Molecular Therapy最新文献_第3页

Machine learning approaches enable the discovery of therapeutics across domains. 机器学习方法可以跨领域发现治疗方法。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-03 DOI: 10.1016/j.ymthe.2025.04.001

Prabal Chhibbar, Jishnu Das

引用次数: 0

An engineered mitoCBE facilitates efficient mitochondrial DNA editing and modified mitochondrial transfer. 工程mitoCBE促进高效的线粒体DNA编辑和修改线粒体转移。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-03 DOI: 10.1016/j.ymthe.2025.03.051

Jie Liu, Jun Chen, Shisheng Huang, Junfan Guo, Xiangyang Li, Ying Yan, Ruijing Chen, Guanglei Li, Ming Liu, Jiao Wei, Xingxu Huang, Yunbo Qiao

{"title":"An engineered mitoCBE facilitates efficient mitochondrial DNA editing and modified mitochondrial transfer.","authors":"Jie Liu, Jun Chen, Shisheng Huang, Junfan Guo, Xiangyang Li, Ying Yan, Ruijing Chen, Guanglei Li, Ming Liu, Jiao Wei, Xingxu Huang, Yunbo Qiao","doi":"10.1016/j.ymthe.2025.03.051","DOIUrl":"https://doi.org/10.1016/j.ymthe.2025.03.051","url":null,"abstract":"Double-stranded DNA (dsDNA) cytosine deaminase DddA orthologs from multiple types of bacteria have been fused with TALE system for mitochondrial DNA (mtDNA) base editing, while the efficiencies remain limited and its nuclear off-targeting activity cannot be ignored yet. Here we identified a DddA ortholog from Burkholderia gladioli (BgDddA) and generated nuclear or mitochondrial DNA cytosine base editors (mitoCBEs), exhibiting higher C•G-to-T•A editing frequencies compared to canonical DdCBE, and fusion with transactivator Rta remarkably improved editing efficiencies by up to 6.4-fold at non-TC targets. Referring to DddA11, we further introduced six substitutions into BgDddA and generated mitoCBE3.2, which efficiently induced disease-associated mtDNA mutations in mouse and human cell lines at both TC and non-TC targets with efficiency reaching up to 99.2%. Using mitoCBE3.2, single clones containing homoplasmic mtDNA mutations or premature stop codons associated with human diseases were generated, and the functions of these mutations have been evaluated upon the treatment of ROS inducers. Importantly, mitochondria harboring these homoplasmic mutations were transplanted into wildtype cells, enabling precise base conversions, without risk of nuclear gene off-targets. Thus, we have engineered an efficient mitoCBE using BgDddA, facilitating mitochondrial disease modeling and potential mutation correction with the aid of mitochondrial transplantation.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":""},"PeriodicalIF":12.1,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143779713","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}

引用次数: 0

AAV Vector Development, Back to the Future. AAV载体发展，回到未来。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-03 DOI: 10.1016/j.ymthe.2025.03.064

Lester Suarez-Amaran, Liujiang Song, Anna P Tretiakova, Sheila A Mikhail, Richard Jude Samulski

{"title":"AAV Vector Development, Back to the Future.","authors":"Lester Suarez-Amaran, Liujiang Song, Anna P Tretiakova, Sheila A Mikhail, Richard Jude Samulski","doi":"10.1016/j.ymthe.2025.03.064","DOIUrl":"https://doi.org/10.1016/j.ymthe.2025.03.064","url":null,"abstract":"Adeno-Associated Virus (AAV) has become a pivotal tool in gene therapy, providing a safe and efficient platform for long-term transgene expression. This review presents a comprehensive analysis of AAV's historical development, from its initial identification as a \"contaminant\" to its current clinical applications. We examine the molecular evolution of AAV, detailing advancements in vector engineering, rational design, directed evolution platforms, and computational modeling, which have expanded its therapeutic potential across diverse disease areas. Additionally, we explore AAV genome regulation, with a particular focus on ITRs and AAV capsid-genome interactions, which play a crucial role in vector transduction efficiency and host adaptation. An assessment of past and present clinical trials as well as future directions is provided to illustrate the field's trajectory. Finally, another unique milestone in AAV research is also reported; namely, a pool of AAV libraries has been successfully administered to human decedents and analyzed, representing a transformative step in AAV evolution and selection for human applications. These studies should pave the way for more refined AAV vector optimization, accelerating the development of next-generation gene therapies with enhanced clinical translatability, potentially accelerating the gene therapy revolution.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":""},"PeriodicalIF":12.1,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788668","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}

引用次数: 0

Therapeutic application of extracellular vesicles in human diseases. 细胞外囊泡在人类疾病中的治疗应用。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-03 DOI: 10.1016/j.ymthe.2025.04.002

Allancer Nunes, Tianpeng Zhang, Xiaodong Mu, Paul D Robbins

引用次数: 0

The advent of clinical self-amplifying RNA vaccines. 临床自我扩增RNA疫苗的出现。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-03 DOI: 10.1016/j.ymthe.2025.03.060

Irafasha C Casmil, Jongwoo Jin, Eun-Jeong Won, Cynthia Huang, Suiyang Liao, Hyunjoo Cha-Molstad, Anna K Blakney

{"title":"The advent of clinical self-amplifying RNA vaccines.","authors":"Irafasha C Casmil, Jongwoo Jin, Eun-Jeong Won, Cynthia Huang, Suiyang Liao, Hyunjoo Cha-Molstad, Anna K Blakney","doi":"10.1016/j.ymthe.2025.03.060","DOIUrl":"https://doi.org/10.1016/j.ymthe.2025.03.060","url":null,"abstract":"Self-amplifying RNA (saRNA) technology is an emerging platform for vaccine development, offering significant advantages over conventional mRNA vaccines. By enabling intracellular amplification of RNA, saRNA facilitates robust antigen expression at lower doses, thereby enhancing both immunogenicity and cost-effectiveness. This review examines the latest advancements in saRNA vaccine development, highlighting its applications in combating infectious diseases, including viral pathogens such as SARS-CoV-2, influenza, and emerging zoonotic threats. We discuss the design and optimization of saRNA vectors to maximize antigen expression while minimizing adverse immune responses. Recent studies demonstrating the safety, efficacy, and scalability of saRNA-based vaccines in clinical settings are also discussed. We address challenges related to delivery systems, stability, and manufacturing, along with novel strategies being developed to mitigate these challenges. As the global demand for rapid, flexible, and scalable vaccine platforms grows, saRNA presents a promising solution with enhanced potency and durability. This review emphasizes the transformative potential of saRNA vaccines to shape the future of immunization strategies, particularly in response to pandemics and other global health threats.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":""},"PeriodicalIF":12.1,"publicationDate":"2025-04-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143788691","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}

引用次数: 0

Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity. 靶向BCL11B在car工程淋巴样祖细胞驱动nk样细胞发育具有持久的抗白血病活性。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-02 Epub Date: 2025-02-15 DOI: 10.1016/j.ymthe.2025.02.024

Franziska Baatz, Arnab Ghosh, Jessica Herbst, Saskia Polten, Johann Meyer, Manuel Rhiel, Tobias Maetzig, Robert Geffers, Michael Rothe, Antonella Lucia Bastone, Philipp John-Neek, Jörg Frühauf, Britta Eiz-Vesper, Agnes Bonifacius, Christine S Falk, Constantin V Kaisenberg, Toni Cathomen, Axel Schambach, Marcel R M van den Brink, Michael Hust, Martin G Sauer

{"title":"Targeting BCL11B in CAR-engineered lymphoid progenitors drives NK-like cell development with prolonged anti-leukemic activity.","authors":"Franziska Baatz, Arnab Ghosh, Jessica Herbst, Saskia Polten, Johann Meyer, Manuel Rhiel, Tobias Maetzig, Robert Geffers, Michael Rothe, Antonella Lucia Bastone, Philipp John-Neek, Jörg Frühauf, Britta Eiz-Vesper, Agnes Bonifacius, Christine S Falk, Constantin V Kaisenberg, Toni Cathomen, Axel Schambach, Marcel R M van den Brink, Michael Hust, Martin G Sauer","doi":"10.1016/j.ymthe.2025.02.024","DOIUrl":"10.1016/j.ymthe.2025.02.024","url":null,"abstract":"Chimeric antigen receptor (CAR)-induced suppression of the transcription factor B cell CLL/lymphoma 11B (BCL11B) propagates CAR-induced killer (CARiK) cell development from lymphoid progenitors. Here, we show that CRISPR-Cas9-mediated Bcl11b knockout in human and murine early lymphoid progenitors distinctively modulates this process either alone or in combination with a CAR. Upon adoptive transfer into hematopoietic stem cell recipients, Bcl11b-edited progenitors mediated innate-like antigen-independent anti-leukemic immune responses. With CAR expression allowing for additional antigen-specific responses, the progeny of double-edited lymphoid progenitors acquired prolonged anti-leukemic activity in vivo. These findings give important insights into how Bcl11b targeting can be used to tailor anti-leukemia functionality of CAR-engineered lymphoid progenitor cells.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1584-1607"},"PeriodicalIF":12.1,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143425833","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}

引用次数: 0

Viral and cellular insulators promote sustained HSV vector-mediated transgene expression in brain. 病毒和细胞绝缘体可促进 HSV 载体介导的转基因在大脑中的持续表达

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-02 Epub Date: 2025-02-28 DOI: 10.1016/j.ymthe.2025.02.039

Selene Ingusci, Justus B Cohen, Joseph C Glorioso

{"title":"Viral and cellular insulators promote sustained HSV vector-mediated transgene expression in brain.","authors":"Selene Ingusci, Justus B Cohen, Joseph C Glorioso","doi":"10.1016/j.ymthe.2025.02.039","DOIUrl":"10.1016/j.ymthe.2025.02.039","url":null,"abstract":"We have developed a gene therapy platform based on non-toxic, high-capacity replication-defective (rd) herpes simplex virus type 1 (HSV-1) vectors. We previously determined that transgene expression from rdHSV-1 vectors requires strategic placement of insulators-small DNA elements that overcome the host's epigenetic silencing of foreign DNA-to maintain transgenes in euchromatin regions. Transgene expression was rescued by replacing either the latency associated transcript (LAT) or the the infected cell protein 4 (ICP4) gene with the transgene cassette close to naturally occurring viral insulators. The ICP4 locus was more permissive for transgene expression than the LAT locus in neurons in vitro. Following in vivo brain delivery, transgene expression from both loci lasted for at least 4 months. However, the level of expression tended to decline over time. To enhance transgene expression, we designed a novel insulator environment by combining cellular insulators with the resident viral insulators. In combination, these elements provided significantly higher levels of transgene expression in the brain than the viral insulators alone, lasting for at least 11.7 months. This new cassette design extends transgene activity in neurons compared with previous designs and holds promise for gene therapy applications in treating brain disorders.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1420-1433"},"PeriodicalIF":12.1,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531681","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}

引用次数: 0

A universal viral capsid protein based one step RNA synthesis and packaging system for rapid and efficient mRNA vaccine development. 一种基于通用病毒衣壳蛋白的一步RNA合成和包装系统，用于快速高效的mRNA疫苗开发。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-02 Epub Date: 2025-02-28 DOI: 10.1016/j.ymthe.2025.02.037

Jiayue Su, Jinsong Zhang, Xiangning Feng, Jinsong Liu, Shan Gao, Xinrui Liu, Mingwei Yang, Zeliang Chen

{"title":"A universal viral capsid protein based one step RNA synthesis and packaging system for rapid and efficient mRNA vaccine development.","authors":"Jiayue Su, Jinsong Zhang, Xiangning Feng, Jinsong Liu, Shan Gao, Xinrui Liu, Mingwei Yang, Zeliang Chen","doi":"10.1016/j.ymthe.2025.02.037","DOIUrl":"10.1016/j.ymthe.2025.02.037","url":null,"abstract":"The success of coronavirus disease 2019 mRNA vaccines highlights the transformative potential of mRNA technology. Current mRNA vaccine development involves complex steps, including plasmid construction, RNA transcription, 5' capping, poly(A) tailing, and lipid nanoparticle encapsulation, yet challenges in vaccine accessibility persist. Here, we present an innovative mRNA platform leveraging the self-assembly capabilities of the MS2 bacteriophage viral capsid protein (VCP). A dual-promoter plasmid has been designed where one promoter drives VCP expression while the other transcribes target RNA containing pac sites, enabling rapid mRNA self-assembly in Escherichia coli. Using an ovalbumin (OVA)-based tumor model, we validate the efficacy of this system. Tumor growth is significantly inhibited, accompanied by robust immune activation. Flow cytometry analyses reveal increased frequencies of OVA-specific CD8+, as well as activated and memory T cells. Additionally, the MS2-OVA vaccine favorably modulated the tumor immunosuppressive microenvironment by reducing myeloid-derived suppressor cells, while sustained antibody responses demonstrated the platform's ability to induce durable humoral immunity. These findings establish the feasibility of one-step mRNA synthesis and packaging in E. coli, providing a versatile and rapid platform for mRNA vaccine development, with broad implications for addressing global vaccination challenges.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1720-1734"},"PeriodicalIF":12.1,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531701","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}

引用次数: 0

A differentiated β-globin gene replacement strategy uses heterologous introns to restore physiological expression. 利用异源内含子恢复生理表达的分化β-球蛋白基因替换策略。

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-02 Epub Date: 2025-02-28 DOI: 10.1016/j.ymthe.2025.02.036

Kirby A Wallace, Trevor L Gerstenberg, Craig L Ennis, Juan A Perez-Bermejo, James R Partridge, Christopher Bandoro, William M Matern, Gaia Andreoletti, Kristina Krassovsky, Shaheen Kabir, Cassandra D Lalisan, Aishwarya R Churi, Glen M Chew, Lana Corbo, Jon E Vincelette, Timothy D Klasson, Brian J Silva, Yuri G Strukov, B Joy Quejarro, Kaisle A Hill, Sebastian Treusch, Jane L Grogan, Daniel P Dever, Matthew H Porteus, Beeke Wienert

{"title":"A differentiated β-globin gene replacement strategy uses heterologous introns to restore physiological expression.","authors":"Kirby A Wallace, Trevor L Gerstenberg, Craig L Ennis, Juan A Perez-Bermejo, James R Partridge, Christopher Bandoro, William M Matern, Gaia Andreoletti, Kristina Krassovsky, Shaheen Kabir, Cassandra D Lalisan, Aishwarya R Churi, Glen M Chew, Lana Corbo, Jon E Vincelette, Timothy D Klasson, Brian J Silva, Yuri G Strukov, B Joy Quejarro, Kaisle A Hill, Sebastian Treusch, Jane L Grogan, Daniel P Dever, Matthew H Porteus, Beeke Wienert","doi":"10.1016/j.ymthe.2025.02.036","DOIUrl":"10.1016/j.ymthe.2025.02.036","url":null,"abstract":"β-Hemoglobinopathies are common monogenic disorders. In sickle cell disease (SCD), a single mutation in the β-globin (HBB) gene results in dysfunctional hemoglobin protein, while in β-thalassemia, over 300 mutations distributed across the gene reduce β-globin levels and cause severe anemia. Genetic engineering replacing the whole HBB gene through homology-directed repair (HDR) is an ideal strategy to restore a benign genotype and rescue HBB expression for most genotypes. However, this is technically challenging because (1) the insert must not be homologous to the endogenous gene and (2) synonymous codon-optimized, intron-less sequences may not reconstitute adequate β-globin levels. Here, we developed an HBB gene replacement strategy using CRISPR-Cas9 that successfully addresses these challenges. We determined that a DNA donor containing a diverged HBB coding sequence and heterologous introns to avoid sequence homology provides proper physiological expression. We identified a DNA donor that uses truncated γ-globin introns, results in 34% HDR, and rescues β-globin expression in in vitro models of SCD and β-thalassemia in hematopoietic stem and progenitor cells (HSPCs). Furthermore, while HDR allele frequency dropped in vivo, it was maintained at ∼15%, demonstrating editing of long-term repopulating HSPCs. In summary, our HBB gene replacement strategy offers a differentiated approach by restoring naturally regulated adult hemoglobin expression.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1407-1419"},"PeriodicalIF":12.1,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143531697","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}

引用次数: 0

Impaired megakaryopoiesis due to aberrant macrophage polarization via BTK/Rap1/NF-κB pathway in sepsis-induced thrombocytopenia. 脓毒症所致血小板减少症中巨噬细胞异常极化通过BTK/Rap1/NF-κB通路导致巨核生成受损

IF 12.1 1区医学

Molecular Therapy Pub Date : 2025-04-02 Epub Date: 2024-12-30 DOI: 10.1016/j.ymthe.2024.12.048

Ziyan Zhang, Meng Zhou, Yaqiong Tang, Jiaqian Qi, Xiaoyan Xu, Peng Wang, Haohao Han, Tingting Pan, Xiaofei Song, Shuhui Jiang, Xueqian Li, Chengyuan Gu, Zhenzhen Yao, Qixiu Hou, Mengting Guo, Siyi Lu, Depei Wu, Yue Han

{"title":"Impaired megakaryopoiesis due to aberrant macrophage polarization via BTK/Rap1/NF-κB pathway in sepsis-induced thrombocytopenia.","authors":"Ziyan Zhang, Meng Zhou, Yaqiong Tang, Jiaqian Qi, Xiaoyan Xu, Peng Wang, Haohao Han, Tingting Pan, Xiaofei Song, Shuhui Jiang, Xueqian Li, Chengyuan Gu, Zhenzhen Yao, Qixiu Hou, Mengting Guo, Siyi Lu, Depei Wu, Yue Han","doi":"10.1016/j.ymthe.2024.12.048","DOIUrl":"10.1016/j.ymthe.2024.12.048","url":null,"abstract":"Sepsis-induced thrombocytopenia (SIT) is a widely accepted predictor of poor prognosis during sepsis, while the mechanism of SIT remains elusive. In this study, we revealed that SIT patients and septic mice exhibited higher levels of pro-inflammatory macrophages and phosphorylated Bruton's tyrosine kinase (p-BTK) expression in macrophages, which were closely correlated with platelet counts. Treatment with the BTK inhibitor BGB-3111 in SIT mice resulted in enhanced production of megakaryocytes and platelets. Depletion of macrophages in SIT mice and coculture experiments further confirmed the critical role of macrophages in the improvement of platelet count induced by BGB-3111. By performing single-cell RNA sequencing on bone marrow-derived cells from SIT mice, we not only confirmed the connection between macrophages and megakaryocytes influenced by BTK but also identified a potential mediation through the Rap1 signaling pathway in macrophages. Subsequent experiments in macrophages demonstrated that inhibition of BTK signaling impeded the pro-inflammatory polarization of macrophages by targeting the Rap1/NF-κB signaling pathway. In conclusion, our study highlights the crucial role of macrophages in SIT, and inhibiting phosphorylation of BTK in macrophages may alleviate SIT through the Rap1/NF-κB signaling pathway.","PeriodicalId":19020,"journal":{"name":"Molecular Therapy","volume":" ","pages":"1769-1784"},"PeriodicalIF":12.1,"publicationDate":"2025-04-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142915312","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}

引用次数: 0