Norman AO, Scaramella C, Biezonski D
… +10 more, Hector RD, Varallo A, Sahni A, Farooq N, Burstein SR, Benito J, Razak KA, Selfridge J, Cobb S, Ethell IM
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42389555
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Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by CGG trinucleotide repeat expansion in the () gene and the resulting loss of fragile X messenger ribonucleoprotein (FMRP). Gene therapy using recombinan...Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by CGG trinucleotide repeat expansion in the () gene and the resulting loss of fragile X messenger ribonucleoprotein (FMRP). Gene therapy using recombinant adeno-associated virus (AAV) to restore FMRP expression, particularly in the brain, is a promising therapeutic strategy targeting the underlying cause of FXS. We examined the impact of AAV serotype 9 (AAV9)-mediated expression of a brain-abundant human FMRP isoform (isoform 7) driven by a fragment of the human FMR1 promoter on circuit and behavioral dysfunctions in the male knockout (KO) mouse, FXS model. Following intracerebroventricular (i.c.v.) injection of AAV9-NG276 into neonatal KO mice at a low (1e11 vg/animal) or high (3e11 vg/animal) dose, we assessed cortical phenotypes using electroencephalography (EEG) recordings and behavioral testing. High-dose AAV9-NG276 normalized baseline gamma power, improved sound-evoked responses, and reduced background neural activity. Analysis of behavioral deficits in adult KO mice showed that high-dose neonatal AAV9-NG276 delivery normalized exploratory behaviors, social preference, and probabilistic reversal learning. Thus, early AAV-mediated delivery of human isoform 7 ameliorates cortical dysfunction and behavioral deficits in a murine FXS model and suggests that widespread cortical biodistribution is required for therapeutic benefit.
Vermeer C, Venema RR, Birnie E
… +4 more, Bolling MC, Knoers N, Bremer J, van den Akker PC
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381708
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Antisense oligonucleotides (ASOs) are increasingly being used as a platform to target various diseases. Currently, there are 13 USFDA- or EMA-approved ASO therapies. Adverse events resulting from ASO treatments are asses...Antisense oligonucleotides (ASOs) are increasingly being used as a platform to target various diseases. Currently, there are 13 USFDA- or EMA-approved ASO therapies. Adverse events resulting from ASO treatments are assessed on a per drug basis, but for many ongoing ASO developments targeting N-of-1 mutations, conventional randomized clinical trials to assess safety cannot be performed. Here, we conducted a systematic review and meta-analysis of adverse events in clinical studies conducting trials of ASO therapies. The study aims to provide better insight into the safety aspects of ASO design choices and to summarize knowledge of the safety aspects of ASOs so that we can better inform researchers and physicians of possible adverse events that can occur during (N-of-1) ASO treatments. Our results provide a list of common adverse events and event rates obtained from pooled data from both approved and non-approved ASO drugs, and we include recommendations that can provide insights into the nature of ASO-induced adverse events in future trials.
Ju S, Lee JH, Yang J
… +10 more, Jeong TY, Choi CG, Lee Y, Cha S, Park J, Kim SP, Kim S, Seong JK, Lee H, Kim K
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381707
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Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome and epigenome editing hold great promise for precision therapy, yet their clinical translation is limited by delivery challenges. Here, we p...Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome and epigenome editing hold great promise for precision therapy, yet their clinical translation is limited by delivery challenges. Here, we present a transient, DNA-free virus-like particle (VLP) platform for the delivery of CRISPR ribonucleoprotein (RNP) across diverse cancer applications. VLP-mediated genome editing achieved high editing efficiency and broad applicability across multiple cancer types. Notably, VLP-mediated mutation-selective targeting of oncogenic alleles induced potent on-target activity and high-fidelity mutation-selective effects in cancer cells, with minimal effects on normal cells. Beyond genome editing, VLP delivery of the epigenome editor CRISPRoff effectively suppressed cancer cell growth and induced stable gene silencing maintained for over 120 days. Moreover, it provided a strategy to overcome knockout escape, a key limitation of conventional genome editing. In comparison with DNA vector-based delivery systems, VLPs enabled transient expression while achieving lower off-target activity and higher efficiency. Furthermore, the VLP platform exhibited a favorable safety profile, characterized by transient exposure and no hepatotoxicity. In a mouse xenograft model, the platform successfully suppressed tumor growth via precision targeting. Collectively, these findings establish VLP-mediated CRISPR delivery as a versatile and safe integrated delivery platform, providing a comprehensive modality for both genome and epigenome editing in precision therapeutics.
Karimi S, Dehpour AR, Hadjighassem M
… +5 more, Khajehnasiri A, Yekta RA, Aghaei M, Majedi H, Hosseindoost S
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381706
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Glioblastoma multiforme (GBM) is the most malignant type of primary brain tumor. Its clinical management is challenging due to its heterogeneity, highly malignant nature, and insensitivity to standard treatments. While c...Glioblastoma multiforme (GBM) is the most malignant type of primary brain tumor. Its clinical management is challenging due to its heterogeneity, highly malignant nature, and insensitivity to standard treatments. While current strategies for GBM treatments are based on inducing apoptosis in GBM cells, some GBM tumors showed resistance to this type of cell death. Recent evidence indicates that pyroptosis is a novel, promising therapeutic method for overcoming tumor cells' resistance to cancer treatment. This inflammatory programmed cell death type is mediated by the cleavage of gasdermin proteins. Based on the evidence, inducing pyroptosis is negatively associated with GBM growth and development; the exact molecular mechanisms and the signaling pathways underlying pyroptosis are not fully understood. This review presents the different pathways of pyroptosis and its role in GBM growth regulating and illustrates various drugs and components that modulate pyroptosis in GBM tumors. It also investigates the regulatory roles of noncoding RNAs in pyroptosis modulation in GBM tumors, providing promising therapeutic approaches that target pyroptosis as a novel strategy for GBM treatment.
Kunrade L, Pleiko K, Sproge L
… +7 more, Goluba K, Pantelejevs T, Wright NT, Akopjana I, Bogans J, Tars K, Riekstina U
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381705
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Malaria remains a major global health concern. The development of point-of-care diagnostic aptasensors could improve malaria detection, patient management, and eradication efforts. We selected and validated single-strand...Malaria remains a major global health concern. The development of point-of-care diagnostic aptasensors could improve malaria detection, patient management, and eradication efforts. We selected and validated single-stranded DNA aptamers against lactate dehydrogenase (Pf-LDH), using systematic evolution of ligands by exponential enrichment (SELEX). The five most abundant aptamers were validated with an enzyme-linked oligonucleotide assay (ELONA). The aptamer binding affinities to Pf-LDH varied between 0.35 and 3.08 nM. Truncation of aptamers enhanced binding affinities in most cases, with all truncated aptamers showing comparable or improved dissociation constants (Kd) relative to full-length aptamers, except Pf-LDH5t, which lost its binding affinity. Selectivity analysis against LDH from , , and showed that Pf-LDH1 and Pf-LDH1t specifically recognized Pf-LDH (Kd = 2.74 ± 0.28 and 1.13 ± 0.26 nM, respectively), while Pf-LDH2, Pf-LDH3, Pf-LDH4, and their truncated forms, as well as Pf-LDH5, recognized both Pf-LDH and LDH (Po-LDH). Crystallography of Pf-LDH4t revealed a binding site overlapping that of the previously characterized aptamer 2008 but involving distinct molecular interactions. These findings demonstrate the structural versatility of LDH-specific aptamers and provide a novel Pf- and Po-LDH co-specific aptamer panel with strong potential for next-generation aptamer-based malaria diagnostics.
Samuels TN, Kakadia JH, Ward C
… +11 more, Abdullatif F, Mendes MI, Smith DE, Heilmann R, Wu F, Swiercz JM, Salamons GS, Hoffman K, Gebregiworgis T, O'Donoghue P, Heinemann IU
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381704
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Pathogenic alleles in the cytoplasmic asparaginyl-tRNA synthetase (NARS1) are associated with infant- and juvenile-onset disease, with no current disease-specific treatments. We developed a tractable human cell system to...Pathogenic alleles in the cytoplasmic asparaginyl-tRNA synthetase (NARS1) are associated with infant- and juvenile-onset disease, with no current disease-specific treatments. We developed a tractable human cell system to study disease-causing NARS1 alleles that can be adapted to investigate NARS1 and other aminoacyl-tRNA synthetase (ARS) alleles. We found that two dominant NARS1 nonsense alleles, R534X and R522X, cause a cytotoxic phenotype and elicit the integrated stress response (ISR). Proteomic and phenotypic changes were rescued by asparagine supplementation in the human cell model. Asparagine supplementation completely restored cell proliferation defects in patient-derived fibroblasts and prevented activation of the ISR. We also tested therapeutic cognate transfer RNA (tRNA) supplementation, which reduced the cytotoxicity of pathogenic NARS1 alleles but did not ameliorate activation of the ISR. A general control nonderepressible 2 (GCN2) inhibitor suppressed ISR activation and reduced cytotoxicity but did not restore changes to the proteome caused by the NARS1 nonsense alleles. The data reveal molecular and cellular defects caused by premature termination codons in NARS1 alleles. Our data also indicate asparagine supplementation as a feasible therapeutic approach to address the underlying cause of NARS1 disease, a rare disease for which currently no treatment is available.
Li CG, Sun Q, Tian T
… +8 more, Wu ZC, Xie YC, Liu XQ, Zhou ZR, Huang LX, Deng XH, Yu SL, Fu QL
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42381703
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Previous studies have demonstrated that miR-146a-5p can effectively inhibit the function of group 2 innate lymphoid cells (ILC2s), which play a pivotal role in the initiation of allergic airway inflammation (AAI), thereb...Previous studies have demonstrated that miR-146a-5p can effectively inhibit the function of group 2 innate lymphoid cells (ILC2s), which play a pivotal role in the initiation of allergic airway inflammation (AAI), thereby alleviating AAI. However, the application of miR-146a-5p faces challenges such as susceptibility to degradation and difficulty in crossing the cellular membrane. Extracellular vesicles (EVs), which naturally transport proteins and nucleic acids, have been proposed as promising RNA delivery vehicles. EVs can protect microRNAs (miRNAs) from degradation and facilitate their penetration across cellular membranes. However, the lack of scalable sources of EVs remains a challenge for their mass production, particularly in clinical application. Bovine milk has emerged as an advantageous source for the large-scale production of EVs because of its low cost and easy accessibility. Moreover, bovine milk-derived EVs (mEVs) have shown low immunogenicity and outstanding cross-species biosafety. In this study, we present the manufacturing methods and immune regulatory effects of mEVs loaded with miR-146a-5p (miR146a-5p-mEVs). Our findings indicated that mEVs exhibited outstanding biosafety and did not elicit significant systemic toxicity. Importantly, miR146a-5p-mEVs exhibited significant immunoregulatory functions in AAI, suggesting that miR146a-5p-mEVs could serve as a novel strategy for the treatment of allergic airway disease.
Phillips MA, Lei L, Kulhankova K
… +6 more, Gingerich MC, Loza LM, Vu A, He B, Thornell IM, McCray PB
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42376650
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Parainfluenza virus 5 (PIV5)-derived amplifying virus-like particles (PIV5-AVLPs) are an efficient gene delivery platform with broad cell tropism. To investigate the feasibility of using PIV5-AVLPs for cystic fibrosis (C...Parainfluenza virus 5 (PIV5)-derived amplifying virus-like particles (PIV5-AVLPs) are an efficient gene delivery platform with broad cell tropism. To investigate the feasibility of using PIV5-AVLPs for cystic fibrosis (CF) gene therapy, we generated AVLPs expressing enhanced green fluorescent protein (AVLP-eGFP) or a codon-optimized human cystic fibrosis transmembrane conductance regulator () coding sequence and an reporter (AVLP-CFTR). We examined the transduction efficiency and persistence of transgene expression of AVLP-eGFP in primary cultures of non-CF and CF human airway epithelia (HAE). When applied to the apical surface of HAE, the AVLP-eGFP mainly transduced ciliated epithelial cells, with lesser targeting to secretory and basal cells. Reporter transgene expression gradually diminished over a 1-month time course. Transducing approximately 15% of CF airway epithelial cells with AVLP-CFTR was sufficient to restore CFTR-dependent short-circuit current to levels similar to non-CF epithelia. Our results demonstrate that PIV5-AVLPs delivered to the apical side of HAE efficiently transduce sufficient epithelial cells to restore functional CFTR expression. PIV5-based AVLPs provide a versatile platform for the delivery of a variety of genetic cargoes to the respiratory tract.
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42367247
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Innate immunity plays a vital role in subverting viral infections via dynamic regulatory networks that calibrate antiviral response and prevent excessive inflammation. These networks are coordinately regulated by coding...Innate immunity plays a vital role in subverting viral infections via dynamic regulatory networks that calibrate antiviral response and prevent excessive inflammation. These networks are coordinately regulated by coding and non-coding elements of the genome. The non-coding elements, particularly circular RNAs (circRNAs), have emerged as a key modulator of various biological processes, yet their precise function in innate immune response during influenza A virus (IAV) infection remains insufficiently defined. Here, we identified circCPSF6, a conserved circRNA that remains suppressed during IAV infection, and . Functional analyses revealed that circCPSF6 acts as an antiviral regulator through a dual regulatory mechanism of RNA-RNA and RNA-protein interactions. circCPSF6 sponges proviral miR-665, thereby alleviating the repression of key antiviral mediators such as MyD88, STAT2, and IKKε via RNA-RNA interactions. At RNA-protein level, circCPSF6 directly interacts with the RNA-binding protein PCBP2, which is a negative regulator of IPS-1, and sustains IPS-1-mediated antiviral signaling. These interactions enhance cytokines and interferon-stimulated genes expression, which strengthen antiviral response. Our findings report circCPSF6 as a crucial regulator of host innate immune response and highlight circRNA-mediated networks as potential therapeutic targets in viral infection.
Kwon JE, Kim YJ, Kim SH
… +9 more, Kwon OS, Jang HK, Choi YM, Na HJ, Han N, Chun Y, Han JW, Park TK, Chung KS
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42367246
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Inherited retinal diseases (IRDs) are major causes of vision loss and often associated with the degeneration of retinal neurons, such as photoreceptors. Adeno-associated virus (AAV) vectors hold therapeutic potential for...Inherited retinal diseases (IRDs) are major causes of vision loss and often associated with the degeneration of retinal neurons, such as photoreceptors. Adeno-associated virus (AAV) vectors hold therapeutic potential for IRDs; however, their off-target expression has prompted the development of refined engineering strategies to enhance cell-type preference without compromising transgene expression. Here, we present G5mP, a streamlined 315-bp synthetic promoter-enhancer construct designed for robust retinal gene expression. G5mP integrates three components: G5, a G protein-coupled receptor kinase 1-derived enhancer; mP, a minimal promoter from phosphodiesterase 6B; and a 5'-untranslated region (UTR) derived from retinoschisin 1 (RS1). Compared with the clinically used 742-bp RIR cassette-comprising the RS1 promoter, interphotoreceptor retinoid-binding protein enhancer, and RS1-derived 5'-UTR-G5mP drove stronger overall retinal expression and showed enhanced activity within photoreceptor cells in retinal cell lines, human retinal organoids, and mouse retina without inducing detectable cytotoxicity . Notably, across retinal cell lines, human retinal organoids, and mouse retina, G5mP induced more robust and distinct photoreceptor-preferential transgene expression than the ubiquitous CAG promoter did. These results highlight its potential as a compact and efficient regulatory element suitable for AAV-mediated gene delivery across retinal cell types, including the effective targeting of photoreceptors.
Penso-Dolfin L, Hauptmann J, Was N
… +3 more, Hehne V, Lindholm MW, Morrison E
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42367245
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Understanding off-target risk is a key element of siRNA design, but predicting hybridization-dependent off targets using alignments is limited by an incomplete understanding of when and how low-homology and seed-mediate...Understanding off-target risk is a key element of siRNA design, but predicting hybridization-dependent off targets using alignments is limited by an incomplete understanding of when and how low-homology and seed-mediated off targets lead to functional downregulation. Here we explore the biological limits of these interactions by measuring the off-target profiles of six "promiscuous" siRNAs with intentional off targets on both an mRNA and protein level, using a cell system featuring catalytically inactive Argonaute 2 (Ago2) to distinguish cleavage-dependent and -independent mechanisms of downregulation. By probing the alignments of the observed off targets with the siRNA sequences, several intriguing patterns emerge; multiple G:U wobbles and target bulges, for example, are more frequently seen than bulges in the siRNA guide strand. Further, a clear sequence-dependence is observed for the overall extent of cleavage-independent downregulation of off targets; this may be explained by impaired unwinding and loading of the fully ribose-modified siRNA into RNA-induced silencing complex (RISC), suggesting an additional nuance to seed-mediated off-target risk associated with other Argonaute proteins. Taken together, this study sheds new light on potential siRNA off-target risk and may help guide and improve the prediction of siRNA:off-target interactions.
Zaborowski MP, Lai CP, Sammarco A
… +15 more, György B, Zhang X, Lee K, Na YJ, Ceppi L, Breyne K, Champagne EM, Cardini J, Wesselhoeft RA, Iwanicki M, Weissleder R, Birrer MJ, Lee H, Tannous BA, Breakefield XO
Mol Ther Nucleic Acids
· 2026 Sep · PMID 42367244
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Circular RNAs (circRNAs) form during splicing as a closed ring and are more resistant to degradation than linear RNAs. Cells release circRNAs within membrane-bound extracellular vesicles (EVs), which transport DNA, RNA,...Circular RNAs (circRNAs) form during splicing as a closed ring and are more resistant to degradation than linear RNAs. Cells release circRNAs within membrane-bound extracellular vesicles (EVs), which transport DNA, RNA, and proteins. Whether circRNAs in EVs can be used as biomarkers remains uncertain. We aimed to identify mutant circRNAs in EVs to develop a new biomarker approach. We demonstrated that ovarian cancer cells release and enrich circRNAs in EVs. We detected mutant circular transcripts from , , and genes in EVs from cancer cells. We noticed that circRNAs in EVs undergo intense rolling circle amplification (RCA), producing multiple copies of the mutant sequence. We revealed that increased reverse transcription time and extended PCR elongation time intensified RCA. Using our RCA protocol, we detected mutant circRNAs derived from the gene in EVs from the plasma of ovarian cancer-bearing animals. In this study, we present a new model of mutant circRNA amplification from EVs, which may provide a basis for a diagnostic test for ovarian cancer patients.
Anwar S, Hay S, Moriyama H
… +3 more, Mir F, Maruyama R, Yokota T
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42327837
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Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder caused by gain-of-function mutations in , most commonly c.617G>A (R206H), leading to progressive heterotopic ossification. In this study, we developed...Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare disorder caused by gain-of-function mutations in , most commonly c.617G>A (R206H), leading to progressive heterotopic ossification. In this study, we developed novel antisense gapmers selectively targeting the mutant transcript while sparing the wild-type allele. We engineered locked nucleic acid (LNA) and 2'-O-methoxyethyl (MOE) gapmers incorporating a single 2'-O-methyl (2'OMe) modification at gap position 2. This is hypothesized to synergize with the wild-type sequence mismatch to restrict RNase H1 cleavage, limiting wild-type degradation while preserving mutant target engagement. In FOP patient-derived fibroblasts carrying the endogenous mutation and in murine-derived C2C12 cells ectopically expressing constructs, 2'OMe-modified gapmers demonstrated robust and preferential suppression of at both RNA and protein levels. Gapmer treatment also reduced osteogenic differentiation, as shown by decreased alkaline phosphatase and Alizarin Red S staining, and lower expression of osteogenic markers. In wild-type mice, 2'OMe modification was associated with higher apparent gapmer levels in skeletal muscle and tendon and lower hepatic and renal stress marker readouts. These findings provide preliminary proof-of-concept that a single-base chemical modification can modulate allele selectivity and biodistribution of gapmers targeting . Further studies in disease-relevant FOP models will be needed to establish therapeutic efficacy and long-term safety.
Naoe Y, Fujimoto N, Makita Y
… +3 more, Li D, Inukai N, Hotta A
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306087
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Ensuring the safety of CRISPR-Cas9 genome editing requires comprehensive assessment of off-target mutagenesis, particularly for therapeutic applications under regulatory review. DNA-free lipid nanoparticle (LNP) delivery...Ensuring the safety of CRISPR-Cas9 genome editing requires comprehensive assessment of off-target mutagenesis, particularly for therapeutic applications under regulatory review. DNA-free lipid nanoparticle (LNP) delivery is expected to minimize insertional risks compared to adeno-associated virus (AAV) vectors, but experimental validation has been limited. Here, on-target amplicon sequencing in mouse muscle demonstrated insertions largely derived from host genomic sequences, with no detectable AAV or transgene integration. Benchmarking 13 prediction tools identified Cas-OFFinder as the most sensitive, though precision remained low, and correlation with CIRCLE-seq data was modest. To strengthen off-target detection, we employed karyotypically normal human iPSCs and developed an "indel cluster" method using high-depth whole-genome sequencing, enabling discrimination between clustered genome editing-induced indels and background variants. Integration of predictions, CIRCLE-seq cleavage sites, and WGS clusters yielded 11 high-confidence off-target candidates, predominantly associated with one gRNA. While sensitivity was maximized, reproducibility across conditions remained limited, and many candidate sites overlapped repetitive or low-mappability regions. Our multilayered framework demonstrates both the utility and current limitations of off-target risk assessment, providing a practical foundation for the safety evaluation of genome editing therapies.
Rezek FO, Sanchez-Pintado B, Eden ER
… +8 more, Corral-Serrano JC, Aychoua N, Webster AR, de Guimarães TAC, Carr AF, Michaelides M, Cheetham ME, van der Spuy J
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306086
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Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033C>T, p.(Arg345Trp) variant in...Doyne honeycomb retinal dystrophy is an incurable juvenile macular dystrophy that leads to visual impairment by early to mid-adulthood. It is an autosomal dominant disorder caused by a c.1033C>T, p.(Arg345Trp) variant in , and is characterized by the early onset of extracellular deposition of drusen between the retinal pigment epithelium and underlying Bruch's membrane. In this study, we developed an antisense oligonucleotide approach to target . We reprogrammed patient-derived renal epithelial cells to induced pluripotent stem cells, followed by directed differentiation to retinal pigment epithelium and compared the phenotype to gene-corrected and knockout patient-derived retinal pigment epithelium. In the patient-derived disease model, remodeling of the extracellular matrix (ECM) occurred with progressive accumulation of the drusen-associated proteins apolipoprotein E and collagen IV, in addition to the intracellular accumulation and extracellular deposition of lipids. We developed an allele-specific antisense oligonucleotide which specifically and effectively promoted the clearance of the c.1033C>T transcript in the patient-derived disease model following assisted or gymnotic delivery. Gymnotic delivery rescued remodeling of the ECM, reduced intracellular accumulation of lipids, and cleared extracellular deposits, even after the onset of the disease phenotype, suggesting that this could be a practical and effective therapeutic approach.
Murmann AE, Ebadi M, Patel M
… +14 more, Ewe A, Barajas S, Xiao S, Ko MJ, Lee S, Cai W, Paudel B, Sun L, Bartom ET, Kocherginsky M, Liu Y, Kim DH, Aigner A, Peter ME
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306085
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RNA interference (RNAi) regulates gene expression through small RNAs that act via Argonaute-containing RNA-induced silencing complexes (RISCs). We previously found that short RNAs with G-rich 6mer seeds (e.g., GGGGGC and...RNA interference (RNAi) regulates gene expression through small RNAs that act via Argonaute-containing RNA-induced silencing complexes (RISCs). We previously found that short RNAs with G-rich 6mer seeds (e.g., GGGGGC and G5C) can kill cells by targeting C-rich 3' UTR seed matches in essential survival genes (SGs), a mechanism termed death induced by survival gene elimination (DISE). To assess therapeutic potential, we systemically delivered two DISE-inducing sRNAs, sG5C and sCAG (based on CAG trinucleotide repeats), using lipopolyplexes (LPPs) composed of low-molecular-weight polyethyleneimines and lipids. In mouse ovarian and prostate cancer models and a rat hepatocellular carcinoma model, LPP-delivered small RNAs (sRNAs) markedly reduced or eliminated tumors without harming normal tissues. Predicted SG targets were engaged in tumors. Transcriptomic analyses across 10 major human cancers showed that many sG5C-targeted SGs are consistently upregulated in tumors and increase with stage, revealing a therapeutic window. These results support LPP-delivered DISE-inducing sRNAs as a promising pan-cancer therapy.
Maximov VV, Shebanov N, Nikitchina N
… +5 more, Rapoport R, Maor Y, Tarassov I, Pines O, Entelis N
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306084
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Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on...Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases, lacking efficient therapies. Direct correction of mtDNA mutations may offer a cure for such diseases. We propose a novel strategy based on double-stranded DNA (dsDNA) oligonucleotide delivery into mitochondria and intrinsic microhomology-mediated end joining (MMEJ) for mtDNA editing. This strategy enables the introduction of multiple predefined nucleotide changes in mtDNA. For this, the presence of MMEJ activity in the human mitochondrial lysates was confirmed. Forty-nine bp DNA oligonucleotide duplexes, fused to an RNA hairpin previously identified as a mitochondrial import signal, were delivered into the mitochondria of cultured human cells. Delivery of these donor dsDNA molecules, homologous to an site of mtDNA and bearing designed nucleotide changes, led to a low but statistically significant introduction of the intended nucleotide changes into mtDNA. Donor dsDNA delivery combined with the CRISPR-mito-AsCas12a system also resulted in a statistically significant number of an expected concomitant change of five nucleotides distributed across a 16 nt site of the mitochondrial genome. The proposed strategy may become an efficient mtDNA editing tool suitable for the correction of near-homoplasmic mutations, such as Leber's hereditary optic neuropathy (LHON)-associated mutations in the gene of mtDNA.
Yang Y, Qiu Y, Wang K
… +5 more, Liu Y, Sanyal G, Whitford PC, Rouhanifard SH, Xie W
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306083
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mRNA lipid nanoparticle (mRNA-LNP) technology has emerged as a cornerstone in vaccine development due to its high delivery efficiency, molecular stability, and favorable safety profile. However, rapid and reliable potenc...mRNA lipid nanoparticle (mRNA-LNP) technology has emerged as a cornerstone in vaccine development due to its high delivery efficiency, molecular stability, and favorable safety profile. However, rapid and reliable potency assessment remains challenging because of limited mechanistic understanding of delivery processes and sparse experimental data. To address these gaps, we introduce a mechanism-informed, multi-scale kinetic modularized modeling framework that quantitatively captures the coupled dynamics of mRNA delivery across nanoparticle, cellular, and macroscopic scales. The model incorporates variability in LNP-cell interactions and integrates key determinants, such as dosage, LNP and cell size distributions, cell proliferation, and membrane properties-factors that critically shape delivery efficiency and response heterogeneity. Its cell-based architecture and modular design enable adaptability to diverse delivery systems and physiological contexts. By leveraging advanced multi-omics assays, including single-molecule fluorescent hybridization (smFISH) for single-cell resolution of mRNA and protein expression, our framework provides mechanistically grounded modeling and robust prediction of therapeutic potency, offering a powerful platform for optimizing mRNA-based interventions.
Philosof NR, Walsh APG, Xu B
… +14 more, Watson AMD, Ren S, Hann L, Nathaniel B, Nguyen A, Liu H, Shi P, Bongcaron V, Nguyen H, Noonan J, Avci-Adali M, Peter K, Vidallon MLP, Wang X
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42306082
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Messenger RNA (mRNA) therapeutics offer a promising strategy for treating inflammatory disease by enabling transient local expression of therapeutic proteins. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) hydro...Messenger RNA (mRNA) therapeutics offer a promising strategy for treating inflammatory disease by enabling transient local expression of therapeutic proteins. CD39 (ectonucleoside triphosphate diphosphohydrolase-1) hydrolyzes pro-inflammatory ATP and ADP and plays a central role in localized immune regulation. We developed cationic nanoliposomes (NLps) for the delivery of CD39 mRNA and evaluated them in a murine model of localized inflammation induced by lipopolysaccharide and matrigel. Biodistribution studies showed substantial retention within the Matrigel matrix, with limited systemic distribution at 24 h. CD39 mRNA-NLps significantly reduced cellular infiltration at day 5, with decreased monocyte and macrophage staining via histological analysis. qPCR confirmed sustained local CD39 mRNA, while flow cytometry demonstrated increased CD39 protein staining in matrigel-derived immune cells, and phosphate release assays showed functional ectonucleotidase activity at the inflammatory site. Interleukin (IL)-6 levels were slightly reduced in matrigel extracts following treatment, supporting suppression of local inflammation. Hemocompatibility was confirmed using in human blood and in treated mice, with liver enzymes and histological analysis supporting formulation's safety and favorable tolerability. These findings establish CD39 mRNA-NLps as a safe, effective strategy for a spatially confined mRNA-based approach to regulate local inflammation.
Xia R, Zhang J, Wang X
… +7 more, Ma J, Li J, Su J, Wong P, Huang D, Meng J, Song B
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42293252
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8-Oxoguanine (oG) is an oxidative RNA modification that plays critical roles in cellular processes including stress responses and RNA integrity regulation. Notably, emerging evidence has implicated oG modifications in ca...8-Oxoguanine (oG) is an oxidative RNA modification that plays critical roles in cellular processes including stress responses and RNA integrity regulation. Notably, emerging evidence has implicated oG modifications in cancer progression and development. However, research progress is limited by the lack of efficient high-throughput detection methods and computational tools for analyzing sequence distribution and regulatory mechanisms. Here, we present OBOE (foundation-model-based prediction of 8-oxoguanine sites), the first computational framework for oG site prediction, developed by fine-tuning multiple pre-trained language models (DNABERT, RNABERT, BERT, and BioBERT). Benchmarking experiments demonstrated that OBOE significantly outperforms conventional machine learning methods, validating its superior capability in capturing RNA sequence features and modification patterns. Furthermore, we applied TF-MoDISCo to extract biologically meaningful motifs from model attributions, followed by validation of sequence similarity and enrichment using STREME and TOMTOM analysis. We identified recurrent GC-rich sequence motifs and CTC-like patterns associated with oG modifications, suggesting potential -regulatory elements involved in oxidative stress responses. To facilitate model accessibility, we implemented two community resources: (1) a user-friendly web platform for online oG prediction and (2) freely available source code and processed datasets.