Molecular Therapy - Nucleic Acids

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

Gene therapy has been the subject of extensive research following the advent of gene-editing technologies. Genetic disorders with difficult-to-target tissues, such as cystic fibrosis (CF), still face many challenges in developing efficacious gene therapy. The potential universal approach of gene replacement involves inserting a functional CFTR gene after generating DNA double strand breaks using gene editors such as CRISPR/Cas9. However, this strategy has not achieved clinical significance, as CRISPR/Cas9-mediated integration of CFTR is limited primarily by the infrequent activity of the homology-directed repair (HDR) pathway. To circumvent this limitation and improve CFTR transgene integration and expression, we explored a method of adding a second integration site, which we termed the dual-locus-targeting method. Using a helper-dependent adenoviral vector (HDAd)-delivered CRISPR/Cas9 system in porcine epithelial cells, we found that sequential delivery of two vectors, one targeting the CFTR locus and the other the genomic safe harbour site GGTA1, enhanced the integration efficiency of lacZ and CFTR donor genes to 16.5% and 3.4%, respectively. These results demonstrated a potential strategy to improve the efficacy of CFTR replacement for the development of a universal and permanent gene therapy treatment for CF lung disease. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=76 SRC="FIGDIR/small/731381v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@1774590org.highwire.dtl.DTLVardef@1782915org.highwire.dtl.DTLVardef@1d13b12org.highwire.dtl.DTLVardef@17d3f93_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.

Messenger RNA (mRNA) is canonically composed of ribonucleotides, with sporadic incorporation of deoxyribonucleotides into natural RNA transcripts being traditionally regarded as a rare, deleterious error arising from transcriptional infidelity. Here, we challenge this paradigm by demonstrating controlled partial substitution of ribonucleotides with deoxyribonucleotides during in vitro transcription (IVT) generates intact, stable and fully translationally competent IVT-mRNA. Unexpectedly, chimeric DNA-RNA backbone modification exhibits markedly enhanced IVT-mRNA translation several fold across multiple cell types and in vivo via diverse dosing routes relative to their ribonucleotide-based counterparts. 25% substitution of cytidine triphosphate with deoxycytidine triphosphate achieved best-performing translational output, surpassing the current gold-standard N1-methylpseudouridine (m1{Psi})-modified IVT-mRNA in a B16-OVA tumor vaccination model. These findings identify nucleotide class composition as a previously unrecognized parameter governing IVT-mRNA function and establish hybrid ribonucleotide-deoxyribonucleotide backbone engineering as a versatile strategy to expand the chemical space for next-generation mRNA therapeutics.

Periodontal disease and COVID-19 are linked by convergent immunoinflammatory pathways, yet the molecular basis of their interaction remains poorly defined. Here, we present a comprehensive salivary microRNA profile from individuals with prior SARS-CoV-2 infection, sampled approximately 3-6 months after diagnosis and meeting criteria for long COVID, providing new insight into the post-viral oral microenvironment. Salivary miRNA sequencing revealed widespread repression in patients with PD, consistent with persistent immune dysregulation. Relative to COVID-19-negative/PD-negative controls, thirty-two miRNAs were differentially expressed in COVID-19-positive/PD-positive individuals, all significantly downregulated. A similar signature was observed in a post-vaccination cohort for the selected dysregulated miRNAs. Integrative pathway analyses identified these miRNAs as regulators of core inflammatory circuits, including Ras, MAPK, and NF{kappa}B signaling, converging on IL-1{beta}- and TNF-centered networks relevant to both PD and COVID-19. Mechanistically, restoration of three downregulated miRNAs, miR- miR-30e-3p 106-3p-3p, and miR-652-3p attenuated NF{kappa}B activation and cytokine release in TLR-stimulated human oral keratinocytes, while their functional suppression using inhibitors potentiates inflammation. These miRNAs were also predicted to target SARS-CoV-2 spike and nucleocapsid transcripts, an interaction validated by dual-luciferase reporter assays. Their overexpression further reduced spike and nucleocapsid expression in Beta- and Omicron-infected epithelial cells, as measured by flow cytometry and RT-qPCR confirming host miRNAs as potent endogenous SARS-CoV-2 restriction factor. Together, these findings identify salivary host miRNAs as mechanistic regulators of oral inflammatory tone and viral persistence, establishing a molecular link between periodontal inflammation and post-COVID oral pathology.

A challenge of "once-and-done" adeno associated virus (AAV)-based gene therapy is the inability to modulate the level of therapeutic protein expression post-administration. Herein, we demonstrate the utility of an adenosine deaminase acting on RNA (ADAR) - mediated gene switch to control AAV-delivered gene expression. Using a premature termination codon (PTC) in the human Factor IX (hFIX) transgene, we established an ON switch, where expression of hFIX is contingent on rescuing the PTC mutation via RNA editing. In vitro and in vivo studies demonstrated silencing of the hFIX transgene by the PTC mutation and induction of protein expression by administration of an ADAR-recruiting trigger RNA. Mice transduced with a hepatotropic AAV capsid encoding an ApoE-hAAT hFIX-PTC transgene expression cassette showed a dose-dependent response between the levels of LNP-delivered trigger RNA and the amount of plasma hFIX expression achieved. We observed predictable and reproducible levels of hFIX expression upon multiple rounds of RNA editing and demonstrated that this system can achieve clinically relevant levels of hFIX. This work suggests that ADAR-mediated RNA editing may be a valuable tool for tunable expression of therapeutic transgenes in applied gene therapies.

Circular RNAs (circRNAs) are ubiquitous in eukaryotes; dysregulated circRNA expression is linked to diseases, including lung cancer. In contrast to canonical circRNAs arising from exon-intron boundaries, noncanonical circRNAs originating within exonic, intronic, and intergenic regions have typically been dismissed as transcriptional noise or technical artifacts. To explore circRNA diversity and appreciate their functions, we developed an algorithm to identify both canonical and noncanonical circRNAs without relying on genome annotation, enabling the identification of circRNAs of all types and in newly sequenced or poorly annotated species. Results from lung cancer cells revealed that noncanonical circRNAs constituted over two-thirds of the circRNA population and were expressed more abundantly than canonical circRNAs, and genes with fewer and shorter exons were hotspots for noncanonical circRNA and circRNA isoform production. Further analyses showed that many noncanonical circRNAs were indeed endogenous circRNAs transcribed within cells rather than experimental artifacts, were potentially translated into proteins or peptides, and were conserved across species. Moreover, we validated 65 noncanonical circRNAs in NCI-H23 cells using multiple bioassays and demonstrated that both exonic and intergenic noncanonical circRNAs influenced cell viability. CircRNA profiles in tumor and tumor-adjacent tissues of lung cancer patients revealed tissue-specific expression and differentially expressed canonical and noncanonical circRNAs from cognate genes involved in cancer-related pathways, indicating their potential clinical relevance. This study confirmed the authenticity of noncanonical circRNAs and provided the first experimental evidence that noncanonical circRNAs influence cancer cell phenotypes. These findings broaden our understanding of circRNA biology, highlighting their widespread genomic distribution, diverse functions, and potential clinical relevance.

Pulmonary hypertension (BPD-PH) associated with bronchopulmonary dysplasia (BPD) in preterm infants associates with high morbidity and mortality within the first two years of life. In a previous unbiased study, we identified a panel miRNAs in tracheal aspirates (TA) that were differentially expressed in extremely low gestational age newborns (ELGANs) with BPD-PH compared to those with BPD but no PH. To explore the predictive potential of these miRNAs, we studied TA exosomes from 7 days old ELGANs and analysed a curated panel of 16 miRNAs through logistic regression and calculated the predictive AUROC to diagnose BPD-PH at 36 weeks PMA. AUROC of TA miRNAs was 0.76 with sensitivity and specificity of 53% and 93%, respectively. Adding sex and gestational age to the variables improved the AUROC to 0.78 with sensitivity and specificity of 61 and 87% respectively. Due to challenges of obtaining TA in non-invasively ventilated infants, we collected saliva samples from ELGANs at 7 days of age and compared the log expression of these 16 miRNAs in both biofluids and found significant correlation in their expression (pearson r=0.92, p<0.001). We calculated the predictive AUROC of the same miRNAs to diagnose BPD-PH at 36 weeks PMA. AUROC of these miRNAs in saliva was = 0.85 with sensitivity and specificity of 82% and 72%, respectively; addition of biological sex and gestational age improved AUROC to 0.86 with sensitivity and specificity of 79% and 76% respectively. Leave-one-sample-out sensitivity analysis demonstrated stable training performance with reduced performance in testing samples, supporting the need for validation in larger independent cohorts. In conclusion, early salivary miRNAs have great potential for risk stratification of ELGANs to develop BPD-PH, while also providing the opportunity to identify target molecules and mechanisms that modulate molecular function.

Prime editing enables the precise modification of genomes, thereby holding great potential for the treatment of genetic diseases. Despite substantial advancements in prime editing technology and the initiation of the first clinical trial for treating chronic granulomatous disease, further enhancement of editing efficiency across edit types is still urgently needed. Here, we developed a compact prime editor, PE2{Delta}R, by deleting the RNase H domain of the MMLV reverse transcriptase (MMLV-RT). We then conducted a saturated mutagenesis screen targeting two DNA interacting regions within the PE2{Delta}R-RT Fingers domain. By integrating three highly effective mutations (I61R, V101R, S67W) into PEmax lacking RNase H domain (termed PEmax{Delta}RM3), we achieved up to a 90% increase in editing efficiency across editing types compared to PEmax. Structural modelling using AlphaFold 3 suggests that these mutations enhance primer-template stabilization and guide the RNA/DNA hybrid into a catalytically favourable trajectory, providing a mechanistic explanation for the enhanced activity. Taken together, our study demonstrates proof-of-concept for the application of unbiased mutagenesis screen to identify novel mutations that enhance prime editor performance. Furthermore, we discovered that RT variants (I61R, V101R, S67W) synergize with PEmax and epegRNA to improve prime editing efficiency across edit types, with the strongest improvement observed in introducing small deletions.

Mucopolysaccharidosis III type C (MPS IIIC) is a rare neurological lysosomal storage disorder caused by genetic deficiency of the lysosomal membrane enzyme, heparan--glucosaminide N-acetyltransferase (HGSNAT). To assess the feasibility of therapeutic strategies based on cross-correction of neurons by HGSNAT secreted from transplanted cells overexpressing the enzyme, we generated induced cortical neurons (iCN) from induced pluripotent stem cells (iPSCs) derived from MPS IIIC patients. The neurons were treated with extracellular vesicles (EV) purified from the culture medium conditioned by human endothelial cells transduced with a lentiviral vector encoding EGFP-tagged HGSNAT (LV-HGSNAT-EGFP). The isolated EV showed supraphysiologic HGSNAT activity levels and efficiently delivered the enzyme to the lysosomes of MPS IIIC iCN reducing lysosomal size and restoring normal synaptic protein levels. EV-mediated delivery of HGSNAT to neurons was further confirmed by the analysis of MPS IIIC iCN either co-cultured with iPSC-derived MPS IIIC microglia (iMGL) transduced with LV-HGSNAT-EGFP or treated with the iMGL conditioned medium. MPS IIIC iCN co-cultured with iMGL overexpressing HGSNAT achieved a complete phenotypic rescue, including normalization of lysosomal size, and the levels of heparan sulfate, GM2-ganglioside, synaptic proteins and brain-derived neurotropic factor. Treatment of MPS IIIC iCNs with conditioned medium led to a partial defects correction. Our findings reveal the translational potential of EV-mediated enzyme delivery in MPS IIIC patients treated with LV-mediated haematopoietic progenitor stem cell gene therapy.

Microplastics are pervasive environmental pollutants increasingly implicated in adverse human health effects, with emerging evidence linking MPLs exposure to elevated cardiovascular risk, including atherosclerosis. However, their specific mechanisms of action remain unknown. Human aortic endothelial cells (HAECs), located in the innermost layer of blood vessels, play a crucial role in maintaining vascular homeostasis and the development of atherosclerosis. This study demonstrates that polystyrene microplastics (80 nm MPLs) can enter HAECs through multiple pathways, including macropinocytosis, clathrin-mediated endocytosis, and caveolin-mediated endocytosis, and co-localize with mitochondria and lysosomes. MPLs exposure resulted in coordinated transcriptional, epitranscriptomic, and metabolomic reprogramming in HAECs, characterized by disruption of mitochondrial genes and an inflammatory response with activation of TNF-a; and NF-kB signaling. Integrative analysis revealed remodeling of the epitranscriptomic profile, demonstrated by an increase in 1-methyladenosine (m1A) modification along with reciprocal regulation (TRMT61A upregulation and ALKBH3 suppression) of its transcriptomic machinery, alongside other enzymes associated with 3-methylcytidine (m3C), pseudouridine (Y), 5-methylcytidine (m5C), and 7-methylguanosine (m7G) pathways. By comparing transcriptomic data from MPLs-treated HAECs with those of human atherosclerotic plaques, several common dysregulated pathways were identified, particularly those related to vascular physiological regulation and cell signaling. Metabolomic profiling further revealed significant remodeling of lipid metabolic networks associated with oxidative stress and inflammatory signaling. In summary, this study reveals that HAECs can internalize MPLs, leading to multiple disturbances in the transcriptome, epigenome, and metabolic networks, suggesting that MPLs exposure may pose a potential hazard to human cardiovascular health.

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@2e4703org.highwire.dtl.DTLVardef@3834d3org.highwire.dtl.DTLVardef@1409c8org.highwire.dtl.DTLVardef@18e463b_HPS_FORMAT_FIGEXP M_FIG Created in BioRender. Schneider, N. (2026) https://BioRender.com/59i0tiy C_FIG

Cardiovascular disease (CVD) remains a leading cause of death worldwide. Dilated cardiomyopathy (DCM), a major cause of heart failure (HF), exhibits ventricular dilation, impaired systolic/diastolic function, arrythmias, and adverse cardiac remodeling. While genetic causes of DCM have been extensively studied, non-genetic and acquired forms of DCM-like HF are less well characterized, especially with respect to non-coding RNA regulation. Circular RNAs (circRNAs) are stable, covalently closed non-coding RNAs that regulate cellular function via sequestering miRNAs, RNA-binding proteins, or translation. Their role in canine HF that recapitulates features of non-genetic DCM remains largely unexplored. To address this, we developed K9HeartCircDB (https://www.k9heartcircdb.com/), a publicly accessible database that catalogs circRNAs expressed in canine left ventricular (LV) tissues under tachypacing-induced HF, a model of non-genetic DCM-like disease, and healthy control conditions. The online interface enables users to query and explore circRNAs based on exon composition, predicted miRNA binding sites, protein-coding potential, siRNA targets, and primer design for experimental validation. By providing an integrated and user-friendly platform for canine heart circRNA exploration, K9HeartCircDB offers a valuable resource to facilitate mechanistic and advance translational studies on non-genetic DCM-like disease.

Background: Endothelial progenitor cells (EPCs) contribute to endothelial repair and neovascularization, and EPC dysfunction is closely associated with oxidative stress-related vascular injury. Forkhead box O3a (FoxO3a) regulates cellular stress responses, whereas miR-34a has been implicated in endothelial dysfunction, senescence, and apoptosis. However, the relationship between FoxO3a and miR-34a-3p in oxidatively injured EPCs remains incompletely defined. Objective: This study investigated the role of FoxO3a in H2O2-induced EPC dysfunction and examined whether miR-34a-3p directly interacts with the FoxO3a 3' untranslated region (3'UTR). Methods: Human umbilical cord blood-derived EPCs were identified by DiI-ac-LDL uptake, FITC-UEA-1 binding, and the expression of EPC-related markers. Oxidative stress was induced by H2O2. Cell viability, apoptosis, and angiogenic capacity were evaluated using CCK-8 assay, Annexin V/7-AAD flow cytometry, and Matrigel tube formation assay, respectively. FoxO3a expression was modulated using adenoviral overexpression or knockdown vectors, and miR-34a was modulated using mimics or antagomir. FoxO3a and miR-34a expression levels were detected by Western blot and qPCR. A dual-luciferase reporter assay was used to verify the interaction between hsa-miR-34a-3p and the FoxO3a 3'UTR. Results: H2O2 reduced EPC viability, increased apoptosis, and impaired tube formation in a concentration-dependent manner. H2O2 increased FoxO3a protein abundance and miR-34a expression, whereas FoxO3a mRNA did not change markedly. FoxO3a overexpression aggravated, whereas FoxO3a knockdown partially alleviated, H2O2-induced EPC dysfunction. Similarly, miR-34a mimics further suppressed EPC viability and tube formation, while miR-34a antagomir exerted a protective effect. Dual-luciferase reporter analysis showed that hsa-miR-34a-3p significantly reduced the activity of the wild-type FoxO3a 3'UTR reporter, while mutation of the predicted binding site abolished this suppression. Conclusion: FoxO3a and miR-34a participate in oxidative stress-induced EPC dysfunction. The dual-luciferase data demonstrate that hsa-miR-34a-3p directly targets the FoxO3a 3'UTR, suggesting the presence of miR-34a-3p-mediated post-transcriptional feedback within the FoxO3a-related stress-response network in EPCs.

Background Idiopathic Pulmonary Fibrosis (IPF) is a fatal chronic lung disease with limited therapeutic options. While alveolar epithelial injury and fibroblast activation are well-studied, endothelial-mesenchymal transition (EndoMT) is emerging as a critical pathogenic mechanism. The regulatory role of exosomal miRNAs in pulmonary fibrosis remains unclear. This study investigates serum exosomal miRNAs, particularly let-7a-5p, in modulating EndoMT during the onset of pulmonary fibrosis. Methods Clinical cohorts of IPF patients and healthy controls were enrolled. Serum exosomal miRNAs were profiled, followed by differential expression and functional enrichment analyses. In vitro experiments involved human pulmonary artery endothelial cells (HPAECs) transfected with let-7a-5p mimic or inhibitor. Dual-luciferase reporter assays confirmed the binding between let-7a-5p and TGFBR1. HPAECs were co-cultured with lung epithelial cells to examine paracrine signaling. In vivo studies used a bleomycin-induced mouse model with let-7a-5p agomir administration. Assessments included histopathological staining, hydroxyproline content, Western blot, qPCR, micro-CT, and pulmonary function tests. Results Let-7a-5p was significantly downregulated in serum exosomes from IPF patients, correlating with clinical indicators. Mechanistically, let-7a-5p directly bound the TGFBR1 3'UTR to inhibit its expression. Inhibition of let-7a-5p upregulated -SMA, FN1, smad2/3 phosphorylation, and collagen I, while downregulating CD31 and VE-cadherin. Therapeutically, let-7a-5p mimic reversed bleomycin-induced EndoMT and suppressed epithelial-mesenchymal transition (EMT) via paracrine signaling. Mice administered agomir showed reduced fibrosis, improved lung function, and suppressed TGF-{beta}/Smad signaling. Conclusion Serum exosomal let-7a-5p suppresses pulmonary fibrosis by targeting TGFBR1 to inhibit EndoMT. Its downregulation in IPF patients correlates with disease progression, highlighting its biomarker potential.

CD8 T cells play a central role in immune responses to infection and cancer. However, the diversity of T cell receptor (TCR) specificities makes it challenging to study the mechanisms that regulate T cell activation, differentiation, and effector function. Beyond TCR transgenic mouse models, various complex genome-editing approaches have been employed to overcome this challenge. However, these strategies are often technically demanding, time-intensive, and difficult to adapt. Investigators who are interested in testing de novo TCRs under their chosen experimental conditions would benefit from a standardized and accessible method. Here, we describe a protocol that combines ribonucleoprotein (RNP)-based CRISPR-Cas9 editing with retroviral transduction to enable efficient genetic manipulation of murine CD8 T cells. We show that T cells engineered via this protocol can be generated at sufficient scale for downstream in vitro assays and in vivo adoptive transfer experiments. We expect this method will be useful for investigators who require a standardized and accessible way to study how TCR specificity impacts CD8 T cell responses.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that disrupt the reading frame and abolish expression of functional dystrophin protein. Antisense oligonucleotides (ASO) can restore production of partially functional dystrophins by inducing exon skipping to restore the reading frame of dystrophin transcripts. While exon skipping is an FDA approved therapeutic strategy, there are currently no approved therapies for patients amenable to exon 44 skipping (8% of DMD patients). Here, we carried out a discovery campaign to identify phosphorothioate (PS) ASOs that efficiently induce exon 44 skipping and to define key sequence and chemistry features associated with activity. A tiling and micro-tiling approach with 18mer fully PS and 2-O-methoxyethyl (2MOE) modified ASOs in patient-derived myotubes identified five exonic target regions that promote skipping. ASO activity was strongly correlated across skeletal muscle and iPSC-derived cardiomyocytes, indicating similar exon 44 splicing regulation across cell types. Optimization studies showed that for 2MOE PS ASOs, 16-20mers were generally most active, while longer ASOs often had reduced activity due in part to impaired productive uptake into cells. Swapping out 2MOE modifications at both terminal positions for locked nucleic acids (LNAs) rarely improved activity and could also reduce it. Finally, promising candidates were tested in a humanized mouse model with an exon 44 skippable deletion, where one ASO outperformed others, inducing dose-dependent exon 44 skipping and dystrophin restoration in mouse models. These findings define practical design rules for exon 44-targeted ASOs and provide a foundation for therapeutic development.

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.

Circular RNAs (circRNAs) are an emerging class of RNAs with biomarker potential, but their detection in liquid biopsies is challenging due to low abundance. We developed Ouro-seq, a novel long-read sequencing protocol optimized for full-length circRNA recovery. Applied to urine, cervico-vaginal self-samples from cervical cancer patients, and plasma from lung cancer patients and controls, Ouro-seq recovered 2-5 times more and substantially longer circRNA molecules than conventional methods. Plasma contained predominantly exonic circRNAs, while urine and cervico-vaginal samples were dominated by previously undercharacterized intergenic circRNAs. We also identified extensive alternative circularization and splicing events. Functional analysis revealed distinct specialization patterns: exonic circRNAs showed enhanced miRNA sponging potential, while circRNAs from unplaced genomic scaffolds demonstrated greater peptide-coding capacity. This study establishes Ouro-seq as a valuable tool for comprehensive circRNA characterization in low-yield clinical samples and advances circRNA biology understanding with potential biomarker discovery and disease monitoring applications. MotivationWhile circular RNAs (circRNAs) constitute a minor fraction of total RNA, they may play critical roles in cancer development. CircRNA concentrations are typically too low for detection by Oxford Nanopore Long-Read Sequencing (LRS), particularly in samples with limited RNA content, such as liquid biopsies. Consequently, LRS-based circRNA analysis from liquid biopsies remains unexplored. To overcome these technical limitations, we developed an optimized circRNA enrichment method utilizing short-amplicon suppression, enabling circRNA profiling from urine, plasma, and cervico-vaginal samples.

Achieving efficient and balanced transgene expression in both rods and cones remains a major challenge in retinal gene therapy. Current promoters either lack specificity or fail to provide sufficient cellular coverage and expression level. To address this limitation, we developed and evaluated two fusion promoters, Pikali and Nocchu, by combining PR1.7, a cone-specific promoter and GRK1, a promoter most active in rods. Here, we show that Pikali and Nocchu outperform their parental promoters, driving broader and more balanced GFP expression in rods and cones of human iPSC-derived retinal organoids. These constructs achieved transduction in 30% to 45% of photoreceptors, with higher expression levels than GRK1 and broader cellular coverage than PR1.7. Our findings establish Pikali and Nocchu as excellent candidates for retinal gene therapy, overcoming the limitations of existing promoters. By combining specificity, efficiency, and extensive photoreceptor targeting, these fusion constructs represent a novel and promising strategy for next-generation gene therapy vectors, addressing inherited retinal dystrophies and advancing clinical translation.