Perrone CD, Raucci L, Papini S
… +5 more, Tosi GM, Galvagni F, Olivucci M, Incarnato D, Orlandini M
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293250
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While targeting angiogenesis represents a key therapeutic strategy in several pathological contexts, it comes with significant challenges associated with therapeutic efficacy and drug resistance. Therefore, developing no...While targeting angiogenesis represents a key therapeutic strategy in several pathological contexts, it comes with significant challenges associated with therapeutic efficacy and drug resistance. Therefore, developing novel, more effective and durable therapeutic modalities is paramount. We have previously reported that antibody-mediated inhibition of CD93, a vascular endothelial cell surface glycoprotein, can inhibit angiogenesis. Here, we describe a novel strategy to efficiently inhibit CD93 expression in cells, based on the targeted degradation of CD93 mRNA using acting hammerhead ribozymes. To pinpoint single-stranded regions in CD93 mRNA that are amenable to ribozyme targeting in living cells, we performed RNA secondary structure mapping via targeted DMS-MaPseq analysis. Next, since exogenous hammerhead ribozymes are easily degraded and lose catalytic activity when expressed in living cells, we developed a novel scaffold RNA based on short stems from the 3' UTR of histone mRNAs to stabilize the active ribozyme structure and promote the CD93 cleavage under physiological conditions. Ectopic expression of these engineered ribozymes in primary endothelial cells resulted in efficient inhibition of CD93 expression, cell migration, and formation of tube-like structures in functional assays. Collectively, our data provides the proof-of-concept for the use of ribozyme-based therapeutics for the treatment of neovascular pathologies.
Zeng D, Zhu L, Liu J
… +5 more, Qiu X, Chen L, Wei C, Wang Y, Deng Z
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293249
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Despite growing recognition of immune involvement in kidney stone disease (KSD), its immunogenetic basis remains unclear. We applied a cell type-stratified immunogenetic framework, integrating single-cell -expression qua...Despite growing recognition of immune involvement in kidney stone disease (KSD), its immunogenetic basis remains unclear. We applied a cell type-stratified immunogenetic framework, integrating single-cell -expression quantitative trait loci (-eQTL) data from the OneK1K immune atlas, to identify gene-cell type associations across four anatomical KSD subtypes. This approach revealed 80 colocalized gene-cell type pairs with strong genetic support. Natural killer (NK) cells and CD4 naive/central memory T cells emerged as major contributors to disease risk, with consistent associations observed for -NK cells and -CD4/CD8 T cells across all KSD subtypes. Functional and network analyses highlighted roles for MHC class II antigen presentation and T cell activation, implicating adaptive immunity in pathogenesis. Prioritized genes showed pleiotropic links to renal function, inflammation, and mineral metabolism-for example, was associated with improved eGFR and reduced urinary citrate; with impaired renal function and elevated CRP; and with higher calcium and uric acid. Clinical complication mapping further implicated these targets in osteoporosis, hypertension, and systemic inflammation. A multi-domain prioritization identified seven top targets-, , , , , , and -as clinically actionable. These insights may inform future strategies for risk stratification, early detection, and immune-targeted intervention in KSD.
Jing M, Law MCY, Phung CD
… +4 more, He Y, Tan YB, Le MTN, Luo D
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293248
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Significant efforts have been made in the burgeoning field of RNA therapies since the COVID-19 pandemic. Self-amplifying RNA (saRNA)-based therapies are considered more promising than conventional messenger RNA due to th...Significant efforts have been made in the burgeoning field of RNA therapies since the COVID-19 pandemic. Self-amplifying RNA (saRNA)-based therapies are considered more promising than conventional messenger RNA due to their lower concentration requirements, long-lasting effects, and self-adjuvanticity. Here, we developed a novel saRNA vector, based on the rubella virus ([RuV], based on the RA27/3 vaccine strain) as an alternative virus-derived replicon that demonstrates effective expression in multiple cell lines and induces immune response. As it is derived from a commonly used vaccine, it potentially offers a better safety profile with reduced risk of adverse events compared with current alphavirus-derived saRNAs. We further optimized this RuV saRNA by screening the capsid region, the nonstructural polyprotein p200 codon, and the 3' untranslated region. This RuV saRNA has great potential to be used as a vaccine vector and in other applications in RNA therapeutics.
Senpuku K, Kunishima Y, Hirose M
… +9 more, Karaki T, Taniguchi K, Kataoka-Nakamura C, Hirai T, Yasuda K, Kuroda E, Kato T, Ito T, Yoshioka Y
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293247
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Enterovirus D68 (EV-D68) primarily causes respiratory illnesses and has been implicated in acute flaccid myelitis. Although virus-like particle (VLP) and traditional inactivated whole-virion (IWV) vaccines have demonstra...Enterovirus D68 (EV-D68) primarily causes respiratory illnesses and has been implicated in acute flaccid myelitis. Although virus-like particle (VLP) and traditional inactivated whole-virion (IWV) vaccines have demonstrated efficacy in mice, their immunological differences remain undetermined. Here, we directly compared the immunogenic and structural properties of VLP and IWV vaccines derived from the same EV-D68 strain under identical conditions. Although VLP induced significantly lower levels of EV-D68-specific IgG than IWV, neutralizing antibody titers and protective effects against viral challenge were comparable between the two groups in mice. Passive transfer experiments in neonatal mice further confirmed protection against lethal infection for both vaccine groups. Notably, in contrast to the IWV vaccine, the VLP vaccine elicited antibodies that preferentially recognized a limited subset of epitopes. Cryo-electron microscopy analyses revealed that VLPs structurally resemble the native virus but display distinct features in regions corresponding to epitopes that show differential antibody reactivity between VLP and IWV vaccines. By integrating structural and immunological analyses, we established a mechanistic framework linking capsid architecture to vaccine-induced antibody specificity. These findings suggest that VLP is a promising EV-D68 vaccine antigen with distinct epitope recognition profiles driven by structural characteristics.
Yi S, Ali SE, Jadeja Y
… +2 more, Davis JW, Metkar M
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293245
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The success of COVID-19 mRNA vaccines has made the in-solution stability optimization of mRNAs a key objective. However, we still lack a complete understanding of sequence metrics that influence mRNA in-solution stabilit...The success of COVID-19 mRNA vaccines has made the in-solution stability optimization of mRNAs a key objective. However, we still lack a complete understanding of sequence metrics that influence mRNA in-solution stability. RNA secondary structure plays a critical role in protecting against hydrolysis, the primary degradation pathway under storage conditions. Yet, the structural metrics that best guide stability-focused mRNA design remain incompletely defined. Global metrics like minimum free energy and average unpaired probability have improved mRNA stability but fail to capture local structural variation relevant to RNA degradation. We demonstrate that base-pairing log odds (LO) provide fine-scale, orthogonal insight that complements global metrics and improves stability modeling. Further, by combining local and global features into a parsimonious four-feature regression model, dubbed stability regression analysis using nucleotide-derived features (STRAND), we achieve a greater than 2-fold reduction in prediction error compared to existing machine learning and deep learning approaches and demonstrate robust generalization across diverse transcript contexts. This compact and interpretable model provides an accurate and reliable framework for predicting mRNA in-solution stability.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293244
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Blood-based transcriptomic profiling provides a minimally invasive approach for disease diagnosis; however, the integration of large-scale, heterogeneous RNA sequencing (RNA-seq) datasets remains challenging. Here, we ma...Blood-based transcriptomic profiling provides a minimally invasive approach for disease diagnosis; however, the integration of large-scale, heterogeneous RNA sequencing (RNA-seq) datasets remains challenging. Here, we manually curated and uniformly reprocessed 134 publicly available human RNA-seq datasets ( = 9,872 samples), covering 88 distinct blood-related diseases from whole blood and peripheral blood mononuclear cell data. To enable robust and scalable multiclass prediction, we employed AutoGluon, an ensemble-based AutoML framework that automates model selection and hyperparameter tuning through multilayer stacking. Our models achieved high accuracy across most disease categories (>90%), demonstrating strong generalizability. To support biological interpretation, we also performed differential expression and pathway enrichment analyses, revealing disease-associated signatures enriched in relevant Gene Ontology terms and Kyoto Encyclopedia of Genes and Genomes pathways. We implemented these capabilities in BRPtools, a web-based platform featuring two modules: (1) a search module for exploring gene- and disease-level expression profiles and (2) a predict module for disease classification using user-uploaded RNA-seq count matrices. The platform supports diagnostic inference and validation and is designed to be accessible to users without programming experience. BRPtools offers an integrated, interpretable, and user-friendly solution for transcriptome-based disease prediction, supporting applications in biomarker discovery, digital diagnostics, and precision medicine.
Lee SY, Lee J, Ahn JH
… +12 more, Lee SY, Choi EJ, Ha D, Oh A, Jo S, Yoon S, Lee S, Bae HJ, Lee NY, Youn H, Kang BC, Nam JH
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42282251
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mRNA vaccines have revolutionized development and continue to advance through clinical and research applications. Circular RNA is emerging as a promising alternative to linear RNA. However, no direct comparison has ident...mRNA vaccines have revolutionized development and continue to advance through clinical and research applications. Circular RNA is emerging as a promising alternative to linear RNA. However, no direct comparison has identified the optimal RNA platform for mRNA-based drug development. This study compares linear and circular RNA platforms and , focusing on expression dynamics, immune responses, and safety. In terms of expression efficiency, the circular RNA showed prolonged expression compared to linear RNA. The immunogenicity was evaluated using influenza HA as an antigen. In humoral immune response, linear RNA with modified nucleosides (Lin-m1Ψ) and circular RNA with unmodified nucleosides (Circ-WT) showed higher neutralizing antibody titers than linear RNA with unmodified nucleosides (Lin-WT). Lin-m1Ψ and Lin-WT induced slightly higher cell-mediated immune responses than Circ-WT. In mice, all RNA types showed a decrease in reticulocytes, monocytes, and alkaline phosphatase on day 2 following high-dose intramuscular injection. Furthermore, Lin-WT and Circ-WT showed greater decreases in platelets than Lin-m1Ψ. However, these changes recovered to normal by day 14 post-vaccination in all groups. These findings provide insights into developing RNA-based immunotherapies and selecting suitable RNA platforms for various applications, including preventive and therapeutic vaccines as well as protein-replacement therapies.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42239496
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Decades of research have cemented antisense oligonucleotides (ASOs) as a cornerstone of molecular medicine. Advancements in synthesis and chemical modification, together with an improved understanding of the human genome...Decades of research have cemented antisense oligonucleotides (ASOs) as a cornerstone of molecular medicine. Advancements in synthesis and chemical modification, together with an improved understanding of the human genome and transcriptome, have enabled their emergence as highly specific and tailorable therapeutics across a wide variety of conditions. Although the greatest strength of ASOs lies in their nucleic acid composition that confers the theoretical ability to target any known genetic sequence, effective ASO design requires a holistic approach considering the molecular mechanism underlying the desired therapeutic outcome. Initially considered straightforward inhibitors of gene expression, ASOs have now evolved into versatile modulators capable of exploiting an increasingly diverse array of molecular processes. Despite this progress, their full therapeutic potential remains far from realized. Emerging research, driven by a deepening understanding of RNA biology, continues to expand the repertoire of mechanisms through which ASOs can modulate gene expression. Collectively, these studies demonstrate that ASOs can be designed to modulate numerous pre-mRNA processing events, including splicing, polyadenylation, microRNA activity, and translation initiation and termination, thereby broadening the range of conditions and patients that may benefit from ASO-based therapeutics.
Chen B, Tian Y, Liang L
… +6 more, Ding P, Yin R, Xu R, Wang T, Chen H, Lu D
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42239495
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Canonical base editors (BEs) enable programmable nucleotide conversions without inducing DNA double-strand breaks (DSBs), yet the significant off-target (OT) effects restrict their applications. The transformer BE (tBE),...Canonical base editors (BEs) enable programmable nucleotide conversions without inducing DNA double-strand breaks (DSBs), yet the significant off-target (OT) effects restrict their applications. The transformer BE (tBE), a BE derivative, minimizes DNA and RNA OT mutations, but its dual single-guide RNA (sgRNAs) requirement limits its targeting scope. To address this, we integrated SpRY, a protospacer-adjacent motif (PAM)-relaxed Cas9 variant, into tBE. The resulting tBE-SpRY achieved robust C-to-T editing across various cell lines in a PAM-unlimited manner, with minimal OT effects at the tested genomic loci and negligible transcriptome-wide RNA editing. We further demonstrated its versatility and . Microinjection of tBE-SpRY components into zygotes efficiently generated albino C57BL/6J mice, while dual adeno-associated virus (AAV)-mediated delivery in mice resulted in modest C-to-T modifications (up to 2.96%) in ocular tissues. Thus, tBE-SpRY provides a safe and PAM-flexible platform for generating genetic mouse models and exploring potential therapeutic applications.
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42233109
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Messenger RNA (mRNA) vaccines using lipid nanoparticles (LNPs) are globally approved with acceptable safety profiles for preventing respiratory diseases. Here, we describe tissue distribution and kinetics of mRNA-LNP vac...Messenger RNA (mRNA) vaccines using lipid nanoparticles (LNPs) are globally approved with acceptable safety profiles for preventing respiratory diseases. Here, we describe tissue distribution and kinetics of mRNA-LNP vaccines after intramuscular dosing using three products formulated with same LNP matrix: mRNA-1273 (Spikevax), mRNA-1647 (a candidate cytomegalovirus [CMV] vaccine), and a reporter mRNA (nascent peptide-luciferase) drug product. Consistent biodistribution patterns were observed: tissues with highest exposures were the injection site, draining lymph nodes, and spleen, with minimal distribution to other organs. Levels of vaccine components declined rapidly in circulation and tissues, with model-based projections indicating concentrations at low or below quantifiable levels by ∼2 weeks. Unbound (free) spike protein was transient after mRNA-1273 vaccination (modeled decline < 5 days) and did not accumulate with repeated dosing. The ionizable lipid in the LNP, lipid H, underwent biotransformation and was excreted renally and hepatically, with no human-specific metabolites. Collectively, these results indicate similar biodistribution patterns across products within this dataset using a shared LNP composition, despite different mRNA cargo. Furthermore, in a SARS-CoV-2 infection-free model, no sustained spike protein was observed. Overall, the data establish a framework that can be used to scientifically justify leveraging data across a shared platform.
Splendiani E, Maiorano G, Spinello Z
… +23 more, Vecchiotti D, Gugliuzza G, Moretti M, Asquino A, Izzo M, Po A, Feverati N, Raia T, Besharat ZM, Verzella D, Mardente S, Zicari A, Ciccarella G, Citarella A, Zazzeroni F, De Smaele E, Venneri MA, Mastronuzzi A, Gigli G, Angelucci A, Catanzaro G, Palamà IE, Ferretti E
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42233108
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MicroRNAs play a crucial role in tumorigenesis, functioning as oncogenes or tumor-suppressors while orchestrating the differentiation states of cancer cells. In medulloblastoma, the most common malignant pediatric brain...MicroRNAs play a crucial role in tumorigenesis, functioning as oncogenes or tumor-suppressors while orchestrating the differentiation states of cancer cells. In medulloblastoma, the most common malignant pediatric brain tumor, the downregulation of miR-326 is associated with a loss of tumor suppression that leads to uncontrolled proliferation and survival or de-differentiation. In this study, we explore an innovative approach for microRNA delivery using polycaprolactone/chitosan-based nanoparticles. We evaluate the biocompatibility of the nanocarrier in medulloblastoma cell lines and report results on nanoparticles administration, demonstrating minimal toxicity in the most exposed organs. findings reveal that nanoparticles-mediated miR-326 delivery significantly reduces stemness markers (Nanog and Sox2) and promotes neuronal differentiation, as evidenced by increased β3-tubulin expression. These results highlight polycaprolactone-based nanoparticles as a promising platform for miR-326 delivery, establishing a foundation for future investigations on therapeutic strategies in medulloblastoma.
de Klerk JA, Slieker RC, Beulens JWJ
… +7 more, Peerlings JHD, Mei H, Elders PJM, van Zonneveld AJ, van Raalte DH, Bijkerk R, 't Hart LM
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42233107
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Chronic kidney disease (CKD) is a common complication of type 2 diabetes, characterized by reduced kidney function and/or albuminuria, yet its progression varies widely among individuals. While sodium-glucose cotransport...Chronic kidney disease (CKD) is a common complication of type 2 diabetes, characterized by reduced kidney function and/or albuminuria, yet its progression varies widely among individuals. While sodium-glucose cotransporter-2 (SGLT2) inhibitors are known to protect against kidney decline, the molecular mechanisms underlying their renoprotective effects remain incompletely understood. Circulating small non-coding RNAs (sncRNAs), particularly microRNAs, have been linked to CKD but the contribution of other sncRNA classes is less explored. We profiled plasma sncRNAs in 263 participants with type 2 diabetes from the Hoorn DCS cohort without CKD at baseline, followed for ∼9 years ( = 122, = 141). sncRNA profiling was also performed before and after treatment of SGLT2 inhibitors in three trials ( = 65, total) to assess drug-induced molecular changes in the circulation. Eleven sncRNAs were nominally associated with incident CKD, most strongly and . In an independent SGLT2 inhibitor trial, exploratory analyses identified 34 sncRNAs that changed following treatment, including . Preliminary analyses linked these snoRNAs to co-regulated proteins, suggesting potential functional relevance. Our results identify snoRNAs, particularly , as potential novel markers of CKD risk and SGLT2 inhibitor response in type 2 diabetes, revealing an unexplored sncRNA axis and providing a foundation for future functional studies into their mechanistic role in CKD progression.
Slattery S, Huo W, Arif A
… +2 more, Gordon J, Takeuchi R
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42221546
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Gene editing technologies have opened the possibility of directly targeting viral DNA for therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA...Gene editing technologies have opened the possibility of directly targeting viral DNA for therapeutic applications. In chronically infected hepatocytes with hepatitis B virus (HBV), covalently closed circular DNA (cccDNA) serves as the master template for viral transcripts and gene products. In the present study, we evaluated anti-HBV multiplex gene editing with the CRISPR-Cas9 endonuclease from (SaCas9) using primary human hepatocytes (PHHs) and HBV mouse models. Nonviral delivery of SaCas9-encoding mRNA and a pair of HBV-targeting guide RNAs (gRNAs) substantially reduced viral biomarkers and intrahepatic HBV DNA copies and . Hybridization capture sequencing analyses showed that small insertions and deletions (indels) and structural variants including excisions and inversions of the viral sequences were introduced in the residual HBV DNA. These assays also demonstrated that transient expression of the HBV-targeting SaCas9 significantly suppressed random integration of HBV DNA into the host genome, while leading to no detectable increase in chromosomal translocations involving viral copies. Lastly, our gene editing approach blocked viral rebound after stopping treatment with a nucleos(t)ide analogue (NA), entecavir. Our results suggest that anti-HBV multiplex gene editing removes viral DNA from chronically infected hepatocytes, potentially reducing the risk of hepatocarcinogenesis associated with HBV DNA integration.
Chen S, Peng Y, Yan D
… +3 more, Lyu L, Scherngell T, Hu Y
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42211693
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Small interfering RNA (siRNA) represents a transformative modality in next-generation therapeutics, yet challenges in delivery efficiency still limit its clinical translation. Systematic evaluation tools are needed to re...Small interfering RNA (siRNA) represents a transformative modality in next-generation therapeutics, yet challenges in delivery efficiency still limit its clinical translation. Systematic evaluation tools are needed to reveal technological progress in this area. Given the innovation momentum inherent in patents, this study develops a patent-based evaluation model for siRNA delivery technologies from a technology management perspective. By integrating the Hierarchical Decision Model (HDM) with patent landscape analysis, the model incorporates 15 multidimensional criteria across technological, commercial, and legal perspectives, with the commercial perspective assigned the greatest weight (41%), followed by technical (32%) and legal (27%) perspectives. A large-scale dataset comprising 20,319 siRNA delivery-related patent documents was quantitatively assessed using this model. The results reveal that high-value patents are primarily concentrated in lipid-based carriers and ligand-siRNA conjugates, with firms such as Alnylam and Arbutus emerging as dominant innovation leaders. Furthermore, the analysis highlights that therapeutic indication, assignee's technological accumulation, technological impact, and drug delivery method are key drivers of patent value. Overall, the proposed model supports strategic decision-making in patent portfolio management and technology forecasting by identifying high-value innovations.
Sasset L, Cameron AD, Sussman C
… +8 more, Rubinelli L, Maji D, Miller R, Thompson A, Campbell D, Walker MR, Drozdz MM, Liberatore RA
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42211692
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Incretin receptor agonists (IRAs) have recently come to prominence as powerful weight and glucose control drugs and are approved for the treatment of type 2 diabetes (T2D) and obesity. Semaglutide and tirzepatide, curren...Incretin receptor agonists (IRAs) have recently come to prominence as powerful weight and glucose control drugs and are approved for the treatment of type 2 diabetes (T2D) and obesity. Semaglutide and tirzepatide, currently the most widely prescribed within this class, are both potent molecules but have a short half-life, requiring weekly dosing. This requirement can negatively affect quality of life and the adherence to therapy, as well as create a significant financial burden for patients who may have a life-long need for treatment. The MYO Technology platform was developed to overcome these barriers. It consists of therapeutic-encoding plasmid DNA, and a proprietary medical device for intramuscular injection and electroporation of muscle cells. This leads to the uptake of injected DNA, expression of the therapeutic, and its secretion into peripheral circulation. Here, we demonstrate that MYO Technology-delivered IRAs are efficacious in promoting long-lasting weight and glucose control in mouse models of diet-induced obesity. Moreover, engineering the IRAs to facilitate blood-brain barrier penetration further enhances treatment efficacy, with benefits persisting beyond 1 year following a single administration. Together, these findings highlight MYO Technology's potential to transform standard of care by enabling long-lasting therapeutic effects with minimal dosing.
Dolgin V, Eremenko E, Narkis G
… +3 more, Mazor-Oring M, Gradstein L, Birk OS
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42211691
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The development of antisense oligonucleotide (AON) therapy for monogenic diseases such as retinitis pigmentosa (RP) is effective but labor-intensive. Rapid, efficient technologies to identify optimal AONs are needed. We...The development of antisense oligonucleotide (AON) therapy for monogenic diseases such as retinitis pigmentosa (RP) is effective but labor-intensive. Rapid, efficient technologies to identify optimal AONs are needed. We present a novel approach focusing on intronic splicing mutations. We identified a novel heterozygous intronic non-canonical splice variant in causing dominant RP in a four-generation pedigree. Using a Dual Fluorescence Reporter Assay, we transfected HEK293T cells with a reporter plasmid containing either the wildtype intron (resulting in GFP fluorescence due to normal splicing) or the mutant intron (showing no fluorescence due to splicing defects). As an alternative to standard AONs, we used uridine-rich 7 (U7) small nuclear RNA (snRNA) derivatives with optimized Sm protein-binding site and a modified antisense sequence (U7 Sm OPT snRNA) as modulators of pre-mRNA splicing. Eight AONs were designed to target the aberrant transcript, to restore normal splicing and gene function. To evaluate treatment efficacy, HEK293T cells with the reporter GFP plasmid were co-transfected with U7snRNA cassettes, each containing an incorporated AON sequence. FACS analysis quantified splicing modulation and gene expression rescue, identifying the most effective AON. Our study broadens the genetic understanding of RP, highlighting the significance of personalized medicine in genetic disorder treatment.
Diaz JA, Chilcott EM, Barbanoj AA
… +11 more, Keegan A, Gurung S, Pauzuolyte V, Waddington Z, Kyriacou M, McTague A, Cross JH, Schorge S, Lignani G, Waddington SN, Karda R
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42181696
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Dravet syndrome (DS) is a severe childhood developmental and epileptic encephalopathy. Symptoms usually manifest in the first year of life and include prolonged severe seizures, developmental delay, severe intellectual d...Dravet syndrome (DS) is a severe childhood developmental and epileptic encephalopathy. Symptoms usually manifest in the first year of life and include prolonged severe seizures, developmental delay, severe intellectual disability, and increased mortality. Approximately, 90% of patients carry a heterozygous loss-of-function mutation in , encoding a voltage-gated sodium ion channel, Na1.1. Na1.1 is expressed in the brain and at a lower level, in the heart. Previous studies have identified a long non-coding RNA (lncRNA), which specifically downregulates expression. This natural antisense transcript (NAT) can be modulated by AntagoNATs, small synthetic oligonucleotides. AntagoNATs have shown to improve seizure frequency in DS mice after repeated administration. Here, we have developed new AntagoNATs and incorporated these into an adeno-associated virus serotype 9 (AAV9) gene therapy vector. We administered two new AAV9-AntagoNAT-H and AntagoNAT-K vectors to newborn mice via intracerebroventricular (i.c.v.) and intravenous (i.v.) injection to deliver vector to the brain and heart. AAV9-AntagoNAT-H significantly increased survival, decreased the frequency of febrile and spontaneous seizures. In this proof-of-concept study, we have demonstrated for the first time the delivery of AntagoNAT via an AAV9 vector. Thus, offering the possibility of a one-time treatment for DS patients.