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

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.

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.

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.

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.

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

Hypertrophic cardiomyopathy (HCM) is an inherited disease characterized by the thickening of the heart muscle. Mutations in genes encoding contractile proteins, such MYBPC3 and MYH7 account for over half of patients with familial HCM. MicroRNAs (miRNAs) are pivotal regulators of gene expression that may influence disease progression. Despite their potential significance, there is a scarcity of published data on miRNAs in HCM. Induced pluripotent stem cells (iPSCs) have emerged as valuable tools for modeling various cardiovascular diseases, including HCM. However, the miRNA expression profiles associated with HCM remain largely unexplored within iPSC models. Investigating the differential expression of miRNAs in HCM patients offers a promising avenue to identify potential therapeutic targets for individuals affected by this condition. In this study, we performed an unbiased screen using a microarray (NanoString Technologies) that contained 798 miRNAs to identify differentially expressed miRNAs in iPSC-cardiomyocytes (CM) and myocardial tissues derived from healthy donors and HCM patients with MYBPC3 and MYH7 mutations, identifying miR-9-5p as significantly downregulated in all HCM iPSC-CM and myectomy tissue compared to control. KEGG pathway analysis of gene targets using miRTarBase and g:Profiler revealed that miR-9-5p regulates genes in the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor resistance pathway, which is involved in hypercontractility associated with HCM. Modulation of miR-9-5p expression in vitro using mimics significantly reduced cardiomyocyte size and contractility of MYBPC3 mutant iPSC-CMs whereas healthy control iPSC-CMs treated with antagomiRs exhibited hypertrophy. Our findings identify miR-9-5p as a regulator of cardiomyocyte hypertrophy and contractility, and suggest the miR-9-5p/EGFR tyrosine kinase inhibitor resistance axis as a potential therapeutic target to mitigate hypertrophy and hypercontractility exhibited in HCM.

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 a fatal disorder caused by dystrophin mutations, leading to progressive muscle degeneration and cardiac failure. Although AAV-based DMD therapies with innovative designs to overcome the cargo limits of the virus are effective in animal models, these current systems may face clinical translation challenges related to efficiency, off-target effects, and immunogenicity. We developed a multi-vector RNA End Joining (REJ) system to split large genes into multiple co-delivered AAVs that engage the cell's intrinsic spliceosome to precisely reassemble RNA segments that encode very large scar-free proteins. We show the system is efficient and has negligible off-target interactions. In vivo, REJ vectors expressing the adenine base editor Abe8e, mini-dystrophin Dp253, or native full-length dystrophin Dp427 each prevented muscle degeneration. Machine-learning histopathology of more than 120,000 myofibers revealed robust reductions in nuclear infiltration, improved centronucleation, and normalized hypertrophy, supported by functional, transcriptomic, and behavioral analyses. These findings establish REJ as a clinically viable AAV-based strategy for DMD that enables expression of large therapeutic proteins while avoiding potentially antigenic bacterial protein- or DNA-recombinases.

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.

Transfer RNA (tRNA) is highly modified and difficult to profile using reverse-transcription-based sequencing. Here, we established a direct RNA sequencing (tRNA-DRS) workflow to simultaneously characterize tRNA abundance and modification signals in mouse liver. The workflow included small-RNA enrichment, deacylation, splint-adapter ligation, barcoding, reverse transcription, and nanopore sequencing on PromethION with modification-aware basecalling. From 1,678,666 raw reads, 1,421,279 (84.67%) reads were retained after 20-200 nt length filtering, and 17,094 uniquely mapped reads (MAPQ [≥] 10) were obtained. These reads covered 244 tRNA genes and represented all 23 tRNA isotypes. tRNA abundance was strongly skewed, with Asp, Gly, Ser, and Lys accounting for 56.6% of uniquely mapped reads. Read-body coverage indicated that most uniquely mapped reads spanned substantial portions of tRNA molecules (mean 71.1%, median 75.9%; 83.3% reads covering [≥]50% of the reference). Modification analysis identified 41,013 candidate sites, of which 711 passed stringent filtering (modification fraction [≥]50%, coverage [≥]5), spanning eight modification types and dominated by pseudouridine and m5C. Filtered sites showed position-specific clustering and isotype-dependent patterns. Together, these results support tRNA-DRS as a practical single-assay framework for simultaneous tRNA expression and epitranscriptomic profiling in mammalian tissue.

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.

Background & Aims: Adeno-associated virus (AAV) vectors are attractive delivery vehicles for therapeutic gene delivery, and a notable feature of most AAVs is their natural tropism for the liver, which leads to significant hepatic uptake following systemic administration. In previous work, we identified 266G as a conserved motif on a variable region on the capsid of many commonly used AAV variants that controls liver uptake in both mice and non-human primates. This single amino acid could be functionally leveraged to engineer AAVs to either de-target from or enhance tropism to the liver. Here, we explored whether these observations extended to the human context. Methods: Two human hepatocyte models were tested: Fah-/-/Rag2-/-/Il2rg-/- (FRG) mice with humanized livers and a bioengineered human microliver platform in vitro. A barcoded AAV capsid library including standard control serotypes were used to assess the role of the 266G motif on gene transfer and transgene expression in both liver systems. Results: In vivo, 266G containing AAVs indeed targeted human hepatocytes superiorly, with some noted dependency on the degree of human-hepatocyte replacement in the chimeric mouse model. Initial studies in the micropatterned primary human hepatocyte co-culture model however demonstrated enrichment of heparin-binding AAVs, and not 266G variants. Notably, incorporation of polyethylene glycol (PEG) into the system modified the AAV transduction potential of those capsids including the liver-targeting motif, recapitulating the hepatocyte transduction pattern observed in vivo. Importantly, when PEG was used, the two human models, both at the DNA and RNA level, did correlate significantly. Conclusions: Our results showed the potential of a combinatorial AAV library for model validation and revealed the human microliver platform-PEG as a reliable system for the development of AAV therapeutics.

Facioscapulohumeral muscular dystrophy (FSHD) is a rare neuromuscular disease caused by aberrant re-expression of the embryonic transcription factor DUX4 in skeletal muscle, which activates a toxic transcriptional program that drives progressive muscle wasting. No approved disease-modifying therapies currently exist. Prior work in mammalian and zebrafish models has shown that a truncated form of DUX4 retaining only its DNA-binding domain (DBD) lacks transactivation capacity and can suppress DUX4-FL-driven pathology; separately, dCas9/KRAB-based epigenetic repressors have demonstrated efficacy in silencing DUX4 expression, though CRISPR-based strategies face challenges from the repetitive nature of the D4Z4 locus, the immunogenicity associated with bacterial Cas proteins, and the payload limitations of gene delivery vehicles. Building on these findings, we corroborate that the DUX4 DBD, comprising both homeodomains, acts as a non-toxic competitive inhibitor of full-length DUX4 (DUX4-FL) at its genomic target sites, and extend this strategy by fusing the DBD to a human KRAB(ZNF10) domain, converting DUX4 from a transcriptional activator into a fully humanized epigenetic silencer of its own targets. Using a fluorescent DUX4-responsive reporter, we show that DBD alone produces dose-dependent repression of DUX4-FL transcriptional activity in HEK293T cells (200-fold at the highest inducible dose tested), while a constitutively expressed DBD-KRAB fusion produces significantly greater repression than DBD alone (949-fold versus 17-fold at a 25x molar ratio), with a similar trend observed in C2C12 myoblasts (47-fold versus 3.3-fold knockdown). To contextualize these findings and explore dosing considerations, we developed three complementary computational models - a transcription factor competitive binding model, a myotube diffusion model, and an ordinary differential equation (ODE) compartmental model - that illustrate how DBD concentration, intracellular diffusion, and population-level cell state transitions may relate to therapeutic efficacy. Together, these results corroborate and extend existing approaches into a single, fully humanized construct that may help circumvent the immunogenicity and delivery limitations of Cas-based systems.

Diabetes is a highly prevalent complication of cystic fibrosis (CF), affecting 50% of adults with CF and over 80% of those with exocrine pancreatic insufficiency (PI) by age 50 years. Development of cystic fibrosis-related diabetes (CFRD) is associated with increased morbidity and mortality mostly due to advancement of chronic obstructive lung disease. Highly effective modulator therapy (HEMT), using precision medications targeting the cystic fibrosis transmembrane conductance regulator (CFTR), improves CFTR function and CF lung disease, but its impact on diabetes pathogenesis remains uncertain. We sought to determine whether two types of HEMT, ivacaftor and elexacaftor/tezacaftor/ivacaftor (ETI), alter diabetes prevalence in two large cohorts of individuals with CF and exocrine PI. For comparison, a non-highly-effective modulator, lumacaftor/ivacaftor (LUM/IVA), was also assessed. Data were provided by the CFTR2 project, a multinational CF registry (for ivacaftor and LUM-IVA), and by the CF Genome Project (CFGP), a predominantly US-based CF cohort (for ETI). Among 32,753 individuals with CF (2,803 treated), ivacaftor was associated with reduced diabetes prevalence (age-adjusted OR=0.55). In contrast, lumacaftor/ivacaftor (not highly effective) was not associated with diabetes prevalence (n=32,749). Among 2,854 individuals with CF (2,458 treated), ETI was associated with reduced diabetes prevalence (age-adjusted OR=0.47). Overall, HEMT (ivacaftor and ETI) was associated with a 25-39% reduction in diabetes prevalence in CF, while a non-highly-effective modulator (lumacaftor/ivacaftor) showed no difference. Precision targeted amelioration of CFTR dysfunction can delay onset of diabetes in a high-risk CF population.

Very short RNAs play critical roles in modern biology, and are thought to have been crucial for genome replication during the origin of life. Next-generation sequencing is an essential tool for characterizing pools of small RNAs, but current library preparation methods suffer from strong size and sequence biases. Here we present tinyRNA-seq, an optimized library preparation method designed to minimize length- and sequence-dependent capture bias enabling the sequencing of RNA fragments as short as 2 nucleotides. We use degenerate adaptor regions to reduce ligation sequence bias and facilitate unique molecular identifier (UMI) installation. We benchmarked tinyRNA-seq against commercial kits using a model primordial RNA genome consisting of hundreds of defined oligonucleotides ranging from 2 to 12 nucleotides. tinyRNA-seq reproduced the input RNA distribution without the size and sequence bias of the commercial kits. tinyRNA-seq also enables the detection of de novo oligonucleotide generation, an important process for the origins of life. Applied to biologically derived small RNAs including miRNAs, piRNAs, and cityRNAs, tinyRNA-seq showed significantly lower capture bias and recovered a wider range of sequences than commercial kits. tinyRNA-seq may thus provide a more complete and quantitatively accurate representation of small RNAs from both biological and chemical sources. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/743385v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@95ee64org.highwire.dtl.DTLVardef@155fb06org.highwire.dtl.DTLVardef@1d3665forg.highwire.dtl.DTLVardef@1e61404_HPS_FORMAT_FIGEXP M_FIG C_FIG

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.

T7 RNA polymerase is widely used to produce RNA using a canonical T7 promoter; however, it will also bind to low-affinity sites to generate cryptic transcription and produce RNA byproducts, which reduce full-length mRNA purity and yield. When manufacturing therapeutic RNAs for clinical applications, RNA byproducts must be removed using costly downstream purification and can cause adverse immunogenicity. To predict T7 transcription rates and reduce cryptic transcription, we designed 11588 T7 promoters and measured their mRNA levels, spanning a 6300-fold range within in vitro transcription reactions. We developed the T7 Promoter Calculator, a sequence-to-function machine learning model that predicts the T7 transcription rate on arbitrary DNA sequence across a 500-fold range with high accuracy (R2 = 0.80), accounting for both core and flanking motif sequences. We combined the model with generative design to remove low-affinity T7 sites from a therapeutic T7 expression system, resulting in a 2-fold increase in full-length mRNA purity. The automated design of T7 expression systems to remove undesired RNA byproducts increases mRNA purity and lowers downstream separation costs, while reducing adverse immunogenicity.

In terms of cancer-related death, non-small cell lung cancer (NSCLC), the worlds leading cause, highlights the need for continued research into the genetic factors that influence tumor growth. Long non-coding RNAs (lncRNAs) are now well recognized as essential regulators of oncogenic signaling cascades; nevertheless, the specific role and molecular basis of the SOX2 overlapping transcript (SOX2OT) in NSCLC are not entirely understood. This study examined the functional importance of SOX2OT and its regulatory interactions with tumor-suppressive microRNAs in NSCLC cells. In A549 and Calu-3 cells, RNA interference-mediated SOX2OT silencing dramatically reduced cellular proliferation, migration, and invasiveness. Moreover, SOX2OT knockdown was associated with inhibition of epithelial-mesenchymal transition (EMT), alongside induction of cell cycle arrest and activation of apoptotic pathways. Integrated transcriptomic profiling and bioinformatic prediction analyses identified miR-143 as a putative downstream effector of SOX2OT activity. Consistently, depletion of SOX2OT resulted in marked elevation of miR-143 expression, which corresponded with downregulation of oncogenic mediators, including STAT3, EZH2, and CXCL13. As a result of SOX2OT suppression, both the transcript and the protein levels of PTEN were restored. Further functional characterization demonstrated that SOX2OT knockdown inhibits EMT progression by decreasing mesenchymal markers and EMT-related transcription factors (TFs) while concomitantly enhancing epithelial marker expression. Collectively, these findings suggest that SOX2OT contributes to NSCLC pathogenesis through regulation of a miR-143-centered signaling network that influences oncogenic signaling, cellular survival, and metastatic potential. Targeting the SOX2OT/miR-143 regulatory axis may therefore represent a promising therapeutic approach for NSCLC, while also underscoring the broader importance of lncRNA-mediated post-transcriptional regulation in lung cancer biology.