Pub Date : 2025-11-19DOI: 10.1126/scitranslmed.adr1777
Dingkang Liu, Jing Tian, Lichao Yu, Hong Luo, Haibo Rong, Yue Tong, Xiangdong Gao, Jun Yin
Immunogenic cell death (ICD) is a promising approach for generating antitumor immune responses to treat patients with cancer. However, the stereotactic induction of ICD and spatiotemporal synchronized activation of the tumor-specific immune response pose two critical challenges. Here, we report the basic principles and systematic development of a modular metalloprotein platform designated the protease-activated PSTAGylated in situ tumor vaccine (PPTV). Ferritin was used as a vaccine framework, with its outer surface fused with an optimal mito-disrupt peptide and its inner cavity loaded with manganese ions (Mn2+). Moreover, a protease-activated PSTAGylated prodrug strategy was developed to circumvent the major issues associated with ferritin, such as liver interception and drug leakage. We also demonstrated the activation of prodrugs in tumor lysates from patients. In subcutaneous and orthotopic tumor transplantation mouse models, the PPTV effectively codelivered mito-disrupt peptides and Mn2+ into tumors, leading to a combined effect of ICD and cGAS-STING cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS-STING) pathway activation, thereby achieving a potent antitumor immune response. The combined application of PPTV and anti–programmed death-ligand 1 resulted in the eradication of established tumors. These results underscore the applicability of the PPTV as an innovative in situ vaccine nanoplatform for establishing an antitumor immune response. This modular metalloprotein vaccine has clinical translational potential because of its simple generation and safety profile.
{"title":"A modular metalloprotein in situ vaccine for cancer immunotherapy in mouse models of breast cancer","authors":"Dingkang Liu, Jing Tian, Lichao Yu, Hong Luo, Haibo Rong, Yue Tong, Xiangdong Gao, Jun Yin","doi":"10.1126/scitranslmed.adr1777","DOIUrl":"10.1126/scitranslmed.adr1777","url":null,"abstract":"<div >Immunogenic cell death (ICD) is a promising approach for generating antitumor immune responses to treat patients with cancer. However, the stereotactic induction of ICD and spatiotemporal synchronized activation of the tumor-specific immune response pose two critical challenges. Here, we report the basic principles and systematic development of a modular metalloprotein platform designated the protease-activated PSTAGylated in situ tumor vaccine (PPTV). Ferritin was used as a vaccine framework, with its outer surface fused with an optimal mito-disrupt peptide and its inner cavity loaded with manganese ions (Mn<sup>2+</sup>). Moreover, a protease-activated PSTAGylated prodrug strategy was developed to circumvent the major issues associated with ferritin, such as liver interception and drug leakage. We also demonstrated the activation of prodrugs in tumor lysates from patients. In subcutaneous and orthotopic tumor transplantation mouse models, the PPTV effectively codelivered mito-disrupt peptides and Mn<sup>2+</sup> into tumors, leading to a combined effect of ICD and cGAS-STING cyclic GMP–AMP synthase–stimulator of interferon genes (cGAS-STING) pathway activation, thereby achieving a potent antitumor immune response. The combined application of PPTV and anti–programmed death-ligand 1 resulted in the eradication of established tumors. These results underscore the applicability of the PPTV as an innovative in situ vaccine nanoplatform for establishing an antitumor immune response. This modular metalloprotein vaccine has clinical translational potential because of its simple generation and safety profile.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 825","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145545439","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1126/scitranslmed.adn3993
Xiaoshu Pan, Peixin Huang, Samantha S. Ali, Bryan Renslo, Zachary Greenberg, Nina Erwin, Yanjun Li, Zuo Ding, Tarun E. Hutchinson, Athanasia Warnecke, Natalia E. Fernandez, Hinrich Staecker, Mei He
The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet–based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current–controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the Myo7aWT/Sh1 mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old Myo7aWT/Sh1 mice resulted in a reduction in Myo7aSh1 messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only–injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.
{"title":"Extracellular vesicle–mediated gene editing for the treatment of nonsyndromic progressive hearing loss in adult mice","authors":"Xiaoshu Pan, Peixin Huang, Samantha S. Ali, Bryan Renslo, Zachary Greenberg, Nina Erwin, Yanjun Li, Zuo Ding, Tarun E. Hutchinson, Athanasia Warnecke, Natalia E. Fernandez, Hinrich Staecker, Mei He","doi":"10.1126/scitranslmed.adn3993","DOIUrl":"10.1126/scitranslmed.adn3993","url":null,"abstract":"<div >The clinical translation of gene therapy has been challenging in part because of the limitations of current delivery approaches. Herein, we report an efficient nonviral genome editor delivery approach using extracellular vesicles (EVs) carrying single-guide RNA (sgRNA): CRISPR-Cas9 ribonucleoprotein (RNP) complexes for in vivo gene therapy. By leveraging a high-throughput microfluidic droplet–based electroporation system (μDES), we achieved a 10-fold enhancement in loading efficiency and more than 1000-fold increase in processing throughput for loading RNP complexes into EVs compared with conventional high-voltage pulsed electroporation. μDES generated uniform microdroplets containing EVs and RNPs by applying direct current–controlled low voltage (up to 60 V) to transiently permeabilize membranes and enable efficient cargo encapsulation while maintaining EV integrity at both the protein and morphological levels. In the <i>Myo7a<sup>WT/Sh1</sup></i> mouse model of autosomal dominant progressive hearing loss, which may model MYO7A-associated DFNA11 hearing loss in humans, we demonstrated the effective delivery of RNPs by EVs into cochlear hair cells by cross-sectional and whole-mount confocal imaging. The injection of RNP-EVs via the posterior semicircular canal in 4-week-old <i>Myo7a<sup>WT/Sh1</sup></i> mice resulted in a reduction in <i>Myo7a<sup>Sh1</sup></i> messenger RNA expression and evidence of hearing preservation, as measured by auditory brainstem responses, compared with untreated ears and EV only–injected mice. This study highlights the potential of μDES-produced RNP-EVs for gene editing as a treatment for progressive nonsyndromic hearing loss in patients.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492714","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1126/scitranslmed.adq6645
Cheng-Chia Wu, Luca Szalontay, Antonios N. Pouliopoulos, Sua Bae, Xander Berg, Hong-Jian Wei, Andrea Webster Carrion, Danae Kokossis, Chankrit Sethi, Jessica Fino, Halina Shatravka, Jennifer Lipina, Robin Ji, Keyu Liu, Omid Yousefian, Matthew Gallitto, Nina Yoh, Zachary Englander, Nicholas McQuillan, Masih Tazhibi, Genesis De Los Santos, Peter Canoll, Zhezhen Jin, James Garvin, Robyn D. Gartrell, Jovana Pavisic, Alexis Maddocks, Angela Lignelli, Neil Feldstein, Elisa E. Konofagou, Stergios Zacharoulis
Focused ultrasound (FUS)–mediated blood-brain barrier (BBB) opening with microbubbles is an emerging technology that enables drug delivery for central nervous system diseases. To date, most clinical trials assessing BBB opening in adults were designed to deliver US with a frequency of one treatment over several weeks. Little is known about the feasibility of shorter intervals of US delivery or whether this can be achieved in a pediatric population using a mobile device. Here, FUS and panobinostat were shown to have additive therapeutic effects in a syngeneic orthotopic model of diffuse midline glioma (DMG). We then conducted a single-arm first-in-pediatric trial to investigate the safety and feasibility of delivering neuronavigation-guided FUS treatment in combination with oral panobinostat in children with relapsed DMGs. We included an intrapatient escalation of FUS delivery to assess the feasibility of opening multiple sites in the brain. We demonstrated successful BBB opening using neuronavigation-guided FUS as frequently as every 2 days. Magnetic resonance imaging with contrast was used to identify the region of BBB opening. Three patients were accrued; 22 FUS procedures were delivered for 1 NOTS (number of tumor sites) treated, and four FUS procedures were delivered for 2 NOTS. All three patients received 1 NOTS, without serious adverse events, and two of the patients received 2 NOTS, all without sedation. For 2 NOTS, prolonged BBB opening and one grade 5 event, unlikely related to FUS, were observed. This study demonstrates feasibility of FUS for BBB opening and drug delivery without sedation in pediatric patients.
{"title":"Blood-brain barrier opening with neuronavigation-guided focused ultrasound in pediatric patients with diffuse midline glioma","authors":"Cheng-Chia Wu, Luca Szalontay, Antonios N. Pouliopoulos, Sua Bae, Xander Berg, Hong-Jian Wei, Andrea Webster Carrion, Danae Kokossis, Chankrit Sethi, Jessica Fino, Halina Shatravka, Jennifer Lipina, Robin Ji, Keyu Liu, Omid Yousefian, Matthew Gallitto, Nina Yoh, Zachary Englander, Nicholas McQuillan, Masih Tazhibi, Genesis De Los Santos, Peter Canoll, Zhezhen Jin, James Garvin, Robyn D. Gartrell, Jovana Pavisic, Alexis Maddocks, Angela Lignelli, Neil Feldstein, Elisa E. Konofagou, Stergios Zacharoulis","doi":"10.1126/scitranslmed.adq6645","DOIUrl":"10.1126/scitranslmed.adq6645","url":null,"abstract":"<div >Focused ultrasound (FUS)–mediated blood-brain barrier (BBB) opening with microbubbles is an emerging technology that enables drug delivery for central nervous system diseases. To date, most clinical trials assessing BBB opening in adults were designed to deliver US with a frequency of one treatment over several weeks. Little is known about the feasibility of shorter intervals of US delivery or whether this can be achieved in a pediatric population using a mobile device. Here, FUS and panobinostat were shown to have additive therapeutic effects in a syngeneic orthotopic model of diffuse midline glioma (DMG). We then conducted a single-arm first-in-pediatric trial to investigate the safety and feasibility of delivering neuronavigation-guided FUS treatment in combination with oral panobinostat in children with relapsed DMGs. We included an intrapatient escalation of FUS delivery to assess the feasibility of opening multiple sites in the brain. We demonstrated successful BBB opening using neuronavigation-guided FUS as frequently as every 2 days. Magnetic resonance imaging with contrast was used to identify the region of BBB opening. Three patients were accrued; 22 FUS procedures were delivered for 1 NOTS (number of tumor sites) treated, and four FUS procedures were delivered for 2 NOTS. All three patients received 1 NOTS, without serious adverse events, and two of the patients received 2 NOTS, all without sedation. For 2 NOTS, prolonged BBB opening and one grade 5 event, unlikely related to FUS, were observed. This study demonstrates feasibility of FUS for BBB opening and drug delivery without sedation in pediatric patients.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.science.org/doi/reader/10.1126/scitranslmed.adq6645","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492697","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Therapeutic vaccines against cervical cancer caused by human papillomavirus (HPV) are still an unmet medical need, despite a prophylactic HPV vaccine being available, and the now-licensed systemic vaccines may have a limited effect on the reproductive tract. To specifically inhibit cervical cancer development, the concept of mucosal immunity based on the reproductive-respiratory axis was adopted to develop a nasal HPV therapeutic vaccine. We used a cationic nanogel for the nasal vaccine delivery system and targeted HPV16 E7, an oncoprotein in HPV-driven cervical cancer to demonstrate the feasibility of a nasal therapeutic vaccine. The vaccine was combined with cyclic di–adenosine monophosphate as a cell-mediated immunity-inducing adjuvant. Intranasal immunization with the nanogel vaccine induced E7-specific CD4+ and CD8+ T cells in mouse cervicovaginal tissue. An antitumor effect due to the infiltration of vaccine-induced E7-specific T cells was also observed in an orthotopic tumor model in mice. Furthermore, intranasal immunization of nonhuman primates with the nanogel vaccine using a spray device that is also applicable to humans induced E7-specific T cells in the reproductive tissues. Our findings demonstrated that this nasal therapeutic vaccine effectively controlled cervical cancer and will contribute to preclinical evidence for clinical testing in the near future.
{"title":"Cationic nanogel–based nasal therapeutic HPV vaccine prevents the development of cervical cancer","authors":"Rika Nakahashi-Ouchida, Hiromi Mori, Yoshikazu Yuki, Tomonori Machita, Yuko Katakai, Shingo Umemoto, Yohei Uchida, Tomoyuki Yamanoue, Shin-ichi Sawada, Kazuya Ishige, Takashi Miyazaki, Kohtaro Fujihashi, Kazunari Akiyoshi, Yasuhiro Yasutomi, Kei Kawana, Hiroshi Kiyono","doi":"10.1126/scitranslmed.ado8840","DOIUrl":"10.1126/scitranslmed.ado8840","url":null,"abstract":"<div >Therapeutic vaccines against cervical cancer caused by human papillomavirus (HPV) are still an unmet medical need, despite a prophylactic HPV vaccine being available, and the now-licensed systemic vaccines may have a limited effect on the reproductive tract. To specifically inhibit cervical cancer development, the concept of mucosal immunity based on the reproductive-respiratory axis was adopted to develop a nasal HPV therapeutic vaccine. We used a cationic nanogel for the nasal vaccine delivery system and targeted HPV16 E7, an oncoprotein in HPV-driven cervical cancer to demonstrate the feasibility of a nasal therapeutic vaccine. The vaccine was combined with cyclic di–adenosine monophosphate as a cell-mediated immunity-inducing adjuvant. Intranasal immunization with the nanogel vaccine induced E7-specific CD4<sup>+</sup> and CD8<sup>+</sup> T cells in mouse cervicovaginal tissue. An antitumor effect due to the infiltration of vaccine-induced E7-specific T cells was also observed in an orthotopic tumor model in mice. Furthermore, intranasal immunization of nonhuman primates with the nanogel vaccine using a spray device that is also applicable to humans induced E7-specific T cells in the reproductive tissues. Our findings demonstrated that this nasal therapeutic vaccine effectively controlled cervical cancer and will contribute to preclinical evidence for clinical testing in the near future.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492699","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Rheumatoid arthritis (RA) is a chronic autoimmune condition characterized by aggressive fibroblast-like synoviocytes (FLSs). Small nucleolar RNAs (snoRNAs), traditionally implicated in ribosome biogenesis, are now recognized as disease regulators. However, their involvement in RA-FLSs remains poorly understood. Here, we identified small nucleolar RNA, C/D box 3 (SNORD3), a specific snoRNA up-regulated by tumor necrosis factor–α and interleukin-17, as a key driver of the aggressive transformation of RA-FLSs in vitro. Using an FLS-specific aptamer-functionalized siRNA delivery system, we demonstrated that silencing Snord3 alleviated arthritic symptoms in collagen-induced arthritis (CIA) mice. Transcriptome analyses revealed that SNORD3 up-regulated endothelial cell–specific molecule 1 (ESM1) by modulating the polycomb repressive complex 2 (PRC2)–mediated trimethylation of histone H3 at lysine-27 (H3K27me3), driving the aggressive transformation of RA-FLSs. Mechanistically, we found that SNORD3 physically interacted with enhancer of zeste homolog 2 (EZH2) and competitively disrupted the association of EZH2 with retinoblastoma binding protein 4 within PRC2, thus diminishing the H3K27me3 mark on the ESM1 gene promoter to relieve the transcriptional repression of ESM1. We screened an ESM1-specific aptamer 04 (ESMA04) by systematic evolution of ligands by exponential enrichment, which neutralized ESM1 and inhibited the aggressive transformation of RA-FLSs in vitro. When administered either alone or in combination with a biologic disease-modifying antirheumatic drug, etanercept, ESMA04 demonstrated therapeutic efficacy in CIA mice. Overall, our findings identified SNORD3-EZH2-ESM1 signaling as a driver of RA-FLS pathogenesis and underscored the promise of aptamer-based therapies for RA treatment.
{"title":"snoRNA Snord3 promotes rheumatoid arthritis by epigenetic regulation of ESM1 in fibroblast-like synoviocytes in mice","authors":"Jie Huang, Xuekun Fu, Runrun Zhang, Zhuqian Wang, Fang Qiu, Xinxin Chen, Junyu Fan, Chunhao Cao, Xu Yang, Jie Li, Yiying Liang, Dongyi He, Aiping Lu, Chao Liang","doi":"10.1126/scitranslmed.adt5340","DOIUrl":"10.1126/scitranslmed.adt5340","url":null,"abstract":"<div >Rheumatoid arthritis (RA) is a chronic autoimmune condition characterized by aggressive fibroblast-like synoviocytes (FLSs). Small nucleolar RNAs (snoRNAs), traditionally implicated in ribosome biogenesis, are now recognized as disease regulators. However, their involvement in RA-FLSs remains poorly understood. Here, we identified <i>small nucleolar RNA, C/D box 3</i> (<i>SNORD3</i>), a specific snoRNA up-regulated by tumor necrosis factor–α and interleukin-17, as a key driver of the aggressive transformation of RA-FLSs in vitro. Using an FLS-specific aptamer-functionalized siRNA delivery system, we demonstrated that silencing <i>Snord3</i> alleviated arthritic symptoms in collagen-induced arthritis (CIA) mice. Transcriptome analyses revealed that <i>SNORD3</i> up-regulated endothelial cell–specific molecule 1 (ESM1) by modulating the polycomb repressive complex 2 (PRC2)–mediated trimethylation of histone H3 at lysine-27 (H3K27me3), driving the aggressive transformation of RA-FLSs. Mechanistically, we found that <i>SNORD3</i> physically interacted with enhancer of zeste homolog 2 (EZH2) and competitively disrupted the association of EZH2 with retinoblastoma binding protein 4 within PRC2, thus diminishing the H3K27me3 mark on the <i>ESM1</i> gene promoter to relieve the transcriptional repression of <i>ESM1</i>. We screened an ESM1-specific aptamer 04 (ESMA04) by systematic evolution of ligands by exponential enrichment, which neutralized ESM1 and inhibited the aggressive transformation of RA-FLSs in vitro. When administered either alone or in combination with a biologic disease-modifying antirheumatic drug, etanercept, ESMA04 demonstrated therapeutic efficacy in CIA mice. Overall, our findings identified <i>SNORD3</i>-EZH2-ESM1 signaling as a driver of RA-FLS pathogenesis and underscored the promise of aptamer-based therapies for RA treatment.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492695","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1126/scitranslmed.adz2276
Wataru Akahata, Mai Komori, Amber L. Morey, Andrés A. Quiñones-Molina, James B. Hood, Josiane Fofana, Luis Romero, Elizabeth Peters, Jonathan D. Webber, Tyler Meeks, Paulina A. Przygonska, Isabel Steinberg, Ellison Ober, Tae Kim, Daniel C. Rogan, Kenta Matsuda, Jonathan F. Smith, Suryaram Gummuluru, Mark Connors, Hisashi Akiyama
To improve existing synthetic RNA-based vaccines, we previously developed a self-amplifying RNA (saRNA)–based vaccine expressing a membrane-anchored (TM) receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein (S-RBD-TM) and demonstrated that a low dose of this saRNA vaccine elicits robust immune responses. Moreover, a recent clinical trial with an saRNA vaccine incorporating 5-methylcytidine (5mC) (saRNA-5mC) has demonstrated reduced vaccine reactogenicity while maintaining robust humoral responses. In this study, we investigate the mechanisms by which 5mC incorporation attenuates adverse effects while maintaining immunogenicity. We found that incorporation of 5mC into the saRNA platform led to prolonged and robust expression of antigen and attenuated induction of type I interferon, a key driver of reactogenicity, specifically in plasmacytoid dendritic cells (pDCs). As a result, saRNA-5mC alleviated excessive innate immune responses in vivo without impairing B cell and T cell responses against the SARS-CoV-2 RBD. Mechanistically, we demonstrated that the detection of unmodified saRNA in pDCs was mediated by a host cytosolic RNA sensor, RIG-I, and this sensing was abolished with 5mC incorporation. In contrast, saRNA-5mC induced robust innate activation in professional antigen-presenting cells, such as macrophages, in a RIG-I–independent manner, highlighting distinct host sensing mechanisms for synthetic RNAs. Our study provides support for the potential clinical use of saRNA-5mC vaccine platforms.
{"title":"Incorporation of 5-methylcytidine alleviates RIG-I–mediated innate immune responses to a self-amplifying RNA vaccine","authors":"Wataru Akahata, Mai Komori, Amber L. Morey, Andrés A. Quiñones-Molina, James B. Hood, Josiane Fofana, Luis Romero, Elizabeth Peters, Jonathan D. Webber, Tyler Meeks, Paulina A. Przygonska, Isabel Steinberg, Ellison Ober, Tae Kim, Daniel C. Rogan, Kenta Matsuda, Jonathan F. Smith, Suryaram Gummuluru, Mark Connors, Hisashi Akiyama","doi":"10.1126/scitranslmed.adz2276","DOIUrl":"10.1126/scitranslmed.adz2276","url":null,"abstract":"<div >To improve existing synthetic RNA-based vaccines, we previously developed a self-amplifying RNA (saRNA)–based vaccine expressing a membrane-anchored (TM) receptor binding domain (RBD) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein (S-RBD-TM) and demonstrated that a low dose of this saRNA vaccine elicits robust immune responses. Moreover, a recent clinical trial with an saRNA vaccine incorporating 5-methylcytidine (5mC) (saRNA-5mC) has demonstrated reduced vaccine reactogenicity while maintaining robust humoral responses. In this study, we investigate the mechanisms by which 5mC incorporation attenuates adverse effects while maintaining immunogenicity. We found that incorporation of 5mC into the saRNA platform led to prolonged and robust expression of antigen and attenuated induction of type I interferon, a key driver of reactogenicity, specifically in plasmacytoid dendritic cells (pDCs). As a result, saRNA-5mC alleviated excessive innate immune responses in vivo without impairing B cell and T cell responses against the SARS-CoV-2 RBD. Mechanistically, we demonstrated that the detection of unmodified saRNA in pDCs was mediated by a host cytosolic RNA sensor, RIG-I, and this sensing was abolished with 5mC incorporation. In contrast, saRNA-5mC induced robust innate activation in professional antigen-presenting cells, such as macrophages, in a RIG-I–independent manner, highlighting distinct host sensing mechanisms for synthetic RNAs. Our study provides support for the potential clinical use of saRNA-5mC vaccine platforms.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492691","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1126/scitranslmed.ady0210
Shady Younis, Salvinaz I. Moutusy, Sajede Rasouli, Shaghayegh Jahanbani, Mahesh Pandit, Xiaohao Wu, Suman Acharya, Orr Sharpe, Tilini U. Wijeratne, Marlayna L. Harris, Emily Y. Yang, Yashaar Chaichian, Shima Parsafar, Matthew C. Baker, John B. Harley, Eric Meffre, Lawrence Steinman, Ann Marshak-Rothstein, Judith A. James, Olivia M. Martinez, Paul J. Utz, Dana E. Orange, Tobias V. Lanz, William H. Robinson
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by antinuclear antibodies (ANAs). Epstein-Barr virus (EBV) infection has been epidemiologically associated with SLE, yet its role in pathogenesis remains incompletely defined. Here, we developed an EBV-specific single-cell RNA-sequencing platform and used it to demonstrate that EBV infection reprograms autoreactive antinuclear antigen B cells to drive autoimmunity in SLE. We demonstrated that, in SLE, EBV+ B cells are predominantly CD27+CD21low memory B cells that are present at increased frequencies and express ZEB2, TBX21 (T-bet), and antigen-presenting cell transcriptional pathways. Integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin sequencing (ATAC-seq), and RNA polymerase II occupancy data revealed EBV nuclear antigen 2 (EBNA2) binding at the transcriptional start sites and regulatory regions of CD27, ZEB2, and TBX21, as well as the antigen-presenting cell genes demonstrated to be up-regulated in SLE EBV+ B cells. We expressed recombinant antibodies from SLE EBV+ B cells and demonstrated that they bind prototypical SLE nuclear autoantigens, whereas those from healthy individuals do not. We further found that SLE EBV+ B cells can serve as antigen-presenting cells to drive activation of T peripheral helper cells with concomitant activation of related EBV− antinuclear double-negative 2 B cells and plasmablasts. Our results provide a mechanistic basis for EBV being a driver of SLE through infecting and reprogramming nuclear antigen-reactive B cells to become activated antigen-presenting cells with the potential to promote systemic disease–driving autoimmune responses.
{"title":"Epstein-Barr virus reprograms autoreactive B cells as antigen-presenting cells in systemic lupus erythematosus","authors":"Shady Younis, Salvinaz I. Moutusy, Sajede Rasouli, Shaghayegh Jahanbani, Mahesh Pandit, Xiaohao Wu, Suman Acharya, Orr Sharpe, Tilini U. Wijeratne, Marlayna L. Harris, Emily Y. Yang, Yashaar Chaichian, Shima Parsafar, Matthew C. Baker, John B. Harley, Eric Meffre, Lawrence Steinman, Ann Marshak-Rothstein, Judith A. James, Olivia M. Martinez, Paul J. Utz, Dana E. Orange, Tobias V. Lanz, William H. Robinson","doi":"10.1126/scitranslmed.ady0210","DOIUrl":"10.1126/scitranslmed.ady0210","url":null,"abstract":"<div >Systemic lupus erythematosus (SLE) is a systemic autoimmune disease characterized by antinuclear antibodies (ANAs). Epstein-Barr virus (EBV) infection has been epidemiologically associated with SLE, yet its role in pathogenesis remains incompletely defined. Here, we developed an EBV-specific single-cell RNA-sequencing platform and used it to demonstrate that EBV infection reprograms autoreactive antinuclear antigen B cells to drive autoimmunity in SLE. We demonstrated that, in SLE, EBV<sup>+</sup> B cells are predominantly CD27<sup>+</sup>CD21<sup>low</sup> memory B cells that are present at increased frequencies and express <i>ZEB2</i>, <i>TBX21</i> (T-bet), and antigen-presenting cell transcriptional pathways. Integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq), assay for transposase-accessible chromatin sequencing (ATAC-seq), and RNA polymerase II occupancy data revealed EBV nuclear antigen 2 (EBNA2) binding at the transcriptional start sites and regulatory regions of <i>CD27</i>, <i>ZEB2</i>, and <i>TBX21</i>, as well as the antigen-presenting cell genes demonstrated to be up-regulated in SLE EBV<sup>+</sup> B cells. We expressed recombinant antibodies from SLE EBV<sup>+</sup> B cells and demonstrated that they bind prototypical SLE nuclear autoantigens, whereas those from healthy individuals do not. We further found that SLE EBV<sup>+</sup> B cells can serve as antigen-presenting cells to drive activation of T peripheral helper cells with concomitant activation of related EBV<sup>−</sup> antinuclear double-negative 2 B cells and plasmablasts. Our results provide a mechanistic basis for EBV being a driver of SLE through infecting and reprogramming nuclear antigen-reactive B cells to become activated antigen-presenting cells with the potential to promote systemic disease–driving autoimmune responses.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492694","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-12DOI: 10.1126/scitranslmed.aec0501
Guy Gorochov, Alexis Mathian
Epstein-Barr virus–mediated B cell reprogramming may initiate systemic lupus erythematosus (Younis et al., this issue).
Epstein-Barr病毒介导的B细胞重编程可能引发系统性红斑狼疮（Younis等，本期）。
{"title":"EBV and the making of lupus","authors":"Guy Gorochov, Alexis Mathian","doi":"10.1126/scitranslmed.aec0501","DOIUrl":"10.1126/scitranslmed.aec0501","url":null,"abstract":"<div >Epstein-Barr virus–mediated B cell reprogramming may initiate systemic lupus erythematosus (Younis <i>et al.</i>, this issue).</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 824","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145492698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-05DOI: 10.1126/scitranslmed.adu6015
Qin Zeng, Lin Li, Xue Li, Linmang Qin, Tianxiao Feng, Yunfeng Zhu, Jieying Wang, Yaoyao Zou, Jianling Su, Guangfu Dong, Wuzheng Xia, Ting Xu, Guangfeng Zhang, Yang Cui, Haobo Lin, Xin Li, Yang Li
Extrafollicular age-associated B cells (ABCs) excessively expand and produce autoantibodies in systemic lupus erythematosus (SLE), and the regulatory mechanism remains elusive. We found that the m6A demethylase fat mass and obesity-associated protein (FTO) was highly expressed in ABCs from patients with SLE, which was positively associated with renal immune damage. FTO overexpression in murine and human B cells facilitated ABC expansion and exacerbated SLE in lupus-prone mice, whereas FTO ablation ameliorated ABC-driven autoimmunity. FTO expression was up-regulated upon activation of the toll-like receptor 7–myeloid differentiation primary response protein 88 (TLR7-MyD88) signaling pathway. FTO, in turn, promoted TLR7-driven ABC differentiation by targeting ATPase H+ transporting V1 subunit G1 (ATP6V1G1), a subunit of the vacuolar H+-ATPase (V-ATPase), in an m6A-dependent manner. Mechanistically, FTO deficiency impaired lysosomal autophagy by reducing ATP6V1G1-mediated V-ATPase activity. The accumulation of damaged mitochondria led to mitochondrial dysfunction in human and murine B cells, characterized by reduced oxidative phosphorylation and elevated reactive oxygen species. This dysfunction limited cell proliferation and blocked ABC differentiation by dampening cellular responsiveness to interleukin-12. Thus, TLR7-FTO-ATP6V1G1 signaling metabolically shapes extrafollicular ABCs in SLE, providing a potential therapeutic target.
系统性红斑狼疮（SLE）患者滤泡外年龄相关B细胞（ABCs）过度扩增并产生自身抗体，其调控机制尚不明确。我们发现m6a去甲基化酶脂肪质量和肥胖相关蛋白（FTO）在SLE患者的abc中高度表达，这与肾免疫损伤呈正相关。小鼠和人B细胞中FTO的过度表达促进了ABC扩增，加重了狼疮易感小鼠的SLE，而FTO消融则改善了ABC驱动的自身免疫。激活toll样受体7-髓样分化主要反应蛋白88 （TLR7-MyD88）信号通路后，FTO表达上调。反过来，FTO通过靶向ATPase H +转运V1亚基G1 (ATP6V1G1)，促进tlr7驱动的ABC分化，atph + -ATPase （V-ATPase）的一个亚基，以依赖于m6a的方式。机制上，FTO缺乏通过降低atp6v1g1介导的V-ATPase活性来损害溶酶体自噬。受损线粒体的积累导致人和小鼠B细胞线粒体功能障碍，其特征是氧化磷酸化减少和活性氧升高。这种功能障碍通过抑制细胞对白细胞介素-12的反应性来限制细胞增殖和阻断ABC分化。因此，TLR7-FTO-ATP6V1G1信号在SLE中代谢塑造滤泡外的abc，提供了一个潜在的治疗靶点。
{"title":"The m6A demethylase FTO links TLR7 to mitochondrial oxidation driving age-associated B cell formation in systemic lupus erythematosus","authors":"Qin Zeng, Lin Li, Xue Li, Linmang Qin, Tianxiao Feng, Yunfeng Zhu, Jieying Wang, Yaoyao Zou, Jianling Su, Guangfu Dong, Wuzheng Xia, Ting Xu, Guangfeng Zhang, Yang Cui, Haobo Lin, Xin Li, Yang Li","doi":"10.1126/scitranslmed.adu6015","DOIUrl":"10.1126/scitranslmed.adu6015","url":null,"abstract":"<div >Extrafollicular age-associated B cells (ABCs) excessively expand and produce autoantibodies in systemic lupus erythematosus (SLE), and the regulatory mechanism remains elusive. We found that the m<sup>6</sup>A demethylase fat mass and obesity-associated protein (FTO) was highly expressed in ABCs from patients with SLE, which was positively associated with renal immune damage. FTO overexpression in murine and human B cells facilitated ABC expansion and exacerbated SLE in lupus-prone mice, whereas FTO ablation ameliorated ABC-driven autoimmunity. FTO expression was up-regulated upon activation of the toll-like receptor 7–myeloid differentiation primary response protein 88 (TLR7-MyD88) signaling pathway. FTO, in turn, promoted TLR7-driven ABC differentiation by targeting ATPase H<sup>+</sup> transporting V1 subunit G1 (ATP6V1G1), a subunit of the vacuolar H<sup>+</sup>-ATPase (V-ATPase), in an m<sup>6</sup>A-dependent manner. Mechanistically, FTO deficiency impaired lysosomal autophagy by reducing ATP6V1G1-mediated V-ATPase activity. The accumulation of damaged mitochondria led to mitochondrial dysfunction in human and murine B cells, characterized by reduced oxidative phosphorylation and elevated reactive oxygen species. This dysfunction limited cell proliferation and blocked ABC differentiation by dampening cellular responsiveness to interleukin-12. Thus, TLR7-FTO-ATP6V1G1 signaling metabolically shapes extrafollicular ABCs in SLE, providing a potential therapeutic target.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 823","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145441238","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2025-11-05DOI: 10.1126/scitranslmed.adr6207
Shailesh Singh, Claudia Weindorfer, Ajeya Nandi, Chermakani Panneer Selvam, Marcelo Mendes Götze, Megha Das, Andres Falaschini, Youley Tjendra, Melinda M. Boone, Helmut Dolznig, Qing Zhang, Robert Clarke, Christoforos Thomas, Rumela Chakrabarti
Estrogen receptor–positive (ER+) luminal breast cancer comprises 75% of patients with breast cancer and presents notable treatment challenges because of endocrine resistance. The effectiveness of immunotherapy in endocrine therapy–resistant luminal breast cancer remains unclear. This limitation is due in part to a lack of immunocompetent preclinical models investigating the comprehensive involvement of immune cells in the tumor microenvironment (TME) in the context of endocrine resistance. In this study, we identified a subtype of immunosuppressive (M2-like) programmed death ligand 1–positive (PD-L1+) tumor-associated macrophages (TAMs) critically fostering resistance to tamoxifen (TMX) and fulvestrant (FV) through maintaining cancer stem cell (CSC) activity in new mouse models. These TAMs are recruited by Delta-like ligand 1 (DLL1), a Notch signaling ligand expressed in luminal tumor cells, through the CCR3/CCL7 axis. Combination therapy with anti-DLL1 and anti–PD-L1 antibodies with TMX reduced tumor growth and associated CSCs and reprogrammed the immunosuppressive TME in both preclinical mouse models and patient-derived explants, thus laying the foundation for a future combined immune-endocrine therapy in these patients.
{"title":"DLL1-responsive PD-L1+ tumor-associated macrophages promote endocrine resistance in breast cancer","authors":"Shailesh Singh, Claudia Weindorfer, Ajeya Nandi, Chermakani Panneer Selvam, Marcelo Mendes Götze, Megha Das, Andres Falaschini, Youley Tjendra, Melinda M. Boone, Helmut Dolznig, Qing Zhang, Robert Clarke, Christoforos Thomas, Rumela Chakrabarti","doi":"10.1126/scitranslmed.adr6207","DOIUrl":"10.1126/scitranslmed.adr6207","url":null,"abstract":"<div >Estrogen receptor–positive (ER<sup>+</sup>) luminal breast cancer comprises 75% of patients with breast cancer and presents notable treatment challenges because of endocrine resistance. The effectiveness of immunotherapy in endocrine therapy–resistant luminal breast cancer remains unclear. This limitation is due in part to a lack of immunocompetent preclinical models investigating the comprehensive involvement of immune cells in the tumor microenvironment (TME) in the context of endocrine resistance. In this study, we identified a subtype of immunosuppressive (M2-like) programmed death ligand 1–positive (PD-L1<sup>+</sup>) tumor-associated macrophages (TAMs) critically fostering resistance to tamoxifen (TMX) and fulvestrant (FV) through maintaining cancer stem cell (CSC) activity in new mouse models. These TAMs are recruited by Delta-like ligand 1 (DLL1), a Notch signaling ligand expressed in luminal tumor cells, through the CCR3/CCL7 axis. Combination therapy with anti-DLL1 and anti–PD-L1 antibodies with TMX reduced tumor growth and associated CSCs and reprogrammed the immunosuppressive TME in both preclinical mouse models and patient-derived explants, thus laying the foundation for a future combined immune-endocrine therapy in these patients.</div>","PeriodicalId":21580,"journal":{"name":"Science Translational Medicine","volume":"17 823","pages":""},"PeriodicalIF":14.6,"publicationDate":"2025-11-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145441236","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":1,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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