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.
Ozaki A, Kawai A, Akiba R
… +13 more, Okayama S, Ohno N, Kajita K, Masuda T, Yokota S, Ito SI, Peiyan D, Onoue K, Yonemura S, Kondo M, Kurimoto Y, Fu Y, Mandai M
Retinal organoids represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but the capacity of host cone bipolar cells in the primate macula to rewire with transplanted photorece...Retinal organoids represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but the capacity of host cone bipolar cells in the primate macula to rewire with transplanted photoreceptors has not been established. In this study, we transplanted genome-edited ISL1 human retinal organoids lacking ON-bipolar cells into an acute laser-induced macular photoreceptor ablation nonhuman primate model. Using immunohistochemistry, ultrastructural imaging, and focal macular electroretinography (FMERG), we demonstrate that host rod and cone bipolar cells actively extend dendrites toward grafted photoreceptors and form synaptic contacts, with evidence of functional signal transmission in a subset of transplanted eyes. Longitudinal, per-eye analyses revealed that host ON-bipolar responses improved in two of four eyes with ISL1 graft by up to 21.6% and remained stable for up to 2 years post-transplantation. Moreover, OFF-pathway connectivity showed potential progressive maturation, with delayed increase in d-wave after 13 months in one of those eyes. These findings provide the first demonstration of long-term anatomical host-graft synaptic integration in the primate macula, establishing that central cone bipolar circuits retain the capacity for durable rewiring with human stem cell-derived grafts. Our results highlight ISL1 retinal organoids as a promising approach for central vision restoration in macular degeneration.
Deng R, De Oliveira Silva T, Liu X
… +16 more, Fan C, Young Seok H, Wu J, Wickramage I, Guo H, Lin T, Zhang S, Semerci N, Taheri S, Lu YW, Huang ZP, Mably JD, Teng M, Chen H, Diniz GP, Wang DZ
Duchenne Muscular Dystrophy (DMD) is caused by mutations in dystrophin, leading to degeneration and weakness of skeletal and cardiac muscle. Despite great progress in the development of gene replacement therapies, DMD re...Duchenne Muscular Dystrophy (DMD) is caused by mutations in dystrophin, leading to degeneration and weakness of skeletal and cardiac muscle. Despite great progress in the development of gene replacement therapies, DMD remains a devastating disease. In our recent work, we demonstrated that loss of the cardiac Isl1-interacting protein (CIP), which interacts with dystrophin in sarcolemma of cardiomyocytes, accelerates the progression of dystrophic cardiomyopathy and identified Nox4 as one of the downstream mediators of this process. Here, we report that Setanaxib, a Nox1/4 inhibitor, protected the heart of CIP/Mdx double knockout (dKO) mice from heart failure; significantly, this compound also reduced cardiac fibrosis and protected against heart failure in Mdx/Utrn dKO mouse. At a molecular level, Nox1/4 inhibition reduced the expression of genes associated with cardiomyopathy in these animals. Further transcriptomic analysis of the hearts treated with Setanaxib revealed an enrichment in genes associated with fatty acid metabolism, oxidative phosphorylation, and protein binding, while genes related to epithelial-mesenchymal transition were downregulated. Collectively, these findings suggest that oxidative stress plays a key role in development of myocardial fibrosis and heart failure caused by dystrophin deficiency and suggest that Nox1/4 inhibitor treatment could be a novel therapy to treat DMD-associated cardiomyopathy.
Insufficient microwave ablation (iMWA) of hepatocellular carcinoma (HCC) often leads to rapid and aggressive recurrence, and effective intervention remains a formidable challenge. Here, we show that the DNA methylation m...Insufficient microwave ablation (iMWA) of hepatocellular carcinoma (HCC) often leads to rapid and aggressive recurrence, and effective intervention remains a formidable challenge. Here, we show that the DNA methylation machinery is aberrantly activated in residual HCC following iMWA. The heat-responsive stress protein HSP90α collaborates with the deubiquitinase USP7 to stabilize and upregulate DNA methyltransferase DNMT1, thereby transducing the sublethal heat stress input from iMWA into an epigenetic output and elevating DNA methylation levels. Sublethal heat stress-induced DNMT1 upregulation significantly promotes the growth and invasive potential of post-iMWA residual HCC cells, a process that involves disruption of propionate metabolism. Mechanistically, the propionate-metabolizing enzyme ACSS3 in residual HCC is found to be silenced by DNMT1-mediated promoter hypermethylation, impairing propionate-to-propionyl-CoA conversion. Reduction in propionyl-CoA production enhances the activity of the fatty acid β-oxidation (FAO) rate-limiting enzyme CPT1, ultimately stimulating FAO-dependent ATP regeneration. Critically, we developed a liposomal CRISPR/dCas9-based locus-specific demethylation system for precise epigenetic reactivation of ACSS3 and demonstrated its ability to effectively prevent post-iMWA HCC recurrence and metastasis, with more pronounced efficacy when combined with propionate supplementation. Our findings identify a novel DNMT1-driven "epigenetic-metabolic cascade" as a central mechanism promoting HCC recurrence following iMWA, emphasizing the therapeutic potential of ACSS3-targeted methylation editing.
Chronic inflammation mediated by innate immune signaling contributes to retinal degeneration, pathological angiogenesis, and ocular surface damage in age-related macular degeneration (AMD) and dry eye disease (DED). Myel...Chronic inflammation mediated by innate immune signaling contributes to retinal degeneration, pathological angiogenesis, and ocular surface damage in age-related macular degeneration (AMD) and dry eye disease (DED). Myeloid differentiation primary response 88 (MyD88), a central adaptor downstream of Toll-like and IL-1 receptors, integrates NF-κB-dependent cytokine induction and inflammasome priming. In this study, we developed OLX10212, a chemically optimized palmitoyl-conjugated, cell-penetrating asymmetric siRNA (cp-asiRNA) that silences MyD88 without an external delivery vehicle. Following intravitreal administration, OLX10212 achieved broad retinal distribution and sustained MyD88 knockdown in mice and rabbits. In sodium iodate- and Alu RNA-induced geographic atrophy models, OLX10212 preserved retinal architecture and electroretinographic function while suppressing NLRP3-associated transcripts, consistent with the inhibition of inflammasome signaling. A single intravitreal dose reduced lesion volume in laser-induced choroidal neovascularization models in mice and cynomolgus monkeys. Vascular endothelial growth factor (VEGF) expression was also decreased in mice, suggesting that OLX10212 modulates pro-angiogenic inflammatory pathways. Topical administration further ameliorated corneal epithelial injury and inflammatory cytokine expression in a benzalkonium chloride-induced DED model. A phase 1 clinical trial of intravitreal OLX10212 in patients with neovascular AMD is ongoing, and phase 2 studies are planned.
mRNA-lipid nanoparticle (LNP) and adenoviral delivery of vaccines have proven effective during the COVID-19 pandemic. Herein, we explored whether antigen-presenting cell (APC)-targeting of antigens could further enhance...mRNA-lipid nanoparticle (LNP) and adenoviral delivery of vaccines have proven effective during the COVID-19 pandemic. Herein, we explored whether antigen-presenting cell (APC)-targeting of antigens could further enhance immunogenicity of these delivery formats. Experiments were performed in mouse models for malaria (Plasmodium falciparum reticulocytes-binding protein homolog 5, PfRH5, antigen) and influenza (hemagglutinin, HA, antigen). We used genetic constructs encoding bivalent fusion proteins that target antigens to MHC class II (MHCII) molecules on professional APCs. We demonstrate that MHCII-targeted fusion proteins bound to professional APCs and that such APC-targeting increased antibody and T cell responses as well as protection against influenza virus. The results suggest that the injected mRNA-LNP and adenoviral vectors resulted in secretion of fusion proteins that targeted APCs. Employing the APC-targeting principle could enhance efficiency of adenoviral and mRNA-LNP vaccines against a variety of diseases.
Circadian clock-driven orchestration of metabolic pathways in adipocytes is required to maintain nutrient homeostasis, and clock disruption predisposes to the development of obesity and insulin resistance. However, pharm...Circadian clock-driven orchestration of metabolic pathways in adipocytes is required to maintain nutrient homeostasis, and clock disruption predisposes to the development of obesity and insulin resistance. However, pharmacological means to promote beneficial clock actions for metabolic disease interventions remain to be explored. Here we report the identification and characterization of clock-activating molecules with developmental stage-dependent inhibition of adipocyte development and hypertrophy, leading to robust anti-obesity efficacy in vivo. Both chlorhexidine and a new derivative CM002 augmented clock oscillation in adipocytes with induction of core clock components and shortening of period length. Clock activation by these molecules blocked adipogenesis mediated by transcriptional induction of the Wnt signaling pathway, preventing the lineage commitment and terminal differentiation of adipogenic progenitors. In mature adipocytes, CM002 attenuated lipid storage in a clock-dependent manner via inhibition of the lipogenic program. Furthermore, in vivo administration of CM002 in normal or obese mice enhanced clock output in distinct adipose depots with markedly suppressed adipogenic and lipogenic pathways, abrogating adipocyte hypertrophy while promoting insulin sensitivity. Collectively, our study provides the mechanistic underpinning to develop clock activators as a novel avenue for anti-obesity therapy with potential therapeutic benefits against Type II diabetes.
Qiao K, Huang Y, Chua D
… +7 more, Qu J, Liao Q, Wang G, Tan NS, Liao S, Li L, Zhang G
Mol Ther Oncol
· 2026 Sep · PMID 42358378
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Lung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality, driven by extensive tumor heterogeneity and therapy resistance. Conventional models fail to capture the dynamic interactions between cancer cells...Lung adenocarcinoma (LUAD) is a leading cause of cancer-related mortality, driven by extensive tumor heterogeneity and therapy resistance. Conventional models fail to capture the dynamic interactions between cancer cells and the tumor microenvironment (TME), limiting therapeutic advancements. Here, we present a tricomponent organoid platform integrating patient-derived lung cancer tumoroids (LCTs), normal lung organoids (LOs), and field-cancerized fibroblasts to dissect LUAD heterogeneity and treatment resistance mechanisms. This platform uniquely enables the simultaneous investigation of tumor-specific vulnerabilities, stromal contributions to drug resistance, and normal tissue toxicity. Spatial and bulk transcriptomic analyses reveal LUAD-specific gene signatures and highlight cancer-associated fibroblasts (CAFs) as key drivers of a pro-tumorigenic TME, reinforcing tumor survival and recurrence. Functional drug screening demonstrates that LCTs exhibit selective sensitivity to pemetrexed while fibroblasts remain universally resistant, underscoring stromal barriers to therapy. The inclusion of LOs allows direct assessment of drug toxicity on healthy lung tissue, bridging a critical gap in preclinical modeling. By capturing the tumor-stroma interplay with patient specificity, this organoid platform advances precision oncology, paving the way for more effective therapeutic strategies targeting both LUAD cells and the supportive microenvironment.
Choudhury PR, Chakravarti M, Bera S
… +20 more, Arora P, Sultana J, Khan MA, Biradar R, Guha A, Dhar S, Ghosh I, Ganguly N, Lasure A, Kashyap P, Das J, Mandal B, Das P, Sarkar A, Datta D, Nandi S, Alam N, Baral R, Banerjee S, Bose A
Mol Ther Oncol
· 2026 Sep · PMID 42358377
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Early inception of hypercholesterolemia has led to prevalent statin pre-exposure among cancer patients. Furthermore, emerging associations between increased circulating cholesterol, dyslipidemia, and cancer recurrence ha...Early inception of hypercholesterolemia has led to prevalent statin pre-exposure among cancer patients. Furthermore, emerging associations between increased circulating cholesterol, dyslipidemia, and cancer recurrence have spurred the repurposing of statins to combat cancer. However, the mechanistic impact of statin pre-exposure on cancer progression remains unclear. Here, our retrospective study with triple-negative breast cancer (TNBC) patients showed that atorvastatin pre-exposure promotes recurrence-free survival. Atorvastatin pre-exposure in TNBC patients and a preclinical high-fat diet-induced hypercholesterolemic murine tumor model lowered intratumoral 27-hydroxycholesterol (27HC) concentration, as estimated by LC-MS/MS. Within the tumor microenvironment, elevated 27HC interacted with estrogen receptor alpha (ERα) on dendritic cells (DCs), hindering DC maturation, migration, and antigen presentation. Atorvastatin reduced ERα expression on DCs and remediated 27HC-ERα-induced DC dysfunctions, improved DC-mediated T cell priming, thereby facilitating tumor restriction when adoptively transferred to 4T1-bearing NOD/SCID/IL2Rγ mice. Targeted intratumoral knockdown of (rate-determining enzyme for 27HC production) alongside atorvastatin pre-exposure uplifted cDC1 functionality. Atorvastatin pre-treatment exerts its action by reducing the availability of intratumoral 27HC and expression of ERα on DCs, which eventually enhances DC functionality and restores their efficiency to prime anti-tumor CD8 T cell responses. Therefore, atorvastatin pre-exposure adjunct to 27HC-ERα inhibition may serve as an effective immunotherapeutic intervention in TNBC.
Wang J, Fang Z, Wang J
… +7 more, Han X, Feng F, Zhang R, Han B, Sun G, Zhang Y, Ni S
Mol Ther Oncol
· 2026 Sep · PMID 42358376
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Pituitary corticotroph tumors are the primary cause of Cushing's disease. Even after transsphenoidal surgery, residual tumor cells frequently persist, leading to a recurrence rate of approximately 20%. However, safe and...Pituitary corticotroph tumors are the primary cause of Cushing's disease. Even after transsphenoidal surgery, residual tumor cells frequently persist, leading to a recurrence rate of approximately 20%. However, safe and effective adjuvant treatments for these residual lesions are lacking. In this study, we analyzed single-cell RNA sequencing data to characterize the transcriptional profile of corticotroph tumors. On the basis of the high activity of the promoter and its transcriptional regulators, we designed an adeno-associated virus vector carrying the pro-apoptotic gene , with expression controlled by the promoter for lineage-specific targeting. We also developed an injectable, self-assembling peptide hydrogel for the sustained local delivery of the gene and tested its therapeutic efficacy in subcutaneous and post-resection mouse models. Results showed the promoter drove selective expression in corticotroph tumor cells, inducing mitochondrial membrane potential loss and apoptosis . , the virus suppressed tumor growth and lessened systemic tumor effects; when delivered via the hydrogel into surgical cavities, it completely eliminated residual tumors without detectable toxicity in major organs. This targeted, localized strategy, thus has translational potential as an adjuvant therapy for reducing the recurrence of Cushing's disease.
Epilepsy arises from disruption of excitation-inhibition (E/I) balance, typically due to excessive excitatory activity. Despite available therapies, a substantial proportion of patients remain treatment resistant. Enhanc...Epilepsy arises from disruption of excitation-inhibition (E/I) balance, typically due to excessive excitatory activity. Despite available therapies, a substantial proportion of patients remain treatment resistant. Enhancing inhibitory neuron activity via gene therapy can restore E/I balance and may therefore provide a therapeutic strategy for treatment-resistant epilepsy. Here, we developed a compact 410-bp glutamic acid decarboxylase 67 promoter (cmGAD67) that enables strong, selective transgene expression in inhibitory neurons while preserving adeno-associated virus (AAV) packaging capacity. Systemic delivery of AAV vectors carrying cmGAD67 preferentially targeted parvalbumin interneurons and enabled efficient circuit modulation. To evaluate therapeutic potential, we expressed glutamic acid decarboxylase 65 (GAD65) under the control of cmGAD67 (AAV-GAD65). AAV-GAD65 suppressed abnormal delta oscillations, reduced seizure-like activity, normalized anxiety-like behavior, and improved survival in seizure models. Biochemical analyses confirmed increased GABA levels in the cortex and hippocampus, linking functional improvements to enhanced inhibitory neurotransmitter synthesis. Together, these findings establish the cmGAD67 promoter as a versatile platform for inhibitory neuron-targeted AAV gene delivery and identify AAV-GAD65 as a promising strategy for seizure control and disorders associated with E/I imbalance.