molecular severity when interpreting longitudinal SI change, particularly in the context of clinical trials.

Pitt-Hopkins syndrome (PTHS) is a severe neurodevelopmental disorder caused by haploinsufficient expression of transcription factor 4 (TCF4). Increasing expression from the remaining functional TCF4 allele could provide a mutation-independent strategy for patients with diverse pathogenic variants. We integrated genomic datasets with strand-specific RNA analysis to identify transcribed regions of the TCF4 promoter suitable for targeting by activating double-stranded RNAs (dsRNAs). Screening of overlapping promoter-targeted dsRNAs identified a hotspot for increased TCF4 expression. Activation was retained by a lead dsRNA containing extensive 2'-fluoro, 2'-O-methyl, and phosphorothioate modifications used in therapeutic oligonucleotides. Anti-AGO2 immunoprecipitation showed association with RNA transcribed from the targeted promoter region. dsRNA-mediated activation was associated with increased RNA polymerase II occupancy. These findings establish promoter-targeted dsRNAs as chemically tractable starting points for increasing TCF4 expression and support their investigation as a mutation-independent strategy for haploinsufficient disease.

Mitochondrial RNA (mtRNA) released into the cytosol functions as a damage associated molecular pattern that activates pattern-recognition receptor (PRR)-mediated inflammation, yet its release mechanisms and cytoplasmic fate remain poorly understood. Here we report that chemical Abt-373-treatment and Vesicular stomatitis virus (VSV) infection induce mtRNA extrusion through Bax/Bak and VDAC1 channels, accompanied by mtDNA release. Extruded mtRNA in A549 cells activates multiple cytosolic PRRs, including RIG-I, MDA5, TLR3/7/8, and PKR, each contributing differentially to the innate immune signaling. Analysis of GEO datasets and methylated RNA immunoprecipitation (MeRIP) assays further reveals that mtRNA carries methyladenosine (m6A) modification. m6A machinery proteins are involved in the cytoplasmic retention time of mtRNA and its interaction with RIG-I, thereby modulating mtRNA-induced innate immunity. Thus, our work establishes in vitro models of mtRNA extrusion, and highlights m6A-dependent modulation as a potential therapeutic target for mtRNA-driven inflammation.

Vascular endothelial growth factor (VEGF) is a central driver of pathological angiogenesis in cancer and retinal disease. A G-quadruplex (GQ) structure that forms in an Internal Ribosome Entry Site (IRES) of VEGF was proposed to recruit the 40S ribosome and initiate cap-independent translation under hypoxia. We demonstrate that blocking this GQ with dCas13d represses VEGF translation by up to 5-fold, without impacting mRNA abundance, under hypoxia. Similar levels of repression were attained in HeLa and human umbilical vein endothelial cells (HUVECs) using immunofluorescence and western blot assays, as well as a bicistronic dual-luciferase experiment. Single molecule Forster resonance energy transfer (smFRET) and fluorescence enhancement assays demonstrated destabilization of the GQ by CRISPR-dCas13d. We also tested the functional consequences of this regulation using tube formation assay in bovine aortic endothelial cells (BAOECs), which demonstrated consistent results with VEGF expression levels. Our study demonstrates the feasibility of using CRISPR-dCas13d as a sequence-specific and transient translation regulator for a large group of genes that contain translationally-active secondary structures.

The CRISPR-Cas12a system, primarily used for nuclear genome editing, operates through RNA-guided cleavage of target DNA. Given the high copy number of mitochondrial DNA (mtDNA) in cells and the pathogenic potential of mtDNA mutations, there is substantial interest in developing methods to selectively eliminate mutant mtDNA without affecting wild-type molecules. We suggest using AsCas12a, with its shorter CRISPR RNA (crRNA) and proven ability to be targeted into human mitochondria, to perform selective cleavage of mutant mtDNA. Here, we assess the potential of AsCas12a and various engineered crRNAs to discriminate between wild-type and mutant mtDNA in vitro and in living human cells. By exploring chemically synthesized full and split crRNAs with various modifications, we identified crRNAs capable of targeting specific mutant mtDNA. We demonstrated the strong influence of magnesium concentration on the ability of crRNAs to discriminate point mutations. Finally, we show that AsCas12a-fused cytosine base editor can be addressed to mitochondria and can direct low but significant specific base editing of mtDNA in human cells. Our study suggests AsCas12a as a promising tool for targeted mutant mtDNA elimination or editing.

Skeletal muscle dynamically adapts to physiological stimuli such as exercise through coordinated molecular and structural remodeling processes. Circulating microRNAs (miRNAs) represent promising non-invasive biomarkers of exercise responsiveness and skeletal muscle physiological states; however, most analytical frameworks rely solely on annotated miRNAs and overlook novel candidates. Here, we present nf-sarcopipe, a modular Nextflow pipeline that integrates de novo and reference-guided miRNA discovery with transcriptomic analysis and regulatory network reconstruction. The pipeline is organized into three complementary modules: 1) Preprocessing, 2) miRNA Discovery, and 3) Target Prediction & mRNA Integration. Using publicly available datasets from active and sedentary young women, the pipeline identified reproducible miRNA signatures and prioritized a small set of structurally supported, high-confidence de novo candidates. Previously reported exercise-associated miRNAs compiled from the literature were additionally incorporated for comparative candidate evaluation. Although the available datasets were derived from different tissues, confounding-aware analyses enabled the identification of coherent transcriptional signatures associated with exercise responsiveness. Integrative miRNA-mRNA analysis uncovered consistent regulatory interactions linking circulating miRNAs--both novel and known--to pathways involved in immune response, extracellular matrix remodeling, autophagy, and skeletal muscle adaptation. Together, these results establish nf-sarcopipe as a robust and scalable framework for complementary miRNA discovery and for investigating regulatory mechanisms associated with exercise-induced skeletal muscle adaptation.

Circular RNAs (circRNAs) are generated by backsplicing of eukaryotic protein-coding transcripts and can regulate microRNAs and RNA binding proteins, or serve as translation templates. Their covalently closed structure confers resistance to exonuclease-mediated degradation, extending their half-life and supporting their development as RNA therapeutics. However, existing overexpression methods often yield substantial contaminating linear RNAs, limiting their utility. Here, we systematically benchmarked plasmid-based circRNA overexpression strategies in human cells, comparing spliceosome- and ribozyme-based mechanisms across constructs incorporating widely used flanking sequences. The ribozyme-based Tornado system produced the highest circRNA yield but introduced extraneous "molecular scars" into the mature product. By contrast, spliceosome-mediated circularization using introns from the Drosophila Laccase2 gene, which contain imperfect complementary repeats, produced scarless circRNA with substantially lower linear RNA contamination. Linear RNA was further reduced by engineering the primary transcript to terminate in a non-polyadenylated end, increasing its susceptibility to exonucleases. Building on this optimized system, we developed a dual-output platform co-expressing a linear fluorescent reporter alongside a circRNA from a single promoter (CIRCUS, circRNA and upstream linear system), enabling efficient screening of circRNA-driven cellular phenotypes, including site-specific A-to-I editing of target mRNAs. Together, this toolkit provides high-purity circRNA production suitable for mechanistic studies and circRNA-based therapeutic development.

Sequence-specific cleavage of viral RNA by deoxyribozymes offers a direct approach for suppressing viral gene expression, although limited cellular accessibility remains a major barrier to their functional application. In this study, a panel of eight deoxyribozymes representing different catalytic motifs and target regions was designed against SARS-CoV-2 Spike RNA and comparatively evaluated through time-dependent in vitro cleavage assays. Substantial target-site-dependent differences were observed even among candidates sharing the same catalytic core, demonstrating that catalytic performance was strongly influenced by the selected RNA target region. Among the tested sequences, 8-17-769 exhibited the fastest and most reproducible cleavage profile and the highest observed rate constants, and was therefore selected as the catalytic module for subsequent chimera development. To evaluate its activity in a cellular context, 8-17-769 was combined with the nucleolin-binding aptamer AS1411 in two opposite linear orientations. In an A549-based Spike expression model, both AS1411-containing chimeras were associated with reduced S gene expression, with 8-17-769-AS1411 producing the strongest response, corresponding to an approximately tenfold reduction relative to the reference group and reaching statistical significance (p < 0.05). The functional difference between the two orientations was further examined structurally. Circular dichroism spectroscopy showed that 8-17-769-AS1411 more closely preserved the spectral characteristics of the individual components, while computational analyses indicated a dynamic organization more similar to the free deoxyribozyme-RNA complex and a nucleolin-compatible docking configuration. Collectively, these findings identify 8-17-769 as a lead Spike RNA-cleaving deoxyribozyme and demonstrate that the linear organization of aptamer and catalytic modules can substantially influence the structural and functional properties of aptamer-deoxyribozyme chimeras.

The ribosome, long regarded as a passive, uniform machine, has only recently been recognised as a direct regulator of translation. Mass spectrometry and sequencing approaches have shown that heterogeneity in ribosome composition exists, which can actively regulate the translational process. One source of this heterogeneity is the modification of ribosomal RNA (rRNA), primarily pseudouridylation (pseU) and 2'-O-methylation (2OMe), mediated by specific H/ACA and C/D box small nucleolar RNAs (snoRNAs). Here, we investigate how the stoichiometry of rRNA modifications varies during embryonic stem cell differentiation. Using the modification basecalling capability of Nanopore direct RNA sequencing, we have identified distinct stoichiometric changes in modification patterns between pluripotent and differentiated cells, revealing highly dynamic, site-specific regulation. Further, profiling of snoRNA expression during trilineage differentiation revealed differential expression of H/ACA and C/D box snoRNAs responsible for a subset of these dynamic modifications. By integrating rRNA and snoRNA sequencing approaches, we have built a comprehensive profile of rRNA modification dynamics during early embryonic cell fate decisions, highlighting potential regulatory mechanisms for ribosome heterogeneity during development. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/743918v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@48ca91org.highwire.dtl.DTLVardef@eb0418org.highwire.dtl.DTLVardef@159fc8corg.highwire.dtl.DTLVardef@d34f19_HPS_FORMAT_FIGEXP M_FIG C_FIG

Actinomycin D (Act D) is a global transcriptional inhibitor widely used in research and clinical practice; however, its effects on RNA-binding protein (RBP) dynamics remain poorly understood. Analysis of an RNA-seq dataset from Act D-treated HeLa cells revealed a compensatory stress response enriched in RNA metabolism, processing, and translation. Here, we investigated the effects of Act D on the subcellular localization of RBPs using HuR as a model mRNA stabilizing RBP. Short-term Act D treatment markedly increased cytoplasmic HuR localization in HCT116 and HeLa cells where the protein is known to be active. Analysis of known pathways regulating HuR nucleocytoplasmic translocation did not fully explain this redistribution, suggesting alternative mechanisms. To identify proteins proximal to HuR following Act D treatment, we performed TurboID labeling followed by LC-MS/MS in HCT116 cells. Several proteins involved in RNA regulation were identified. Probabilistic modeling highlighted FUS, an RBP with established roles in phase-separated granule dynamics, as a candidate proximal protein. The Act D-dependent interaction between HuR and FUS was interrogated using molecular dynamics simulations and validated with proximity ligation assays. Furthermore, increased cytoplasmic localization of RBPs following Act D treatment was accompanied by formation of granular structures that were relatively fluid and could be disrupted by hypotonic shock. Collectively, our findings demonstrate that Act D induces cytoplasmic redistribution of multiple RBPs and their sequestration into dynamic granular structures, revealing a previously unrecognized cellular response to transcriptional inhibition. Graphical AbstractAct D induced cytoplasmic re-localization of HuR along with FUS and other RBPs in dynamic, hypotonic shock-sensitive granular structures. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/745449v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@515322org.highwire.dtl.DTLVardef@128f0d5org.highwire.dtl.DTLVardef@db4302org.highwire.dtl.DTLVardef@10c4a2e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Duchenne muscular dystrophy (DMD) is an X-linked muscle wasting disorder marked by lack of dystrophin expression. Symptoms include loss of ambulation, respiratory problems, and cardiac complications with heart failure being the leading cause of death. Dystrophin transduces force from the actin cytoskeleton to the extracellular matrix to protect cells during muscle contraction. Restoration of dystrophin expression by gene transfer holds promise in addressing the root cause of disease. We compared the changes to transcriptional profiles after gene transfer by adeno-associated virus or lentivirus to examine whether viral treatment alone impacts cell homeostasis. We delivered GFP to cardiomyocytes differentiated from induced pluripotent stem cells (iPSCs) with DMD mutations. Global transcriptional profiling revealed a downregulation of metabolic genes after lentiviral transduction compared to untreated controls. In both AAV and lentivirus-treated DMD iPSC-cardiomyocytes, we observed an activation of the p53 DNA damage response in addition to a downregulation of cell cycle genes, suggesting stress-induced G2/M checkpoint arrest following viral delivery. These findings demonstrate that gene therapy mediated by viral vectors activates cell stress pathways. Interventions to mitigate these stress responses may be necessary for safe and effective gene transfer in diseased cells.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are two related neurodegenerative disorders that display overlapping features. The hexanucleotide repeat expansion GGGGCC (G4C2) in the C9ORF72 gene is the most common cause of ALS and FTD, which results in the accumulation of dipeptide-repeat protein aggregates. Regulation of protein synthesis at the level of the initiation factor eIF2 has been suggested as a transversal event contributing to neurodegeneration in ALS and FTD. eIF2 phosphorylation blocks protein synthesis to alleviate protein misfolding overload, but conversely it can reduce the expression of synaptic proteins resulting in neuronal dysfunction. Dibenzoylmethane (DBM) is a small molecule that reverses the translational attenuation mediated by eIF2 phosphorylation which has been shown to alleviate neurodegeneration in prion-infected mice and Tau transgenic animals. Here we investigated the efficacy of the oral administration of DBM in protecting a mouse model of C9ORF72 pathogenesis. Treatment of mice with 0.5% of DBM mixture in powdered food ad libitum was sufficient to prevent cognitive impairment in C9ORF72 mice. Unexpectedly, DBM treatment did not modify the content of poly(GA) and poly(GR) protein inclusion in the hippocampus and brain cortex. Proteomic profiling of brain tissue indicated that DBM administration corrected nearly 70% of the changes in gene expression triggered by expanded G4C2, where the main pathways modified by DBM were related to cytoskeleton organization, ALS, and metabolic processes. Most proteins corrected by DBM in our C9ORF72 model were also altered in the brain of human FTD/ALS patients. Overall, our results reinforce the idea that targeting protein synthesis with small molecules in patients carrying C9ORF72 mutations may result in improved cognitive capacity.

Background: Small nuclear RNAs (snRNAs) are RNA components of the major and minor spliceosomes that play a core role in splice-site recognition and control of the splicing process. Variants in genes that produce snRNAs are increasingly recognised as major contributors to rare disorders, including neurodevelopmental disorders (NDD) and retinal dystrophies (collectively termed RNUopathies, a subset of spliceosomopathies). Clinical interpretation of variants in snRNAs is, however, challenging and existing guidance to support clinical variant classification does not adequately capture the unique features of snRNAs that necessitate a bespoke approach. Methods: We quantified the elevated background mutation rate in snRNA genes using de novo variants from 12,007 trios and assessed mutation density in 76,215 genome sequenced individuals in gnomAD. We convened a panel of clinical, research, and industry scientists with wide-ranging expertise in clinical variant interpretation and classification and expert knowledge in snRNA genes to draft and refine a guidance document. Results: We detail important considerations for variant classification in snRNA genes. These include: the difficulties of variant identification which requires genome or targeted sequencing approaches, the large number of gene paralogs with high sequence identity that complicate read mapping and variant calling, and historical inaccuracies in snRNA gene annotation. Further we show a ~50-fold increase in de novo mutation rate in snRNA genes compared to intergenic sequence and discuss the implications of this for variant classification. We provide a set of specific recommendations for classifying variants in snRNA genes. Finally, we introduce RNUdb, an interactive web-based tool to support snRNA variant annotation and classification. Conclusions: We provide the first guidance for clinical variant classification in snRNA genes and anticipate that this will support routine screening and analysis of snRNA genes in clinical genetic testing.

Huntington disease (HD) is caused by a polyglutamine expanded huntingtin protein that exerts progressive cellular toxicity. However, the temporal sequence of pathogenic, particularly early and reversible versus late and irreversible events remain incompletely defined, despite their distinct therapeutic implications. To delineate this trajectory, we profiled the proteome of a huntingtin expressing cell model at early (72 h) and late (144 h) stages. Rather than a linear progression, pathogenicity unfolded in two discrete phases. At the early stage, cells exhibited a broad activation of RNA processing, splicing, and protein synthesis machinery, consistent with an adaptive response aimed at preserving gene expression fidelity under stress. By the late stage, this compensatory program had collapsed, giving rise to a dominant failure in mitochondrial energy metabolism. Notably, 85% of proteins altered at both time points reversed direction of change between stages, indicating that mutant huntingtin reprograms cellular function wholesale rather than amplifying a fixed set of perturbations. Detailed analysis of mitochondrial respiratory complexes revealed that terminal ATP generating components (cytochrome c oxidase and ATP synthase) were severely affected, whereas upstream electron transport elements were retained or upregulated. Leveraging this proteomic map, we applied an AI assisted, direction aware drug repurposing strategy. Of 1,712 differentially expressed proteins, 498 were druggable, and 89 mapped to approved agents with mechanisms concordant with the required correction. These included Complex I targeted agents (metformin, ME 344) and mitochondria directed therapeutics (SS 31, MitoQ), several of which have previously been evaluated in HD. Collectively, these findings define a biphasic course of huntingtin toxicity and highlight an early therapeutic window in which intervention is most likely to be applied, prior to irreversible deterioration of mitochondrial respiratory function.

Emerging and re-emerging zoonotic RNA viruses, including filoviruses, henipaviruses and Rift Valley Fever virus (RVFV), pose a significant threat to human health. Due to their epidemic potential and high pathogenicity, these viruses are categorized as WHO priority pathogens, yet we still lack effective medical countermeasures (MCMs) against most of these dangerous pathogens, supporting that host-directed antivirals will be advantageous for broadly acting MCMs. Here we assessed the antiviral efficacy of SLR14, an immunomodulatory stem-loop RNA that activates RIG-I, for its activity against a panel of highly pathogenic viruses. SLR14 treatment provided broad-spectrum protection, markedly reducing infection with Ebola virus (EBOV), Marburg virus, and Sudan virus in human primary monocyte-derived macrophages, and Nipah virus replication in iPSC-derived endothelial cells, whether administered prophylactically or therapeutically. In vivo, a single dose of SLR14 protected mice against lethal RVFV infection when administered prophylactically, and, notably, retained significant efficacy when administered up to two days post-infection. Therapeutic SLR14 administration also improved survival rates of EBOV-infected mice. Although EBOV infection and SLR14 treatment activated overlapping innate immune pathways in human macrophages, viral infection induced a nonprotective response, whereas SLR14 elicited a well-regulated, markedly stronger and protective interferon-stimulated and inflammatory gene signature while avoiding prolonged IFN gene upregulation. Our data identify SLR14 as a potent host-directed therapeutic with broad antiviral efficacy against some of the most lethal viral pathogens.

As we have entered the endemic phase of COVID-19, there is a need to assess the long-term effects of SARS-CoV-2 infection on the trajectory of neurodegenerative disorders. Herein, we investigate whether the SARS-CoV-2 RNA-binding nucleocapsid protein (NCP; N-protein) may influence molecular processes relevant to motor neuron degeneration in amyotrophic lateral sclerosis (ALS). We detected N-protein immunoreactivity in ALS spinal cord motor neurons, including cytoplasmic N-protein-positive puncta that colocalized with cytoplasmic TAR DNA-binding protein 43 (TDP-43). In vitro, optogenetically induced N-protein biomolecular condensates (BMCs) showed reversible, liquid-like behavior and recovered after photobleaching. Using this model, we found that these condensates colocalized with TDP-43 and the RNA-binding proteins G3BP1/2, PABPC1 and TIA1. Condensate formation was associated with TDP-43 cytoplasmic mislocalization, altered TARDBP expression, and altered exon 3 splicing of POLDIP3. The N-terminal portion of N-protein influenced BMC formation, whereas truncated C-terminal proteoforms did not form detectable BMCs or show detectable TDP-43 colocalization in this assay. Extending these observations to a human cell model, we show that optogenetically induced N-protein BMCs colocalize with TDP-43 in human iPSC-derived motor neurons. Collectively, these findings support further investigation into potential molecular links between SARS-CoV-2 exposure and the trajectory of neurodegenerative disease.