Hao Liu,Nan Liu,Chen Zhou,Ailing Du,Mayank Kapadia,Phillip W L Tai,Erik Barton,Guangping Gao,Dan Wang
{"title":"High-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling.","authors":"Hao Liu,Nan Liu,Chen Zhou,Ailing Du,Mayank Kapadia,Phillip W L Tai,Erik Barton,Guangping Gao,Dan Wang","doi":"10.1038/s44321-025-00248-w","DOIUrl":"https://doi.org/10.1038/s44321-025-00248-w","url":null,"abstract":"Triple transfection of HEK293 cells is the most widely used method for producing recombinant adeno-associated virus (rAAV), a leading gene delivery vector for human gene therapy. Despite its tremendous success, this approach generates several vector-related impurities that could potentially compromise the safety and potency of rAAV. In this study, we introduce a method for high-purity AAV vector production utilizing recombination-dependent minicircle formation and genetic coupling (AAVPureMfg). Compared with traditional triple transfection, AAVPureMfg substantially improves vector purity by reducing prokaryotic DNA contaminants by 10- to 50-fold and increasing the full capsid ratio up to threefold. Mechanistically, Bxb1-mediated excision of the transgene cassette generates a minicircle cis construct devoid of bacterial sequences and ensures synchronized colocalization of trans and cis constructs in productive cells. Furthermore, we developed iterations that enhance vector genome homogeneity and streamline the production of rAAV with various transgenes, serotypes, and ITR configurations. Overall, our findings demonstrate that AAVPureMfg overcomes the inherent limitations associated with triple transfection, offering a broadly applicable and easy-to-implement method for producing high-purity rAAV with reduced plasmid costs.","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":"61 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-05-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144083296","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}
Nadja Rotte, Jessica E M Dunleavy, Michelle D Runkel, Lina Bosse, Daniela Fietz, Adrian Pilatz, Johanna Kuss, Ann-Kristin Dicke, Sofia B Winge, Sara Di Persio, Christian Ruckert, Verena Nordhoff, Hans-Christian Schuppe, Kristian Almstrup, Sabine Kliesch, Nina Neuhaus, Birgit Stallmeyer, Moira K O'Bryan, Frank Tüttelmann, Corinna Friedrich
{"title":"Genotype-specific differences in infertile men due to loss-of-function variants in M1AP or ZZS genes.","authors":"Nadja Rotte, Jessica E M Dunleavy, Michelle D Runkel, Lina Bosse, Daniela Fietz, Adrian Pilatz, Johanna Kuss, Ann-Kristin Dicke, Sofia B Winge, Sara Di Persio, Christian Ruckert, Verena Nordhoff, Hans-Christian Schuppe, Kristian Almstrup, Sabine Kliesch, Nina Neuhaus, Birgit Stallmeyer, Moira K O'Bryan, Frank Tüttelmann, Corinna Friedrich","doi":"10.1038/s44321-025-00244-0","DOIUrl":"https://doi.org/10.1038/s44321-025-00244-0","url":null,"abstract":"<p><p>Male infertility has been linked to M1AP. In mice, M1AP interacts with the ZZS proteins SHOC1/TEX11/SPO16, promoting DNA class I crossover formation during meiosis. To determine whether M1AP and ZZS proteins are involved in human male infertility by recombination failure, we screened for biallelic/hemizygous loss-of-function (LoF) variants in the human genes to select men with presumed protein deficiency (N = 24). After in-depth characterisation of testicular phenotypes, we identified gene-specific meiotic impairments: men with ZZS deficiency shared an early meiotic arrest. Men with LoF variants in M1AP exhibited a predominant metaphase I arrest with rare haploid round or even elongated spermatids. These differences were explained by different recombination failures: deficient ZZS function led to incorrect synapsis of homologous chromosomes, unrepaired DNA double-strand breaks, and incomplete recombination. Abolished M1AP led to a reduced number of recombination intermediates and class I crossover. Medically assisted reproduction resulted in the birth of a healthy child, offering the possibility of fatherhood to men with LoF variants in M1AP. Our study establishes M1AP as an important, but non-essential, functional enhancer in meiotic recombination.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":""},"PeriodicalIF":9.0,"publicationDate":"2025-05-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144076744","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}
Bee Hui Liu, Miao Liu, Sridhar Radhakrishnan, Meng-Yuan Dai, Chaitanya Kumar Jaladanki, Chong Gao, Jing Ping Tang, Kalpana Kumari, Mei Lin Go, Kim Anh L Vu, Junsu Kwon, Hyuk-Soo Seo, Kijun Song, Xi Tian, Li Feng, Justin L Tan, Arek V Melkonian, Zhaoji Liu, Gerburg Wulf, Haribabu Arthanari, Jun Qi, Sirano Dhe-Paganon, John G Clohessy, Yeu Khai Choong, J Sivaraman, Hao Fan, Daniel G Tenen, Li Chai
{"title":"Targeting transcription factors through an IMiD independent zinc finger domain.","authors":"Bee Hui Liu, Miao Liu, Sridhar Radhakrishnan, Meng-Yuan Dai, Chaitanya Kumar Jaladanki, Chong Gao, Jing Ping Tang, Kalpana Kumari, Mei Lin Go, Kim Anh L Vu, Junsu Kwon, Hyuk-Soo Seo, Kijun Song, Xi Tian, Li Feng, Justin L Tan, Arek V Melkonian, Zhaoji Liu, Gerburg Wulf, Haribabu Arthanari, Jun Qi, Sirano Dhe-Paganon, John G Clohessy, Yeu Khai Choong, J Sivaraman, Hao Fan, Daniel G Tenen, Li Chai","doi":"10.1038/s44321-025-00241-3","DOIUrl":"https://doi.org/10.1038/s44321-025-00241-3","url":null,"abstract":"<p><p>Immunomodulatory imide drugs (IMiDs) degrade specific C2H2 zinc finger degrons in transcription factors, making them effective against certain cancers. SALL4, a cancer driver, contains seven C2H2 zinc fingers in three clusters, including an IMiD degron in zinc finger cluster one (ZFC1). Surprisingly, IMiDs do not inhibit the growth of SALL4-expressing cancer cells. To overcome this limit, we focused on a non-IMiD domain, SALL4 zinc finger cluster four (ZFC4). By combining ZFC4-DNA crystal structure and an in silico docking algorithm, in conjunction with cell viability assays, we screened several chemical libraries against a potentially druggable binding pocket, leading to the discovery of SH6, a compound that selectively targets SALL4-expressing cancer cells. Mechanistic studies revealed that SH6 degrades SALL4 protein through the CUL4A/CRBN pathway, while deletion of ZFC4 abolished this activity. Moreover, SH6 treatment led to a significant 87% tumor growth inhibition of SALL4+ patient-derived xenografts and demonstrated good bioavailability in pharmacokinetic studies. In summary, these studies represent a new approach for IMiD independent drug discovery targeting C2H2 transcription factors such as SALL4 in cancer.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":""},"PeriodicalIF":9.0,"publicationDate":"2025-05-14","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144076745","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}
Dana Krauß,Veronica Moreno-Viedma,Emi Adachi-Fernandez,Cristiano de Sá Fernandes,Jakob-Wendelin Genger,Ourania Fari,Bernadette Blauensteiner,Dominik Kirchhofer,Nikolina Bradaric,Valeriya Gushchina,Georgios Fotakis,Thomas Mohr,Ifat Abramovich,Inbal Mor,Martin Holcmann,Andreas Bergthaler,Arvand Haschemi,Zlatko Trajanoski,Juliane Winkler,Eyal Gottlieb,Maria Sibilia
{"title":"EGFR controls transcriptional and metabolic rewiring in KRASG12D colorectal cancer.","authors":"Dana Krauß,Veronica Moreno-Viedma,Emi Adachi-Fernandez,Cristiano de Sá Fernandes,Jakob-Wendelin Genger,Ourania Fari,Bernadette Blauensteiner,Dominik Kirchhofer,Nikolina Bradaric,Valeriya Gushchina,Georgios Fotakis,Thomas Mohr,Ifat Abramovich,Inbal Mor,Martin Holcmann,Andreas Bergthaler,Arvand Haschemi,Zlatko Trajanoski,Juliane Winkler,Eyal Gottlieb,Maria Sibilia","doi":"10.1038/s44321-025-00240-4","DOIUrl":"https://doi.org/10.1038/s44321-025-00240-4","url":null,"abstract":"Inhibition of the epidermal growth factor receptor (EGFR) shows clinical benefit in metastatic colorectal cancer (CRC) patients, but KRAS-mutations are known to confer resistance. However, recent reports highlight EGFR as a crucial target to be co-inhibited with RAS inhibitors for effective treatment of KRAS mutant CRC. Here, we investigated the tumor cell-intrinsic contribution of EGFR in KRASG12D tumors by establishing murine CRC organoids with key CRC mutations (KRAS, APC, TP53) and inducible EGFR deletion. Metabolomic, transcriptomic, and scRNA-analyses revealed that EGFR deletion in KRAS-mutant organoids reduced their phenotypic heterogeneity and activated a distinct cancer-stem-cell/WNT signature associated with reduced cell size and downregulation of major signaling cascades like MAPK, PI3K, and ErbB. This was accompanied by metabolic rewiring with a decrease in glycolytic routing and increased anaplerotic glutaminolysis. Mechanistically, following EGFR loss, Smoc2 was identified as a key upregulated target mediating these phenotypes that could be rescued upon additional Smoc2 deletion. Validation in patient-datasets revealed that the identified signature is associated with better overall survival of RAS mutant CRC patients possibly allowing to predict therapy responses in patients.","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":"14 1","pages":""},"PeriodicalIF":11.1,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143914934","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}
EMBO Molecular MedicinePub Date : 2025-05-01Epub Date: 2025-04-07DOI: 10.1038/s44321-025-00225-3
Chenyi Wang, Yingjun Shi, Da Zhang, Yupeng Sun, Junjie Xie, Bingchen Wu, Cuilin Zhang, Xiaolong Liu
{"title":"Generalization of neoantigen-based tumor vaccine by delivering peptide-MHC complex via oncolytic virus.","authors":"Chenyi Wang, Yingjun Shi, Da Zhang, Yupeng Sun, Junjie Xie, Bingchen Wu, Cuilin Zhang, Xiaolong Liu","doi":"10.1038/s44321-025-00225-3","DOIUrl":"10.1038/s44321-025-00225-3","url":null,"abstract":"<p><p>Neoantigen vaccine is a promising breakthrough in tumor immunotherapy. However, the application of this highly personalized strategy in the treatment of solid tumors is hindered by several obstacles, including very costly and time-consuming preparation steps, uncertainty in prediction algorithms and tumor heterogeneity. Universalization of neoantigen vaccine is an ideal yet currently unattainable solution to such limitations. To overcome these limitations, we engineered oncolytic viruses co-expressing neoantigens and neoantigen-binding major histocompatibility complex (MHC) molecules to force ectopic delivery of peptide-MHC ligands to T cell receptors (TCRs), enabling specific targeting by neoantigen vaccine-primed host immunity. When integrated with neoantigen vaccination, the engineered viruses exhibited potent cytolytic activity in a variety of tumor models irrespective of the neoantigen expression profiles, eliciting robust systemic antitumor immunity to reject tumor rechallenge and inhibit abscopal tumor growth with a favorable safety profile. Thus, this study provides a powerful approach to enhance the universality and efficacy of neoantigen vaccines, meeting the urgent need for universal neoantigen vaccines in the clinic to facilitate the further development of tumor immunotherapy.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":"1118-1152"},"PeriodicalIF":9.0,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081622/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143802706","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}
EMBO Molecular MedicinePub Date : 2025-05-01Epub Date: 2025-03-10DOI: 10.1038/s44321-025-00202-w
Antoine Jalil, Alessia Perino, Yuan Dong, Jéromine Imbach, Colin Volet, Eduard Vico-Oton, Hadrien Demagny, Lucie Plantade, Hector Gallart-Ayala, Julijana Ivanisevic, Rizlan Bernier-Latmani, Siegfried Hapfelmeier, Kristina Schoonjans
{"title":"Bile acid 7α-dehydroxylating bacteria accelerate injury-induced mucosal healing in the colon.","authors":"Antoine Jalil, Alessia Perino, Yuan Dong, Jéromine Imbach, Colin Volet, Eduard Vico-Oton, Hadrien Demagny, Lucie Plantade, Hector Gallart-Ayala, Julijana Ivanisevic, Rizlan Bernier-Latmani, Siegfried Hapfelmeier, Kristina Schoonjans","doi":"10.1038/s44321-025-00202-w","DOIUrl":"10.1038/s44321-025-00202-w","url":null,"abstract":"<p><p>Host-microbiome communication is frequently perturbed in gut pathologies due to microbiome dysbiosis, leading to altered production of bacterial metabolites. Among these, 7α-dehydroxylated bile acids are notably diminished in inflammatory bowel disease patients. Herein, we investigated whether restoration of 7α-dehydroxylated bile acids levels by Clostridium scindens, a human-derived 7α-dehydroxylating bacterium, can reestablish intestinal epithelium homeostasis following colon injury. Gnotobiotic and conventional mice were subjected to chemically-induced experimental colitis following administration of Clostridium scindens. Colonization enhanced the production of 7α-dehydroxylated bile acids and conferred prophylactic and therapeutic protection against colon injury through epithelial regeneration and specification. Computational analysis of human datasets confirmed defects in intestinal cell renewal and differentiation in ulcerative colitis patients while expression of genes involved in those pathways showed a robust positive correlation with 7α-dehydroxylated bile acid levels. Clostridium scindens administration could therefore be a promising biotherapeutic strategy to foster mucosal healing following colon injury by restoring bile acid homeostasis.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":"889-908"},"PeriodicalIF":9.0,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081655/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143596604","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}
EMBO Molecular MedicinePub Date : 2025-05-01Epub Date: 2025-03-26DOI: 10.1038/s44321-025-00223-5
April L Risinger
{"title":"Beyond mitotic arrest: the diverse effects of microtubule-targeting drugs on tumor vasculature.","authors":"April L Risinger","doi":"10.1038/s44321-025-00223-5","DOIUrl":"10.1038/s44321-025-00223-5","url":null,"abstract":"","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":"866-868"},"PeriodicalIF":9.0,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081712/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143718118","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}
{"title":"Liver TET1 promotes metabolic dysfunction-associated steatotic liver disease.","authors":"Hongze Chen, Muhammad Azhar Nisar, Joud Mulla, Xinjian Li, Kevin Cao, Shaolei Lu, Katsuya Nagaoka, Shang Wu, Peng-Sheng Ting, Tung-Sung Tseng, Hui-Yi Lin, Xiao-Ming Yin, Wenke Feng, Zhijin Wu, Zhixiang Cheng, William Mueller, Amalia Bay, Layla Schechner, Xuewei Bai, Chiung-Kuei Huang","doi":"10.1038/s44321-025-00224-4","DOIUrl":"10.1038/s44321-025-00224-4","url":null,"abstract":"<p><p>Global hepatic DNA methylation change has been linked to human patients with metabolic dysfunction-associated steatotic liver disease (MASLD). DNA demethylation is regulated by the TET family proteins, whose enzymatic activities require 2-oxoglutarate (2-OG) and iron that both are elevated in human MASLD patients. We aimed to investigate liver TET1 in MASLD progression. Depleting TET1 using two different strategies substantially alleviated MASLD progression. Knockout (KO) of TET1 slightly improved diet induced obesity and glucose homeostasis. Intriguingly, hepatic cholesterols, triglycerides, and CD36 were significantly decreased upon TET1 depletion. Consistently, liver specific TET1 KO led to improvement of MASLD progression. Mechanistically, TET1 promoted CD36 expression through transcriptional upregulation via DNA demethylation control. Overexpression of CD36 reversed the impacts of TET1 downregulation on fatty acid uptake in hepatocytes. More importantly, targeting TET1 with a small molecule inhibitor significantly suppressed MASLD progression. Conclusively, liver TET1 plays a deleterious role in MASLD, suggesting the potential of targeting TET1 in hepatocytes to suppress MASLD.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":"1101-1117"},"PeriodicalIF":9.0,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081649/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143751613","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}
EMBO Molecular MedicinePub Date : 2025-05-01Epub Date: 2025-03-27DOI: 10.1038/s44321-025-00221-7
Alexandra Helleux, Guillaume Davidson, Antonin Lallement, Fatima Al Hourani, Alexandre Haller, Isabelle Michel, Anas Fadloun, Christelle Thibault-Carpentier, Xiaoping Su, Véronique Lindner, Thibault Tricard, Hervé Lang, Nizar M Tannir, Irwin Davidson, Gabriel G Malouf
{"title":"TFE3 fusions drive oxidative metabolism and ferroptosis resistance in translocation renal cell carcinoma.","authors":"Alexandra Helleux, Guillaume Davidson, Antonin Lallement, Fatima Al Hourani, Alexandre Haller, Isabelle Michel, Anas Fadloun, Christelle Thibault-Carpentier, Xiaoping Su, Véronique Lindner, Thibault Tricard, Hervé Lang, Nizar M Tannir, Irwin Davidson, Gabriel G Malouf","doi":"10.1038/s44321-025-00221-7","DOIUrl":"10.1038/s44321-025-00221-7","url":null,"abstract":"<p><p>The oncogenic mechanisms by which TFE3 fusion proteins drive translocation renal cell carcinoma (tRCC) are poorly characterized. Here, we integrated loss and gain of function experiments with multi-omics analyses in tRCC cell lines and patient tumors. High nuclear accumulation of NONO-TFE3 or PRCC-TFE3 fusion proteins promotes their broad binding across the genome at H3K27ac-marked active chromatin, engaging a core set of M/E-box-containing regulatory elements to activate specific gene expression programs as well as promiscuous binding to active promoters to stimulate mRNA synthesis. Within the core program, TFE3 fusions directly regulate genes involved in ferroptosis resistance and oxidative phosphorylation metabolism (OxPhos). Consequently, human tRCC tumors display high OxPhos scores that persist during their epithelial to mesenchymal transition (EMT). We further show that tRCC tumor aggressiveness is related to their EMT and their associated enrichment in myofibroblast cancer-associated fibroblasts (myCAFs) that are both hallmarks of poor prognostic outcomes. We define tRCC as a novel metabolic subtype of renal cancer and provide unique insights into how broad genomic binding of TFE3 fusion proteins regulates OxPhos and ferroptosis resistance.</p>","PeriodicalId":11597,"journal":{"name":"EMBO Molecular Medicine","volume":" ","pages":"1041-1070"},"PeriodicalIF":9.0,"publicationDate":"2025-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12081665/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143729359","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}
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