Search Results (2,074)

Ocular diseases remain a major global health challenge with substantial unmet therapeutic needs. Small nucleic acid therapeutics offer a precise strategy to regulate disease-related mRNAs or noncoding RNAs through base pairing, thereby modulating protein expression at the RNA level. Major modalities include antisense oligonucleotides, small interfering RNAs, microRNA-based therapeutics, small activating RNAs, and nucleic acid aptamers, which act through RNA degradation, RNA interference, splicing modulation, microRNA regulation, transcriptional activation, or structure-dependent target binding. These properties make them attractive for ocular diseases involving genetic defects, pathological angiogenesis, inflammation, fibrosis, or neurodegeneration. However, their clinical translation in ophthalmology remains limited by poor molecular stability, insufficient tissue retention, immune activation, off-target effects, and inefficient delivery to target ocular tissues. Rational oligonucleotide design, appropriate local administration routes, and optimized delivery platforms are therefore essential for improving stability, tissue penetration, cellular uptake, and therapeutic durability. This review summarizes the major classes, mechanisms, chemical modification strategies, ocular delivery systems, and therapeutic applications of small nucleic acid drugs in ophthalmology. We also discuss lessons from clinical successes and failures and propose future directions for safe, durable, and individualized ocular therapy. Full article

Periodontal bone regeneration depends on the coordinated interplay between osteogenesis and angiogenesis; however, the molecular regulation underlying osteogenic–angiogenic coupling remains incompletely understood. Despite previous evidence that miR-508-5p downregulation enhances the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in vitro, its role in regulating angiogenesis and bone formation in vivo remains unclear. In the present study, miR-508-5p was modulated using synthetic inhibitor and mimic oligonucleotides, and its effects on osteogenesis, angiogenesis, and osteogenic–angiogenic coupling were investigated by the use of alkaline phosphatase and alizarin red S staining, conditioned-medium assays, a chicken chorioallantoic membrane assay, and a nude mouse ectopic bone formation model. The responses to miR-508-5p inhibition differed by cell type: osteogenic differentiation increased in hPDLSCs, while human umbilical vein endothelial cells (HUVECs) showed greater proliferative, migratory, and tube-forming activity. Conditioned medium experiments further revealed that miR-508-5p inhibition enhanced the reciprocal paracrine effects between hPDLSCs and HUVECs, thereby promoting both osteogenic and angiogenic responses. In vivo, miR-508-5p inhibition enhanced bone-like tissue formation and collagen deposition, accompanied by the elevated expression of collagen type I alpha 1 chain, bone morphogenetic protein 2, and the angiogenic marker CD31. Taken together, the findings support a potential regulatory role for miR-508-5p in osteogenic–angiogenic coupling and indicate that this microRNA may warrant consideration as a candidate target in therapeutic strategies for periodontal bone regeneration. Full article

The 4T1 cell line is a widely used model for triple-negative breast cancer (TNBC) research due to its high metastatic potential and resemblance to clinical progression. However, its biological characteristics often limit the efficiency and reproducibility of conventional nucleic acid delivery protocols. Here, we present an optimized non-viral delivery framework for efficient cell-associated uptake in 4T1 cells. Five carrier systems—Xfect RNA polymer (Xfect), polyethyleneimine (PEI), chitosan (CHI), and gold nanoparticle conjugates (AuPEI and AuCHI)—were systematically evaluated using both standard (adherent) and reverse (suspension) delivery approaches. A fluorescently labeled oligonucleotide (5′-TYE563) served as a reporter cargo at final concentrations of 5 nM and 10 nM, comparing complexation times of 15 s and 15 min. The proposed protocol introduces a rapid complexation strategy combined with reverse delivery, significantly reducing preparation time while enhancing cell-associated uptake. Under optimized conditions (10 nM, 15 s, reverse), Xfect achieved near-complete cellular uptake with minimal cytotoxicity. Crucially, the colocalization of dual-fluorescence signals enabled the simultaneous validation of metabolic viability and internalized cargo within a single field of view, bypassing the need for independent bulk cytotoxicity assays. This methodology provides a rapid, reproducible, and cost-effective framework to standardize early-stage delivery conditions for difficult-to-transfect cell lines such as 4T1. Full article

Frontotemporal Dementia and Parkinsonism linked to chromosome 17 (FTDP-17) is a rare, early-onset, autosomal-dominant neurodegenerative tauopathy caused by mutations in the Microtubule-Associated Protein Tau (MAPT) gene. A subset of these mutations selectively disrupts the normal alternative splicing of MAPT exon 10, altering the physiological ratio of 3-Repeat (3R) and 4-Repeat (4R) tau isoforms; this imbalance, driving pathological tau aggregation and progressive neurodegeneration, is a pathogenic hallmark of FTDP-17. This review explores the molecular mechanisms regulating exon 10 splicing, focusing on how exonic and intronic splicing mutations destabilise mRNA structures or alter splicing factor binding, and discusses how these changes can cause disease symptoms in experimental models. It describes different research methods for studying splicing issues, including minigene reporters, cell lines, human induced pluripotent stem cell (iPSC)-derived neurons, brain organoids and mouse models that naturally express only 4R tau. It also covers potential therapeutic approaches applicable to restoring the normal 3R/4R tau ratio. These include Antisense Oligonucleotides (ASOs), RNA interference (siRNA), Small-Molecule Splicing modulators (SMCs), Spliceosome-Mediated RNA trans-splicing (SMaRT), CRISPR-based transcript engineering, genome editing and RNA stem-loop binders. Additionally, the review highlights recent Phase 2 trial results, including the Diranersen (BIIB080) tau-lowering ASO, the development of MAPT-targeted RNA therapies, new tau-PET and plasma p-tau biomarkers, and innovative strategies to deliver CNS treatments, including non-invasive approaches. By integrating advances in RNA biology, disease modelling and targeted therapies, the review outlines potential future strategies for treating FTDP-17 and other tau-related disorders. Full article

A hexanucleotide repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Bidirectional transcription of the repeat expansion generates sense and antisense RNAs that are translated into dipeptide repeat (DPR) proteins, but the mechanisms of translation initiation remain incompletely understood. Here, we used CRISPR-Cas9 genome editing and steric-blocking antisense oligonucleotides (ASOs) to investigate the role of AUG codons within the antisense repeat RNA. Deletion of an AUG-containing region upstream of the antisense repeats markedly reduced poly(GP) production without affecting antisense RNA levels, demonstrating that this sequence is required for efficient poly(GP) synthesis. We further found that unspliced sense transcripts containing the repeat expansion likely serve as templates for poly(GA) and poly(GR) production in motor neurons. Finally, ASOs targeting the antisense AUG-containing region reduced poly(PR) and poly(GP) levels without altering repeat RNA abundance, supporting a role for AUG-dependent translation of the antisense repeat RNA. These findings provide new insights into the mechanisms of DPR production and suggest that translation-blocking ASOs may represent a therapeutic strategy for C9ORF72-associated ALS/FTD. Full article

Temporal lobe epilepsy (TLE) is increasingly conceptualized not as an isolated hippocampal lesion, but as a complex, multiscale limbic network connectomic disorder. Despite advances in pharmacological management, over 30% of patients experience drug-resistant epilepsy, underscoring the urgent need to shift from symptomatic seizure control to mechanism-directed disease modification. This review comprehensively synthesizes the pathophysiological architecture of epileptogenesis in TLE, spanning mitochondrial bioenergetic alterations, chronic neuroinflammation, synaptic reorganization, and ionic plasticity resulting from ion-channel dysregulation. We evaluate diagnostic advancements, highlighting how invasive stereo-EEG disambiguates pathological high-frequency oscillations from physiological ripples, how structural HARNESS-MRI maps anatomical substrates, and how AI-driven algorithms analyze ultra-long-term EEG streams for continuous seizure forecasting. Additionally, peripheral biofluid proteins and microRNAs provide noninvasive windows into active neuroinflammation and network remodeling, serving as valuable tools for longitudinal disease monitoring rather than primary screening. Therapeutically, the field is evolving beyond empirical antiseizure medications toward mechanism-based rational drug design and precision interventions. Dual-mechanism agents enhance seizure freedom, while antisense oligonucleotides, microRNA antagomirs, and cation-chloride cotransporter modulators target underlying genetic and biophysical drivers. Minimally invasive ablation, AI-guided closed-loop neuromodulation, targeted anti-inflammatory biologics, and patient-derived 3D cerebral organoid platforms further expand the translational frontier. Ultimately, bridging these experimental modalities through prospective clinical validation may provide a viable path toward interrupting epileptogenesis and realizing true disease modification in human TLE. Full article

Artificial intelligence is reshaping RNA research by enabling predictive, data-driven workflows across molecular design, therapeutic development, delivery optimization, and analytical quality control. RNA-based medicines, including messenger RNA vaccines, small interfering RNA therapeutics, antisense oligonucleotides, RNA-guided systems, and RNA-targeted small molecules, are programmable but structurally and analytically complex. Their performance depends not only on nucleotide sequence, but also on RNA folding, untranslated regions, chemical modifications, formulation composition, delivery efficiency, stability, manufacturability, and critical quality attributes. This review examines how AI is applied across the RNA therapeutic development pipeline, including RNA sequence and structure prediction, codon optimization, guide RNA design, neoantigen selection, RNA-targeted drug discovery, lipid nanoparticle formulation prediction, and analytical data integration. Particular emphasis is placed on AI-enabled analytics as a translational layer between computational design and therapeutic implementation. Centralized multimodal data platforms that integrate outputs from orthogonal analytical methods can transform fragmented experimental readouts into standardized, model-ready datasets. We argue that future progress will depend on closed-loop systems in which design models, delivery prediction, experimental analytics, and quality-control data continuously inform each other to improve RNA therapeutic translation. Full article

Glioblastoma multiforme (GBM) is an extremely aggressive and lethal brain tumor, characterized by marked molecular heterogeneity, the persistence of glioma stem cells (GSCs), and the limited permeability of the blood–brain barrier (BBB), which collectively hinder therapeutic efficacy. To address these barriers, nucleic acid aptamers, short single-stranded oligonucleotides with high affinity and specificity for molecular targets, have emerged as a promising therapeutic platform. Early unmodified aptamers, such as AS1411 and U2, demonstrated target engagement but showed limited performance due to instability and rapid systemic clearance. Chemical modifications, including 2′-fluoro substitutions and PEGylation, resulted in improved stability, specificity, and pharmacokinetic properties, enabling the development of innovative aptamer drug conjugates (ApDCs) for targeted delivery to GBM cells. In parallel, multivalent aptamer architectures, such as bispecific aptamer targeting entities (BATEs) and aptamer guided nanostructures, have been designed to enhance binding avidity, address tumor heterogeneity, and facilitate BBB transcytosis. More recently, computational strategies ranging from machine learning-guided sequence optimization to structure prediction and generative AI have accelerated the rational design of aptamers tailored to GBM specific challenges. This review examines these advances, the remaining pharmacological limitations, and the potential of computational tools to reshape the future of aptamer based GBM therapeutics. Full article

The long non-coding RNA XIST is a key regulator of X-chromosome inactivation (XCI) and contributes to epigenetic regulation during cell reprogramming. However, whether XIST suppression influences endogenous pluripotency-associated transcriptional programs in porcine somatic cells remains unclear. In this study, we investigated the effects of XIST knockdown on endogenous pluripotency-associated transcription factors (pTFs) in IBRS-2 cells. Chemically modified antisense oligonucleotides (ASOs) targeting XIST were designed and transfected into IBRS-2 cells. XIST expression was efficiently suppressed for up to 120 h after ASO treatment. RT-qPCR analysis showed that XIST knockdown induced sustained upregulation of KLF4 and LIF expression, prolonged intermittent activation of NANOG, and transient activation of G6PD. In contrast, OCT4, SOX2, and c-MYC were significantly upregulated only during the early stage after transfection. KLF4 immunofluorescence was detectable in a subset of ASO-2-treated cells. These findings demonstrate that ASO-mediated XIST knockdown partially facilitates the activation of a subset of endogenous pluripotency-associated genes in porcine female somatic cells. This study provides new insights into the role of XIST as an epigenetic regulator during porcine somatic reprogramming and suggests that optimized XIST interference may represent a potential strategy for enhancing endogenous pluripotency-associated gene activation without relying on exogenous transcription factor delivery. Full article

Background: Thrombin is a central mediator of coagulation and platelet activation and a key anticoagulant target. Direct thrombin inhibitors broadly suppress thrombin proteolytic activity, potentially disrupting procoagulant and anticoagulant signaling. Exosite-targeting aptamers may enable selective modulation and programmable reversal. Methods: We characterize AYA1809002, a 40-nt DNA aptamer targeting thrombin Exosite I. Results: AYA1809002 bound thrombin with nanomolar affinity and inhibited fibrin formation with an IC₅₀ of 25.7 nM and K_i of 10.6 nM. Reduced $\gamma$-thrombin binding supported an Exosite I-dependent mechanism. AYA1809002 preserved thrombin activity toward small peptide substrates and thrombomodulin-dependent protein C activation. It showed no measurable activity against tested coagulation, anticoagulant, fibrinolytic, and control enzymes and inhibited thrombin-mediated platelet activation while preserving responses to non-thrombin agonists. AYA1809002 showed greater functional stability than thrombin-binding aptamer NU172 following serum exposure and remained active under thermal, pH, oxidative, and photostability stress. Anticoagulant activity was rapidly and sequence-specifically reversed by a reverse-complement oligonucleotide, while lipid conjugation preserved function. AYA1809002 also retained high-affinity binding to rat thrombin and anticoagulant activity in rat plasma. Conclusions: These findings identify AYA1809002 as a selective, stable, reversible Exosite I-targeting thrombin aptamer that suppresses key procoagulant functions while preserving thrombomodulin-dependent protein C activation. Full article

Epigallocatechin gallate (EGCG) imparts distinctive health benefits and flavor to tea, and its accumulation is modulated by plant growth regulators (PGRs). However, the regulatory mechanisms of Paclobutrazol (PAC) and Lovastatin (LS) underlying EGCG biosynthesis remain uncharacterized. We integrated quantitative analysis, transcriptomics, and metabolomics to investigate the regulatory effects of exogenous PAC and LS on EGCG accumulation in tea plants and systematically elucidate the underlying molecular mechanism governing EGCG biosynthesis. The results demonstrated that exogenous PAC and LS treatments significantly elevated the accumulation of EGCG, as well as endogenous auxin and jasmonic acid (JA) contents. The variation trend of these key metabolites was highly consistent with the accumulation patterns of upstream flavonoid components, including eriodictyol, delphinidin, and epigallocatechin. Transcriptomic profiling further verified the critical involvement of auxin and JA signal transduction pathways in PGR-induced EGCG differential accumulation, and we screened a total of 14 auxin-related and 8 JA-related core signal regulatory factors. Furthermore, integrated bioinformatic analyses and antisense oligonucleotide (AsODN) functional validation experiments revealed that the core hormone signaling genes CsARF2 and CsMYC2 regulate the expression of CsSCPL16 through the transcription factor CsMYB80, and ultimately promote the conversion of epigallocatechin to EGCG and promote the accumulation of EGCG in tea plants. Collectively, the PGRs boost EGCG biosynthesis by mediating endogenous auxin and JA signal transduction. These results support the development of targeted agronomic practices to improve tea quality and lay a theoretical basis for expanding the industrial exploitation of tea bioactive constituents. Full article

MicroRNA-21 (miR-21) has long been regarded as one of the most promising molecular targets in chronic kidney disease (CKD) because of its consistent upregulation across diverse renal disorders and its involvement in fibrosis, inflammation, oxidative stress, and metabolic dysfunction. Strong preclinical evidence demonstrated that inhibition of miR-21 reduced kidney injury, preserved renal function, and improved survival in multiple experimental models, particularly Alport syndrome, leading to the clinical development of the antisense oligonucleotide lademirsen. However, despite a compelling biological rationale and encouraging animal data, the phase 2 HERA trial failed to demonstrate a clinically meaningful effect on the rate of kidney function decline, resulting in discontinuation of the program. This review examines the biological functions of miR-21 in CKD, summarizes the experimental and clinical evidence that supported its therapeutic development, and critically analyzes the factors that may explain the discrepancy between preclinical success and clinical failure. Particular attention is given to the distinction between disease-associated biomarkers and true therapeutic drivers, the limitations of animal models, disease heterogeneity, timing of intervention, and the complex regulatory networks underlying progressive kidney fibrosis. Rather than representing the end of miR-21 research, the experience with lademirsen provides valuable insights into the challenges of translating RNA-based therapies into clinical practice. Future therapeutic strategies will likely require earlier intervention, improved patient selection, robust biomarkers of target engagement, and combination approaches that address the multifactorial nature of CKD. The lessons learned from the lademirsen program may help guide the development of the next generation of RNA therapeutics for kidney disease. Full article

Grapevine leafroll-associated virus 3 (GLRaV3) and grapevine red blotch virus (GRBV) are two major pathogens associated with significant economic losses, requiring reliable and sensitive diagnostic tools for rapid identification. Current RT-qPCR and qPCR assays are widely used in certification programs; however, the risk of false-negative results due to the genetic diversity of plant viruses is always a concern. Here, we present a new targeted HiPlex assay designed for the detection of GLRaV3 and GRBV. A custom primer panel containing 98 oligonucleotides targeting GLRaV3 and 14 targeting GRBV was evaluated across 45 different grapevine accessions. HiPlex diagnostic performance metrics were evaluated up to the 1:1000 dilution level. The HiPlex assay detected both viruses in 30 ng of total nucleic acids (TNA) and at 1:10 and 1:100 dilutions, exhibiting a strong inverse correlation between read counts and Ct values. Analysis of reads from qPCR-negative samples allowed the determination of a positivity cutoff of 5000 reads, which provided high specificity while maintaining sensitivity. The results presented here support the application of HiPlex amplicon sequencing for the detection of GLRaV3 and GRBV and provides a foundation for future expansion of the primer panel to additional grapevine viruses. To our knowledge, this is the first study evaluating multiplex PCR-based amplicon sequencing specifically for grapevine virus diagnostics. Full article

Gene therapy and genome editing increasingly depend on the safe, effective, and cell-selective delivery of nucleic acids and protein–nucleic acid complexes. Although viral vectors remain important for applications requiring durable gene expression, non-viral vectors offer advantages in cargo capacity, modularity, transient expression, potential repeat dosing, and avoidance of vector–genome integration. Lipid nanoparticles (LNPs), polymeric nanoparticles, inorganic nanomaterials, extracellular vesicles (EVs), and biomimetic hybrid systems have consequently become central platforms for delivery of siRNA, mRNA, plasmid DNA, antisense oligonucleotides, and CRISPR-based genome editors. Among these, ionizable LNPs are currently the most clinically mature non-viral technology, supported by the clinical success of siRNA therapeutics and mRNA vaccines, as well as the emergence of in vivo CRISPR therapies. Nevertheless, efficient endosomal escape, cell-type-selective targeting, extrahepatic delivery, and repeat-dose tolerability remain substantial barriers. Polymeric vectors provide broad chemical tunability, allowing adjustment of charge density, degradability, stimulus responsiveness, intracellular trafficking, and cargo release. However, toxicity and batch-to-batch reproducibility remain key concerns. EVs provide a biologically derived alternative with favorable membrane interfaces and potential advantages for protein and ribonucleoprotein delivery, but their clinical translation is constrained by heterogeneity, loading efficiency, product characterization, and scalable manufacturing. This review summarizes recent advances in non-viral gene-delivery platforms, compares their strengths and limitations, and discusses future directions in cell-selective delivery, endosomal escape, transient delivery of genome-editing machinery, engineered EVs, hybrid vectors, and manufacturing-oriented development. The field is transitioning from organ-level delivery toward delivery of the correct payload to the correct cell type at a clinically relevant exposure and safety margin. Full article

Despite major advances in lipid-lowering therapies, a significant unmet need remains, particularly for patients with homozygous familial hypercholesterolemia (HoFH), severe heterozygous familial hypercholesterolemia (HeFH), and those who fail to achieve guideline-recommended LDL-C targets. Nucleic acid-based therapeutics have emerged as a transformative approach for treating hypercholesterolemia. Antisense oligonucleotides and small interfering RNAs (siRNAs) have demonstrated durable hepatic gene silencing and have led to approved therapies, while gene replacement and in vivo genome-editing strategies offer the potential for long-lasting, and possibly one-time, interventions. In parallel, microRNAs (miRNAs) have attracted increasing interest because of their ability to coordinately regulate multiple genes involved in lipoprotein metabolism, cholesterol transport, and lipid homeostasis. Human genetic studies further support the importance of miRNA-mediated regulation, exemplified by a rare ~2.5 kb deletion in the distal LDLR 3′UTR (“del2.5”) that disrupts miRNA-binding sites and is associated with lifelong low LDL-C levels. This review summarizes recent advances, mechanisms of action, clinical progress, and remaining challenges across antisense oligonucleotides, siRNAs, gene therapy, genome editing, and emerging miRNA-based therapeutics for hypercholesterolemia. As an example of the latter approach, the liver-directed miR-30c analog C2 has demonstrated preclinical activity by coordinately reducing hepatic lipoprotein secretion and lipogenesis while enhancing cholesterol elimination, resulting in reduced LDL-C and atherosclerosis. However, it must be noted that these findings remain preclinical, and further optimization of delivery, pharmacokinetics, safety, and long-term efficacy will be required before clinical evaluation. Continued advances in RNA chemistry, targeted delivery, and genome engineering are expected to further expand the therapeutic landscape for dyslipidemia and cardiovascular disease. Full article