CD8+ T cell targeting of the conserved SARS CoV2 RNA-dependent RNA polymerase (RdRp, nsp12) may offer a means for cross-protection against diverse coronavirus strains. Native RdRp, however, displays primary sequence immu...CD8+ T cell targeting of the conserved SARS CoV2 RNA-dependent RNA polymerase (RdRp, nsp12) may offer a means for cross-protection against diverse coronavirus strains. Native RdRp, however, displays primary sequence immunoevasion (PSI) that inhibits in cis RdRp-derived peptide presentation on major histocompatibility complex class I. We show that this is related to the highly ordered nature of RdRp, which can be reversed by dividing RdRp into two intrinsically disordered, complementary sub-fragments (RdRp). RdRp possesses nearly all of the native peptide epitopes found in the full-length protein. Using a syngeneic MC38 cancer cell mouse model, cellular RdRp expression suppressed tumor growth and markedly increased specific tumor-infiltrating effector T lymphocytes as measured by tetramer assays. mRNA vaccination with WA-1 strain RdRp elicited more potent CD8+ T cell responses than native WA-1 strain RdRp mRNA but did not significantly prevent acute Omicron SARS CoV2 early infection in transgenic hamster challenge studies. These findings suggest highly ordered protein evolution may be an unrecognized PSI mechanism used by viruses to evade T cell recognition. Increasing antigen intrinsic disorder by protein subfragmentation may improve antigenicity for antiviral or antitumor vaccine candidates.
Ligocki AP, Sorets AG, Abdulrahman AM
… +11 more, Francini N, Park JC, Lee JH, Ford WT, Lyons SM, Fritsch EL, Lamantia ZE, Christov PP, Tehrani EE, Duvall CL, Lippmann ES
Aging is the primary risk factor for chronic neurodegenerative diseases and is associated with alterations to cerebrospinal fluid (CSF) flow and clearance. CSF delivery is currently the most clinically advanced route of...Aging is the primary risk factor for chronic neurodegenerative diseases and is associated with alterations to cerebrospinal fluid (CSF) flow and clearance. CSF delivery is currently the most clinically advanced route of administration for oligonucleotide therapeutics, but it remains poorly understood how aging, which is rarely incorporated into clinical trials, impacts biodistribution, gene silencing activity, and potential toxicity of these compounds. Here, we evaluated a lipid-siRNA conjugate (L2-siRNA) for potential age-related changes to CSF-mediated delivery, mRNA silencing, and safety. In the context of aging, we also studied how conjugate chemistry and siRNA structure impact delivery and activity by comparing L2-siRNA to an alternative lipid-siRNA conjugate design. We determined that age had minimal impact on the performance of L2-siRNA and that conjugates exhibit better activity when each lipid is paired with the siRNA structure and chemistry for which it was initially optimized. Collectively, these results provide valuable insight into siRNA conjugate biodistribution and activity in the central nervous system in the context of aging and further establish the performance of L2-siRNA under conditions relevant to the treatment of neurodegenerative diseases.
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.
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.
Mason NJ, Gnanandarajah J, MaloneyHuss M
… +8 more, Reetz J, Agnello KA, Gray F, Engiles J, Olenick L, Hart A, Thamm DH, Paterson Y
Mol Ther Oncol
· 2026 Jun · PMID 42291138
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Ionizing radiation acts synergistically with immunotherapy by inducing immunogenic cell death and modulating the tumor microenvironment, promoting anti-tumor immune responses. In this pilot study, the safety and effectiv...Ionizing radiation acts synergistically with immunotherapy by inducing immunogenic cell death and modulating the tumor microenvironment, promoting anti-tumor immune responses. In this pilot study, the safety and effectiveness of palliative radiation with a expressing (Lm-LLO-HER2), on delaying primary tumor progression in immunocompetent dogs with osteosarcoma was evaluated and correlative immune response biomarkers explored. Fifteen dogs with treatment naive, appendicular osteosarcoma, without evidence of metastatic disease received palliative radiation followed by Lm-LLO-HER2 immunotherapy every 3 weeks for an initial 8 doses then booster treatments. Treatment was well tolerated and delayed primary tumor progression and prolonged overall survival in 5/15 dogs. Peripheral blood mononuclear cell transcriptomic profiling revealed that baseline immune status and immune response to Lm-LLO-HER2 distinguished long-term from short-term survivors. Genes associated with Hedgehog signaling, senescence, DNA damage repair, and cell cycle were downregulated when compared to baseline in long-term survivors. These findings support further exploration of combination palliative radiation and -based immunotherapy in osteosarcoma and suggest that baseline immune status may determine clinical and immunological responses to combination therapy. Pet dogs with osteosarcoma represent a parallel population to pediatric osteosarcoma patients. These findings may inform future pediatric osteosarcoma clinical trials and patient stratification.
Boileau M, Lefebvre A, Marhfor S
… +7 more, Kali Mansouri M, Deleporte P, Paul Grolez G, Dewalle AS, Morales O, Delhem N, Mortier L
Mol Ther Oncol
· 2026 Jun · PMID 42291137
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Rose Bengal (RB), a xanthene dye with established antitumor and immunomodulatory properties, is a cytotoxic molecule evaluated in human melanoma through phase 2 clinical trials. RB demonstrates intrinsic therapeutic acti...Rose Bengal (RB), a xanthene dye with established antitumor and immunomodulatory properties, is a cytotoxic molecule evaluated in human melanoma through phase 2 clinical trials. RB demonstrates intrinsic therapeutic activity; however, its clinical application may be limited at high doses because of potential side effects, including photosensitivity. In this context, and given that RB also functions as a photosensitizer (PS), our objective was to enhance its efficacy by introducing a photodynamic therapy (PDT) approach using green light (550 nm) excitation. PDT relies on activation of a PS in the presence of oxygen, generating reactive oxygen species (ROS) that induce tumor cell death and stimulate immune responses. In terms of relative efficacy, PDT-RB was 121 and 40 times more effective than RB alone in HBL and LND melanoma cell lines, respectively. This reduced the RB concentration from 492 μM to 32 μM for HBL and from 1,043 μM to 74 μM for LND, achieving 80% cell death. This combination of light and low RB concentrations enhances ROS production and induces necrosis in metastatic melanoma cells. Moreover, PDT-RB preserves PBMC proliferation compared with high-dose RB, suggesting reduced immunotoxicity and potential immunogenic properties, supporting further investigation in more advanced models for future validation.
Smith PR, Kabir ME, Zhang J
… +7 more, Maufort JP, Forsberg MH, Sedzro DM, Berres M, Thomson JA, Capitini CM, Slukvin II
Mol Ther Oncol
· 2026 Jun · PMID 42291136
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The SIRPα-CD47 "don't eat me" checkpoint axis plays a critical role in shaping antitumor activities of macrophages within the tumor microenvironment (TME). However, targeting this axis with anti-CD47 antibodies to enhanc...The SIRPα-CD47 "don't eat me" checkpoint axis plays a critical role in shaping antitumor activities of macrophages within the tumor microenvironment (TME). However, targeting this axis with anti-CD47 antibodies to enhance antitumor responses in clinical trials has been challenging. Here, we demonstrated that -knockout (KO) iPSC-derived macrophages (iMacs) exhibit superior antitumor activity against various CD47-expressing tumors when combined with cancer-targeted monoclonal antibodies (mAbs) or chimeric antigen receptors (CARs). Moreover, -KO protected macrophages from mAb- and CAR-driven hypophagia, enabling efficient tumoricidal effects even after serial tumor exposures. Retention of phagocytic activities in -KO iMacs was associated with heightened surface expression of Fc receptors and GD2-CAR compared to their -expressing counterparts. Despite the powerful impact of -KO on iMac antitumor activities , only modest efficacy was observed in human xenograft mouse models of SK-OV3 ovarian carcinoma and CHLA-163 neuroblastoma treated with mAb or CAR-iMac therapy, indicating further engineering or combinatorial therapeutic strategies are needed for potent antitumor efficacy. Together, these findings identify SIRPα as a regulator in macrophage hypophagia and underscore the advantages of using -KO macrophage therapeutic strategies to modulate the SIRPα-CD47 checkpoint to unleash macrophage antitumor activity within the TME.