Lee SY, Lee J, Ahn JH
… +12 more, Lee SY, Choi EJ, Ha D, Oh A, Jo S, Yoon S, Lee S, Bae HJ, Lee NY, Youn H, Kang BC, Nam JH
Mol Ther Nucleic Acids
· 2026 Jun · PMID 42282251
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mRNA vaccines have revolutionized development and continue to advance through clinical and research applications. Circular RNA is emerging as a promising alternative to linear RNA. However, no direct comparison has ident...mRNA vaccines have revolutionized development and continue to advance through clinical and research applications. Circular RNA is emerging as a promising alternative to linear RNA. However, no direct comparison has identified the optimal RNA platform for mRNA-based drug development. This study compares linear and circular RNA platforms and , focusing on expression dynamics, immune responses, and safety. In terms of expression efficiency, the circular RNA showed prolonged expression compared to linear RNA. The immunogenicity was evaluated using influenza HA as an antigen. In humoral immune response, linear RNA with modified nucleosides (Lin-m1Ψ) and circular RNA with unmodified nucleosides (Circ-WT) showed higher neutralizing antibody titers than linear RNA with unmodified nucleosides (Lin-WT). Lin-m1Ψ and Lin-WT induced slightly higher cell-mediated immune responses than Circ-WT. In mice, all RNA types showed a decrease in reticulocytes, monocytes, and alkaline phosphatase on day 2 following high-dose intramuscular injection. Furthermore, Lin-WT and Circ-WT showed greater decreases in platelets than Lin-m1Ψ. However, these changes recovered to normal by day 14 post-vaccination in all groups. These findings provide insights into developing RNA-based immunotherapies and selecting suitable RNA platforms for various applications, including preventive and therapeutic vaccines as well as protein-replacement therapies.
Radic-Sarikas B, Markovic M, Zylka MM
… +8 more, Sturtzel C, Ilg M, Surdez D, Metzelder M, Distel M, Ovsianikov A, Halbritter F, Kovar H
Mol Ther Oncol
· 2026 Jun · PMID 42281954
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Bone sarcomas are rare and aggressive pediatric cancers with limited progress in targeted therapy development, partly due to the poor physiological relevance of conventional two-dimensional (2D) culture systems used for...Bone sarcomas are rare and aggressive pediatric cancers with limited progress in targeted therapy development, partly due to the poor physiological relevance of conventional two-dimensional (2D) culture systems used for preclinical testing. To address this gap, we developed a standardized three-dimensional (3D) culture and drug-testing platform for Ewing sarcoma (ES) and osteosarcoma (OS) that more accurately recapitulates tumor biology. Notably, gene-expression analyses demonstrated that ES and OS spheroids transcriptionally converge toward patient tumor cell states, underscoring their physiological relevance for preclinical testing. Across 3D spheroids, bioprinted constructs, and patient-derived xenograft (PDX) cultures, we observed a consistent activation and dependency on the mevalonate pathway in ES. Leveraging this platform, we identified a selective therapeutic synergy between statins, which inhibit mevalonate pathway flux, and BCL-xL inhibitors-a vulnerability not detectable in 2D cultures. Importantly, this synergistic interaction was tumor-specific and absent in non-malignant fibroblasts, indicating a favorable therapeutic window. Together, these findings highlight the mevalonate pathway as a targetable metabolic dependency in ES and demonstrate how physiologically grounded 3D models can uncover clinically actionable treatment strategies that remain hidden in traditional 2D systems.
Adult T cell leukemia/lymphoma (ATLL), caused by human T cell leukemia virus type 1, is an aggressive malignancy with limited treatment options. Although the combination therapy of azidothymidine (AZT) and interferon-α (...Adult T cell leukemia/lymphoma (ATLL), caused by human T cell leukemia virus type 1, is an aggressive malignancy with limited treatment options. Although the combination therapy of azidothymidine (AZT) and interferon-α (IFN-α) has demonstrated clinical efficacy in ATLL, its underlying mechanism remains unclear. In this study, through comprehensive analyses, we reveal that the combination therapy can enhance CD8+ T cell function both in vitro and in vivo: it promotes cytotoxic T lymphocyte (CTL)-mediated killing of ATLL cell lines, and exhibits antitumor activities in immunocompetent but not immunodeficient mouse xenograft models (MC38 and EL4 were used due to the lack of immunocompetent mouse model for ATLL). Granzyme B, which is downregulated in ATLL patients, was found to be key to the effectiveness of AZT/IFN-α therapy. In addition, AZT promotes IFN-α-mediated antigen processing and presentation, and it further transcriptionally upregulates the expression of the ATLL antigen HBZ. Collectively, AZT could potentiate the immunomodulatory function of IFN-α in ATLL likely via enhancing tumor antigen expression and processing and boosting CTL activities. This study suggests a key role of immunostimulation in AZT/IFN-α-treated ATLL and may provide mechanistic insights into the development of novel therapies.
Story BD, Thomasy SM, Adelman SA
… +19 more, Taghian T, Lopez Y, Ardon M, Shiraishi H, Khan S, Khan M, Do M, Benatti HR, Bertrand S, Prestigiacomo R, Gately R, Martin DR, Sena-Esteves M, Xie J, Gao G, Teixeira LBC, Shaw GC, Marsh-Armstrong N, Gray-Edwards HL
Tay-Sachs disease (TSD) is a fatal neurodegenerative lysosomal storage disease. The Jacob sheep is the only large-animal model of TSD, yet ocular pathology and the therapeutic potential of gene therapy remain poorly defi...Tay-Sachs disease (TSD) is a fatal neurodegenerative lysosomal storage disease. The Jacob sheep is the only large-animal model of TSD, yet ocular pathology and the therapeutic potential of gene therapy remain poorly defined. Sheep cohorts included normal controls (n = 3); untreated TSD-affected (n = 4); intravenous AAV9-Bic_HexA/HexB-treated (n = 3); and intracerebroventricular, cisterna magna, and lumbar intrathecal AAV9- Bic_HexA/HexB-treated sheep (cerebrospinal fluid [CSF] therapy; n = 7). Retinal histopathology and immunohistochemistry, retinal whole-mount analyses for retinal ganglion cell (RGC) morphology and density, optic nerve evaluation with p-phenylenediamine (PPD )semi-thin sections, qPCR assessment for vector genomes, and RNAscope probes for transgene expression were performed. Untreated TSD sheep exhibited RGCs with abundant microvesicular cytoplasmic expansion and optic nerve spheroids, with storage material variably staining with periodic acid-Schiff. Marked astrocytosis, microgliosis, and GM2 accumulation within RGCs were present. Optic nerve axon counts and RGC density were significantly reduced, and optic nerve damage scores increased, in untreated and IV-treated sheep but were rescued with short-term CSF therapy. GM2 volume and signal intensity per RGC were significantly reduced following short-term CSF therapy. Minimal but detectable retinal vector genomes and transgene expression were observed. These findings demonstrate retinal and optic nerve pathology in Jacob sheep with TSD and AAV9 therapy.
Kinna A, Datta P, Bughda R
… +24 more, Parekh F, Robson M, Jha R, Wu P, Scott I, Hogben E, Amin O, Weng-Kit Cheung G, Hazelton W, Akbar Z, Lamb K, Macken E, Allen C, Panovska S, Hogan L, El-Kholy M, Shrestha R, Popplewell J, Kokalaki E, Sillibourne J, Thomas S, Onuoha S, Ferrari M, Pule M
Chimeric antigen receptor (CAR) T cells require an extracellular targeting domain for antigen specificity, typically an scFv. VHH antibodies are emerging as alternative CAR binding regions due to their reduced size, impr...Chimeric antigen receptor (CAR) T cells require an extracellular targeting domain for antigen specificity, typically an scFv. VHH antibodies are emerging as alternative CAR binding regions due to their reduced size, improved stability, and CDR3 loop architecture. Herein, we generated and compared VHH and scFv antibodies targeting CD123, via immunized phage display libraries, selecting ten antibody pairs based on comparable domain targeting and kinetic profiles (K range M 10-10). The VHH antibody fragments showed improved stability (Tm Δ7.85°C), reduced aggregation, and a favorable surface charge. CARs incorporating a VHH demonstrated higher IL2 and IFNγ secretion than scFv-derived CARs during in vitro analysis and substantially enhanced survival and decreased tumor burden in a xenograft murine model of acute myeloid leukemia (AML). This comprehensive comparison of the two most adopted antibody classes provides a rationale for the selection of VHH as preferred CAR binding domains.
Rapid urbanization, migration, and climate change are accelerating the appearance and diversification of respiratory viruses, overwhelming the pace at which conventional drugs can be discovered and manufactured. Here, we...Rapid urbanization, migration, and climate change are accelerating the appearance and diversification of respiratory viruses, overwhelming the pace at which conventional drugs can be discovered and manufactured. Here, we report a fast-response platform based on 15- to 25-residue "clamp peptides" (CPs) that grip conserved receptor-binding domains of viral surface proteins. Developed through rational structure-based docking and supported by retrospective machine learning triage, CPs bind their targets and potently block viral entry across SARS-CoV-2 variants and influenza A strains in human tissue-engineered 3D lung models by 2-4 log, without cytotoxicity or hemolysis at therapeutically relevant concentrations. Unlike antibodies, proteins, or small molecules, CPs are synthetic, reproducible, and rapidly designable to specific domains. This work establishes CPs as a scalable, broad-spectrum, and variant-agnostic platform amenable for use in intranasal delivery to block viral entry of respiratory viruses at the airway mucosa, offering a valuable first-line countermeasure for future respiratory outbreaks.
Three-dimensional hepatocyte-like cells (3D HLCs) derived from human adipose-derived mesenchymal stem cells (ADSCs) represent a scalable and clinically accessible cell source for metabolic liver diseases. Here, the autho...Three-dimensional hepatocyte-like cells (3D HLCs) derived from human adipose-derived mesenchymal stem cells (ADSCs) represent a scalable and clinically accessible cell source for metabolic liver diseases. Here, the authors demonstrate that transplantation (Tx) of ADSC-derived 3D HLCs significantly enhances ammonia detoxification in vivo across multiple urea cycle disorder (UCD) models. 3D HLC Tx attenuated hyperammonemia, accelerated ammonia clearance, and improved encephalopathy-associated behavior in wild-type, ornithine transcarbamylase-deficient, and citrin-deficient mice. Engrafted human cells persisted and contributed to metabolic improvement. These findings highlight ADSC-derived 3D HLCs as a promising, minimally invasive therapeutic strategy and support their potential as an alternative to liver Tx for UCDs.
Nakashima I, Saito S, Zhao J
… +6 more, Tanaka M, Akahoshi E, Hasegawa A, Sugano-Ishihara M, Yagyu S, Nakazawa Y
Mol Ther Oncol
· 2026 Jun · PMID 42256207
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Despite advances in multimodal therapies, outcomes for pediatric patients with relapsed or refractory cancers remain poor. Chimeric antigen receptor (CAR) T cell therapy has demonstrated limited efficacy in solid tumors...Despite advances in multimodal therapies, outcomes for pediatric patients with relapsed or refractory cancers remain poor. Chimeric antigen receptor (CAR) T cell therapy has demonstrated limited efficacy in solid tumors due to the immunosuppressive tumor microenvironment (TME), which promotes T cell exhaustion and restricts CAR T cell expansion. This study evaluated a combinatorial approach to enhance CAR T function by reprogramming the TME to overexpress target antigens (TAs) and co-stimulatory molecules (CSMs) through a lipid nanoparticle-based CAR-T vaccination (CART-Vac). As proof of concept, rhabdomyosarcoma cells (Rh30) engineered to overexpress TAs and CSMs (Rh30-TACS) were examined. EPHB4-directed CAR T cells demonstrated enhanced cytotoxicity, proliferation, and cytokine secretion , and superior tumor control with increased T cell infiltration in Rh30-TACS tumors compared with Rh30 tumors. To induce TA and CSM expression in the TME, CART-Vac was designed to deliver mRNAs encoding truncated EPHB4, CD80, and CD137L. CART-Vac effectively mediated transient expression, significantly enhancing CAR T expansion and antitumor activity in both models. These findings suggest that CART-Vac can modulate the TME, offering a promising strategy to improve the therapeutic efficacy of CAR T cells in solid tumors.
Li Y, Nakkala JR, Akter L
… +4 more, Kang X, Song Y, VanLuinen E, Chen X
Mol Ther Oncol
· 2026 Jun · PMID 42256206
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Personalized neoantigen mRNA vaccines showed good efficacy in treating metastatic melanoma, pancreatic cancer, and breast cancer in early phase clinical trials. Strategies are needed to further enhance neoantigen mRNA va...Personalized neoantigen mRNA vaccines showed good efficacy in treating metastatic melanoma, pancreatic cancer, and breast cancer in early phase clinical trials. Strategies are needed to further enhance neoantigen mRNA vaccine-induced anti-tumor immunity. This study explored conventional adjuvant co-administration to enhance mRNA vaccine-induced anti-tumor immunity. We found a clinically used CpG 1018 adjuvant was highly effective to enhance ovalbumin (OVA) and neoantigen mRNA-induced T cell responses and anti-tumor immunity in B16F10-OVA melanoma models. Mechanistic studies found CpG 1018 mainly enhanced dendritic cell maturation and local cytokine/chemokine release but not mRNA translation. mRNA vaccine in the presence of CpG 1018 induced significantly higher levels of Granzyme B, IFNγ, and TNFα-secreting CD8+ T cells as compared to mRNA vaccine alone. We further found that potent anti-tumor immunity was associated with increased tumor-infiltration of CD8+ T cells and positively correlated with tumor-infiltrating CD8+ to CD4+ T cell ratios. Cell depletion studies found CD8+ T cells rather than CD4+ T cells or NK cells played crucial roles in CpG 1018-augmented mRNA vaccine efficacy. CpG 1018-adjuvanted mRNA vaccine induced transient body weight loss (<6%) with an overall good safety. Our data warrant further investigation of CpG 1018-adjuvanted neoantigen mRNA vaccine for potentially better tumor therapy.
Yang Y, Ronsley R, Kilburn L
… +3 more, Kim A, Wechsler-Reya RJ, Yang ZJ
Mol Ther Oncol
· 2026 Jun · PMID 42256204
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Medulloblastoma (MB), the most common malignant pediatric brain tumor, is treated with intensive multimodal regimens that cause substantial long-term toxicity and fail to prevent recurrence in up to 30% of patients. We i...Medulloblastoma (MB), the most common malignant pediatric brain tumor, is treated with intensive multimodal regimens that cause substantial long-term toxicity and fail to prevent recurrence in up to 30% of patients. We investigated triiodothyronine (T3), an FDA-approved thyroid hormone, as a differentiation-based therapeutic strategy for MB. Using a Sonic Hedgehog (SHH)-driven mouse model, two patient-derived xenografts (SHH and group 3), and drug-resistant TP53-mutant MB cells, we found that T3 and cytotoxic chemotherapy suppress tumor growth through complementary biological mechanisms: T3 promotes terminal differentiation of tumor cells, whereas cyclophosphamide (CTX) and irinotecan (CPT-11) induce caspase-3-dependent apoptosis. , T3 enhanced the tumor inhibitory effects of CTX and CPT-11, suppressed proliferation in chemotherapy-resistant cells, and promoted differentiation across MB subgroups. , sequential administration of T3 following chemotherapy suppressed post-treatment tumor regrowth and prolonged survival without increasing systemic toxicity. T3-induced transient tachycardia was effectively controlled with propranolol without compromising antitumor activity. Together, these findings support the potential of T3 as a clinically accessible differentiation-based therapy that enhances chemotherapy responses and suppresses post-treatment tumor regrowth in medulloblastoma.
Mei X, Li X, Wu Y
… +5 more, Fu D, Zhang F, Yan T, Gu W, Gao B
Mol Ther Oncol
· 2026 Jun · PMID 42256202
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PD-1 is a crucial molecule indispensable for maintaining immune homeostasis. Disrupting the interaction between PD-1 and its ligands can significantly enhance the immune system's ability to combat tumors. By integrating...PD-1 is a crucial molecule indispensable for maintaining immune homeostasis. Disrupting the interaction between PD-1 and its ligands can significantly enhance the immune system's ability to combat tumors. By integrating the PD-1 ligand-binding domain with the CD28 signal fragments (switch molecule) on T cells, we can utilize co-stimulatory signals to boost the function of T cells while removing the inhibition of immune checkpoint molecules. However, the extensive application of anti-PD-1 monoclonal antibodies in clinical settings may neutralize the PD-1 switch molecule, thereby restricting its therapeutic potential. In this study, we used sequential mutation and sorting techniques to rapidly evolve a PD-1 molecule that exhibits high affinity for PD-L1 while avoiding the recognition by five of the six clinically approved anti-PD-1 drugs. Experimental results verify that the Fc fusion protein and switch receptor based on such PD-1 mutant enhance the efficacy of therapeutic T cells.
Mol Ther Oncol
· 2026 Jun · PMID 42256201
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Expression of the αvβ6 integrin is upregulated on multiple solid epithelial tumors while this integrin is largely absent on the healthy human epithelium. In the present study, we sought to compare the biodistributions o...Expression of the αvβ6 integrin is upregulated on multiple solid epithelial tumors while this integrin is largely absent on the healthy human epithelium. In the present study, we sought to compare the biodistributions of three αvβ6-selective agents that could be used for the positron emission tomography (PET)-based detection of malignancies expressing this integrin. A streptavidin-coupled αvβ6-binding peptide (streptavidin-A20), an anti-αvβ6 monoclonal antibody (620W.7) and an αvβ6-targeted protein, derived from a modified adenovirus 5 receptor-binding domain (Ad5.KO1.A20), were radiolabeled with [Zr] and intravenously injected into mice bearing bilateral αvβ6-positive and -negative melanoma xenografts. Micro-PET imaging was performed at 0.33, 24, 48, 72, and (620W.7 group only) 144 h post-injection. Streptavidin-A20-based radiotracers preferentially accumulated in αvβ6-positive tumors, achieving >3.4:1 A375-β6:A375 activity ratios between 24 and 72 h post-injection, with off-target uptake restricted to the kidneys. 620W.7 exhibited roughly 2-fold greater accumulation in A375-β6 than A375 tumors, with lower levels of renal retention and greater activity detected in the liver and spleen. Despite having demonstrated selective binding to αvβ6-expressing cells , the Ad5.KO1.A20 protein failed to selectively traffic to tumors expressing this integrin . Nevertheless, streptavidin-A20 and 620W.7 demonstrated promise as αvβ6-selective agents for applications.
The loss in segmental inhibitory GABAergic tone plays the key role in the development of spinal injury-induced muscle spasticity. We use a subpial segment-targeted delivery of adeno-associated virus vector(s) expressing...The loss in segmental inhibitory GABAergic tone plays the key role in the development of spinal injury-induced muscle spasticity. We use a subpial segment-targeted delivery of adeno-associated virus vector(s) expressing GAD65 (glutamic acid decarboxylase-65) and VGAT (vesicular GABA transporter) transgenes in rats with spinal transection-induced spasticity. In treated animals, a significant suppression in spasticity was seen at 5-8 weeks after treatment. Naive rats, pigs, and non-human primates (NHPs) injected with human equivalent dose of treatment vectors and surviving for 3 weeks to 4.5 years showed normal motor function and pinch-evoked response. A significant increase in the number of VGLUT2 terminals co-expressing GAD65 and VGAT protein in vector-injected segment was seen. This corresponded with the presence of transgene-specific rat Gad2 or human GAD2 and rat Slc32a1 or human SLC32A1 mRNA signal. No spinal toxicity was noted in NHPs at 4.5 years post vector delivery. Analysis of peripheral organs (liver, spleen, and skeletal muscle) showed minimal or no detectable transgenes in pigs and NHPs. These data demonstrate that a single-time-point spinal-segment-targeted subpial delivery of GAD65/VGAT transgenes is effective in suppressing spinal injury-induced spasticity and has a favorable long-term safety profile as defined by normal neurological function and histopathology in naive pigs and NHPs.