Baum R, Telugu N, Bruyneel AAN
… +9 more, Kay M, Nair P, Perea-Gil I, Termglinchan V, Bharucha N, Lee E, Mercola M, Diecke S, Karakikes I
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41674724
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CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in...CRISPR-Cas9 is widely used to model genetic disorders by introducing or correcting disease-associated mutations in induced pluripotent stem cells (iPSCs) through homology-directed repair (HDR). However, HDR efficiency in iPSCs remains low and is highly dependent on the target locus. Here, we developed CRISPR-MiX, an improved protocol to enhance HDR efficiency in human iPSCs. Using a GFP-to-BFP reporter system, we identified key single-stranded oligodeoxynucleotide (ssODN) donor design parameters, including homology arm symmetry, CRISPR/Cas-blocking mutations, and strand complementarity, which significantly influence HDR outcomes. We applied this approach to introduce pathogenic variants into five genes related to genetic cardiomyopathies. Quantitative analysis of HDR events showed that both the target locus and ssODN design strongly affect HDR efficiency. To address the locus- and design-specific limitations, we established CRISPR-MiX, a pooled ssODN-based method for scarless genome editing using ribonucleoproteins (RNPs) that does not require selection. CRISPR-MiX consistently improved HDR efficiency across multiple loci. This strategy offers a simple, robust, and versatile approach for precise genome engineering in iPSCs, supporting broad applications in disease modeling and functional genomics.
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41646887
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Common ophthalmic diseases, including age-related macular degeneration (AMD), generally have a complex pathogenesis involving multiple pathways and varying involvement of specific cell types. This provides a strong ratio...Common ophthalmic diseases, including age-related macular degeneration (AMD), generally have a complex pathogenesis involving multiple pathways and varying involvement of specific cell types. This provides a strong rationale for developing novel gene therapy platforms that allow cell-specific up- and down-regulation of multiple targets while contained within standard adeno-associated viral vectors (AAVs). Hence, we engineered a tunable expression cassette with two pri-miR-embedded, Ago2-dependent shRNAs (miR-agshRNAs) units enabling dual target silencing, and intron embedment allowing downstream protein expression. With this platform, we demonstrated additive knockdown, concurrent silencing of and , and simultaneous expression of pigment epithelium-derived factor (PEDF) from a single promoter. Following the subretinal injection of AAV5 vectors encoding -targeting miR-agshRNAs and PEDF into the murine retina, profound suppression and strong PEDF expression were observed. Notably, laser-induced choroidal neovascularization (CNV) was significantly reduced in the therapeutic groups, with the multi-targeting vector achieving the highest level of CNV suppression. Collectively, our data demonstrated robust anti-angiogenic effects of multiple gene therapies, suggesting a "one-and-done" AAV-based delivery of cross-species anti-VEGFA RNAi therapeutics together with PEDF as a valuable tool for the management of neovascular AMD (nAMD) and other complex neovascular ocular diseases.
Zhang H, Liu Y, Ma J
… +7 more, Huang H, Zhang W, Xiu D, Ma T, Zhang M, Yu F, Song G
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41646886
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Necessary chemical modifications can enhance the metabolic stability and therapeutic efficacy of oligonucleotide-based drugs. Herein, a series of mononucleotides with different functional group modifications at the ribos...Necessary chemical modifications can enhance the metabolic stability and therapeutic efficacy of oligonucleotide-based drugs. Herein, a series of mononucleotides with different functional group modifications at the ribose 2'-position were designed and synthesized for reducing off-target effects of oligonucleotide drugs. After incorporating them into the antisense strand of small interfering RNAs (siRNAs), we assessed their biophysical and biological properties. The results revealed that, following the substitution of the conventional 2'-O-methyl or 2'-O-fluoro ribose modifications with our newly designed functional groups, although the thermal stability of the duplexes decreased, the silencing activity against proprotein convertase subtilisin/kexin type 9 (PCSK9) gene was significantly enhanced and the off-target effects of specific genes could be effectively mitigated. These data prove that 2'-ribose-modified nucleotides can be effectively utilized in preclinical and clinical disease model studies for gene silencing and reducing off-target effects.
Ding T, Li Y, Xue L
… +6 more, Xiong C, Yu L, He Q, Liu J, Hao X, Zhao D
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41646885
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Prostate cancer (PCa) lacks convenient, non-invasive, and highly specific diagnostic markers. Aptamers have emerged as preferred probes for biosensors that target extracellular vesicles (EVs). This study aimed to explore...Prostate cancer (PCa) lacks convenient, non-invasive, and highly specific diagnostic markers. Aptamers have emerged as preferred probes for biosensors that target extracellular vesicles (EVs). This study aimed to explore the diagnostic value of PCa-specific EVs aptamer probes. We used EV-SELEX to identify aptamers that selectively target PCa small EVs (sEVs). Surface plasmon resonance (SPR) and nanoflow cytometry were used to verify aptamer affinity. The diagnostic value of PCa was evaluated using clinical samples from patients. We screened and validated an aptamer, seq25, which exhibited high specificity for PCa-derived sEVs. The SPR assay revealed a strong binding affinity, with a KD of 24.02 nM and a dose-dependent binding response. Nanoflow cytometry demonstrated that seq25 could distinguish sEVs from PCa and normal prostate cell lines. In clinical specimens, the proportion of seq25-positive sEVs isolated from urine samples was significantly higher in patients with PCa than in those with benign prostatic hyperplasia. Our study integrated the diagnostic advantages of EVs with the technical benefits of aptamers to develop a PCa-specific sEVs aptamer probe that offers a promising non-invasive approach for PCa diagnosis.
Hoque MM, Kong M, Yuan X
… +5 more, Zhou X, Ting-Yuan Yeh S, Long K, Lin H, Yip Cheng KK
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41646884
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Metabolic-dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of pathogenic conditions ranging from steatosis, inflammation, and fibrosis with limited treatment options. We previously demonstrat...Metabolic-dysfunction-associated steatotic liver disease (MASLD) encompasses a spectrum of pathogenic conditions ranging from steatosis, inflammation, and fibrosis with limited treatment options. We previously demonstrated an upregulation of hepatic murine double minute 2 (MDM2) in human subjects with MASLD. Genetic deletion of hepatic MDM2 and pharmacological inhibition of systemic MDM2 improves steatosis and fibrosis in MASLD mouse models. In this study, we further developed and investigated the therapeutic potential of a hepatocyte-specific triantennary N-acetylgalactosamine (GalNAc)-conjugated antisense oligonucleotide targeting MDM2 (GalNAc-Mdm2ASO) in two distinct dietary-induced mouse models of MASLD: a high-fat-high-cholesterol (HFHC) diet and a choline-deficient, L-amino-acid-defined, high-fat diet (CDAHFD). In the HFHC-induced MASLD model, GalNAc-Mdm2ASO not only alleviated liver injury, steatosis, and fibrosis but also improved obesity-related insulin resistance and hyperlipidemia. The hepatoprotective effects of GalNAc-Mdm2ASO treatment were associated with a reduced accumulation of hepatic cholesterol, diacylglycerol, and ceramide, which are known to trigger MASLD. In CDAHFD-induced MASLD mouse model, GalNAc-Mdm2ASO significantly mitigated hepatic inflammation, cholesterol accumulation, and fibrosis but not triglyceride accumulation. Overall, we prove hepatic inhibition of MDM2 using GalNAc-Mdm2ASO as a promising therapeutic agent for MASLD in two rodent models with distinct pathogenesis.
Blitek M, Gastaldi C, Doisy M
… +8 more, Le Coz O, David M, Phongsavanh X, Ben Aicha S, Garcia L, Rotini A, Pagès G, Goyenvalle A
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41630990
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Duchenne muscular dystrophy (DMD) is a severe X-linked disease caused by pathogenic variants in the gene, resulting in the absence of functional dystrophin. Antisense oligonucleotide (ASO)-based therapies aim to restore...Duchenne muscular dystrophy (DMD) is a severe X-linked disease caused by pathogenic variants in the gene, resulting in the absence of functional dystrophin. Antisense oligonucleotide (ASO)-based therapies aim to restore the open reading frame and produce a truncated but functional dystrophin protein. Although several ASOs are approved in the United States and Japan via accelerated approval procedures, dystrophin restoration in patient biopsies remains low, underlining the need to improve ASO potency. One major limitation is poor ASO biodistribution to skeletal muscle, influenced by both ASO chemistry and pathological features of dystrophic tissue. In DMD patients and mice, microvascular abnormalities and impaired angiogenesis likely restrict ASO delivery. Here, we hypothesized that enhancing muscle vascularization could improve ASO biodistribution and therapeutic outcomes. mice were treated with a pro-angiogenic treatment prior to ASO administration targeting exon 23 of dystrophin pre-mRNA. Angiogenic stimulation increased capillary density and improved ASO delivery to muscles (3.8-fold), exon skipping (1.8-fold), and dystrophin expression (1.5-fold) compared to ASO alone. These molecular improvements were associated with increased myofiber size, larger mean cross-sectional area, and decreased serum myomesin levels, without signs of toxicity. This study provides proof of concept that promoting angiogenesis can enhance the potency of ASO-based treatments, offering a complementary strategy to improve therapeutic outcomes in DMD.
Gaddam RR, Pathuri M, Deb P
… +10 more, Dwivedi S, Vikram A, Kasina V, Amalkar VS, Lira V, Kaur H, Dhanesha N, Mangalam AK, Bahal R, Vikram A
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41630989
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MicroRNA-122-5p (miR-122) is primarily expressed in the liver and is increasingly released into the bloodstream during obesity. It impacts the function of non-liver tissues, such as vascular endothelial cells, and increa...MicroRNA-122-5p (miR-122) is primarily expressed in the liver and is increasingly released into the bloodstream during obesity. It impacts the function of non-liver tissues, such as vascular endothelial cells, and increases the risk of diabetic vasculopathy. Using a gamma-peptide-nucleic acid-based miR-122 inhibitor (γP-122-I), we show that miR-122 regulates blood glucose levels and endothelial function in high-fat diet-fed mice. Targeting γP-122-I to endothelial cells retains its ability to improve vascular function but reduces metabolic benefits compared to the non-targeted version. Our results show that endothelial cells take up miR-122 through a neuropilin-1-dependent mechanism. Aortic transcriptomic analysis implicates miR-122 role in mitochondrial function. The aortas of high-fat diet-fed mice receiving an inhibitor of miR-122 were more efficient in oxygen consumption despite a decline in the expression of mitochondrial electron transport chain complexes. Supporting these findings, the overexpression of miR-122 under hyperglycemic conditions decreases mitochondrial electron transport chain respiration and mitochondria with high membrane potential, indicating its detrimental impact on mitochondrial function. These findings support miR-122 as a therapeutic target for diabetic vasculopathy and support γPNA-based miR-122 inhibition as a potentially safer and more effective therapy.
Gaci A, Menchon G, Bruce J
… +3 more, Salnot V, Pedeux R, Goyenvalle A
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41630988
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Cellular uptake and intracellular distribution of phosphorothioate-modified antisense oligonucleotides (PS-ASOs) are mediated by protein interactions. While several PS-ASOs-binding proteins have been identified, mainly u...Cellular uptake and intracellular distribution of phosphorothioate-modified antisense oligonucleotides (PS-ASOs) are mediated by protein interactions. While several PS-ASOs-binding proteins have been identified, mainly using gapmer designs with 2'-O-methoxyethyl (2'MOE) modifications, less is known about protein partners of splice-switching oligonucleotides (SSOs) with alternative ribose modifications. Here, using affinity purification mass spectrometry (AP-MS), we identified the intracellular protein partners of PS-SSOs of the same sequence with three distinct ribose modifications: tricyclo-DNA (tcDNA), locked nucleic acid (LNA), and 2'MOE. Interestingly, we found previously reported PS interactors, such as GRSF1, NONO, and NCL, as well as uncharacterized protein partners. Four shared interactors identified in this study, ERC1, SPIRE1, THRAP3, and GOLGA2, were selected based on functional relevance and tested for their impact on exon skipping efficacy using PS-SSOs targeting the human Duchenne muscular dystrophy (DMD) transcript. RNA interference-mediated knockdown of each protein led to a reduction of exon skipping efficiency, suggesting that these proteins may contribute to PS-ASOs activity regardless of their sugar modifications. Overall, our results provide a set of intracellular protein interactors of different PS-ASOs, representing a valuable resource to explore mechanisms underlying their activity and offering potential leads for the optimization of oligonucleotide therapeutics.
Allaire NE, Armstrong MS, Yoon JS
… +13 more, Walker MC, Bhatt P, Harrington JM, Cheng Y, Wong EI, Mahoney JE, Valley HC, Macadino CM, Bihler HJ, Sivachenko AY, Cotton CU, Carulli JP, Mense M
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41624335
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Nonsense or premature termination codon (PTC) variants of the CFTR gene are pathogenic and found in ∼10% of North American people with cystic fibrosis. In addition to encoding incomplete proteins, PTC variants induce non...Nonsense or premature termination codon (PTC) variants of the CFTR gene are pathogenic and found in ∼10% of North American people with cystic fibrosis. In addition to encoding incomplete proteins, PTC variants induce nonsense-mediated mRNA decay (NMD), leading to ∼80%-90% reduction in full-length mRNA. This reduction is a contributor to PTC mutation-related pathology. E22 trunc is a naturally occurring truncated CFTR mRNA that terminates before the W1282X PTC variant and is resistant to NMD. To induce its expression, antisense oligonucleotides (ASOs) were tiled across intron 22 splice donor (SD) and splice acceptor (SA) sites. Top SD/SA ASO pairs were assessed for their impact on e22 trunc mRNA, e22 trunc protein, and CFTR-mediated chloride (Cl) transport in immortalized and primary human bronchial epithelial (hBE) cell cultures. We demonstrate that e22 trunc mRNA generates a truncated CFTR protein whose Cl transport function can be enhanced with elexacaftor/tezacaftor/ivacaftor (ETI) treatment. ASO and ETI treatment in combination restore ∼20% and 25% of wild-type CFTR Cl transport function in immortalized epithelial and primary hBE cells homozygous for CFTR W1282X, respectively. This study provides a foundation for advancing ASO-mediated upregulation of e22 trunc mRNA and protein as a therapeutic approach for cystic fibrosis caused by 3'-terminal CFTR PTC mutations.
Smith VL, Gidi BZ, Bragg RM
… +15 more, Cantle JP, Ben-Varon A, Noble B, Prades S, Compton A, Greenfield J, Korecka JA, Gemos A, Yu T, Khurana V, Kordasiewicz HB, Zhao HT, Barker-Haliski M, Child DD, Carroll JB
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41624332
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Dentatorubral-pallidoluysian atrophy (DRPLA) is a fatal neurodegenerative disease arising from a CAG repeat expansion in the atrophin-1 () gene. Because DRPLA, like many repeat expansion disorders (REDs), arises predomin...Dentatorubral-pallidoluysian atrophy (DRPLA) is a fatal neurodegenerative disease arising from a CAG repeat expansion in the atrophin-1 () gene. Because DRPLA, like many repeat expansion disorders (REDs), arises predominantly from toxic gain-of-function mechanisms, we hypothesized that knockdown would have therapeutic potential. To test this, we established the first fully humanized mouse model of a RED, in which one allele of mouse is completely replaced by human , including 112 pure CAG repeats. This novel approach to exploring RED biology provides significant advantages, notably the ability to test sequence-specific therapeutics targeting human sequences, even in introns and untranslated regions of pre-mRNA. We found that our model-the mouse-recapitulates key features of human DRPLA, including behavioral alterations, reduced brain size, and aggregate accumulation. We treated mice with antisense oligonucleotides (ASOs) targeting mouse (to probe for loss of function concerns), human , or a combination. Treatment with human, but not mouse, -targeting ASOs provides remarkable protection from a range of disease-related behavioral phenotypes and marked rescue of transcriptional dysregulation in the cerebellum. These results have helped motivate an ongoing human clinical study of ASOs targeting for DRPLA.
Tang Q, Yu Y, Shen M
… +3 more, Zhang L, Jia X, Kang J
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41583561
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Certain RNAs exhibit both protein-coding and regulatory non-coding functions, termed bifunctional RNAs or coding and non-coding RNAs. Long non-coding RNAs (lncRNAs), which play crucial roles in gene regulation and cellul...Certain RNAs exhibit both protein-coding and regulatory non-coding functions, termed bifunctional RNAs or coding and non-coding RNAs. Long non-coding RNAs (lncRNAs), which play crucial roles in gene regulation and cellular processes, represent a major subset of bifunctional RNAs. Accurate identification of bifunctional lncRNAs is critical for advancing RNA biology and uncovering opportunities for biomarker discovery and therapeutic development. Here, we present cncFinder, a graph-attention-network-based model for predicting bifunctional lncRNAs. It transforms lncRNA sequences into k-mer graphs, encodes node features with Word2Vec, and employs graph attention network to capture higher-order sequence dependencies. On the testing dataset, cncFinder achieved superior performance, significantly outperforming state-of-the-art models. Its robustness and broad applicability were further confirmed through validation on cross-species datasets from mouse and fruit fly. Interpretability analysis revealed that cncFinder captured biologically meaningful motifs, including canonical start codons and Kozak-like elements. In a case study of LINC00961, cncFinder precisely detected an experimentally validated translation initiation motif, highlighting its biological relevance. To support broad accessibility, we developed a user-friendly web server. In summary, cncFinder advances predictive accuracy and interpretability, providing a powerful tool for systematic discovery of bifunctional lncRNAs and enabling new insights into RNA multifunctionality.
Pechnikova NA, Zafeiriou-Chatziefraimidou M, Poimenidou M
… +4 more, Patsidou C, Iliadis I, Ostankova YV, Yaremenko AV
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41583559
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Hepatitis B virus (HBV) remains a leading cause of cirrhosis and hepatocellular carcinoma, while functionally curative therapies remain scarce. Durable remission is hindered by the persistence of covalently closed circul...Hepatitis B virus (HBV) remains a leading cause of cirrhosis and hepatocellular carcinoma, while functionally curative therapies remain scarce. Durable remission is hindered by the persistence of covalently closed circular DNA (cccDNA) and viral genome integration, both of which contribute to impaired immune responses. mRNA-based technologies provide a versatile platform for prevention and treatment, owing to their rapid development cycle and intrinsic immunostimulatory properties. In preclinical HBV-carrier mouse models, lipid-nanoparticle (LNP) mRNA vaccines encoding hepatitis B surface antigen (HBsAg) or polyvalent Ags have achieved HBsAg clearance and HBV-DNA reduction, outperforming protein-based comparators. Clinically, two phase I therapeutic trials have been initiated to date. An ARCUS nuclease delivered as LNP-mRNA entered first-in-human testing in 2025, with only preliminary safety data available from a small patient cohort and no published efficacy data. Similarly, HBx-biased mRNA vaccines, such as WGc-0201, have recently entered early clinical evaluation without reported clinical efficacy. Despite these advances, several challenges impede effective therapy, including innate-immune overactivation, HBV genotypic diversity, and the need for rational therapeutic combinations. This review summarizes current preclinical findings and emerging clinical evidence and outlines future strategies toward durable viral control and effective prophylaxis, including self-amplifying or cyclic RNAs, receptor-targeted LNPs, and multimodal therapeutic regimens.
Zhou D, Chen X, Jia M
… +8 more, Liu X, Huang L, Xie Y, Li L, Fu Y, Feng Y, Yang Z, Tang L
Mol Ther Nucleic Acids
· 2026 Mar · PMID 41583555
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Ferroptosis, an iron-dependent form of cell death characterized by lipid peroxidation, has been recognized as a critical mechanism contributing to podocyte injury in diabetic kidney disease (DKD). Although interferon-sti...Ferroptosis, an iron-dependent form of cell death characterized by lipid peroxidation, has been recognized as a critical mechanism contributing to podocyte injury in diabetic kidney disease (DKD). Although interferon-stimulated gene 20 (ISG20) has been identified as a novel regulator of RNA oxidation stress in acute kidney disease, its specific role and mechanism in DKD remain to be elucidated. In this study, we investigated the role of ISG20 in ferroptosis and its potential as a therapeutic target in DKD. We found that ISG20-deficient mice exhibited excessive iron accumulation, increased lipid peroxidation, and markedly higher proteinuria compared to wild-type mice. Nevertheless, the administration of a ferroptosis inhibitor, ferrostatin-1(Fer-1), mitigated kidney injury. Consistent results were observed , where the detrimental effects of ISG20 gene silencing could be mitigated by Fer-1 treatment in mouse podocytes under high glucose (HG) conditions. To further investigate the protective effects of ISG20, we administered two distinct vectors into diabetic mice: an adenovirus vector overexpressing ISG20 and a novel mRNA-loaded lipid nanoparticle (LNP) therapeutic vector expressing ISG20 mRNA. Both approaches effectively mitigated podocyte ferroptosis, alleviated podocyte injury, and reduced proteinuria in DKD. Similar results were also found in mice with adriamycin-induced nephropathy. Overall, this study establishes that ISG20 safeguards podocyte viability by suppressing ferroptosis, which extends ISG20's biological role. These findings suggest that ISG20 may be a potential therapeutic strategy for treating patients with proteinuric kidney disease.