{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,22]],"date-time":"2026-01-22T01:31:15Z","timestamp":1769045475010,"version":"3.49.0"},"reference-count":51,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T00:00:00Z","timestamp":1754524800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Poliomyelitis Research Foundation"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Viruses"],"abstract":"<jats:p>Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-\u03b1 and nucleoside\/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently.<\/jats:p>","DOI":"10.3390\/v17081090","type":"journal-article","created":{"date-parts":[[2025,8,7]],"date-time":"2025-08-07T10:07:38Z","timestamp":1754561258000},"page":"1090","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Lipid Nanoparticle-Encapsulated TALEN-Encoding mRNA Inactivates Hepatitis B Virus Replication in Cultured Cells and Transgenic Mice"],"prefix":"10.3390","volume":"17","author":[{"given":"Tiffany","family":"Smith","sequence":"first","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Prashika","family":"Singh","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ridhwaanah","family":"Bhana","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dylan","family":"Kairuz","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5796-7720","authenticated-orcid":false,"given":"Kristie","family":"Bloom","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2249-7615","authenticated-orcid":false,"given":"Mohube Betty","family":"Maepa","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1205-2436","authenticated-orcid":false,"given":"Abdullah","family":"Ely","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4149-8995","authenticated-orcid":false,"given":"Patrick","family":"Arbuthnot","sequence":"additional","affiliation":[{"name":"Wits\/SAMRC Antiviral Gene Therapy Research Unit, Infectious Diseases and Oncology Research Institute (IDORI), School of Pathology, Faculty of Health Sciences, University of the Witwatersrand, Parktown 2193, South Africa"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,8,7]]},"reference":[{"key":"ref_1","unstructured":"World Health Organization (WHO) (2024, April 10). Hepatitis B, Key Facts. Available online: http:\/\/www.who.int\/news-room\/fact-sheets\/detail\/hepatitis-b."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"524","DOI":"10.1038\/s41575-023-00760-9","article-title":"Global burden of hepatitis B virus: Current status, missed opportunities and a call for action","volume":"20","author":"Hsu","year":"2023","journal-title":"Nat. Rev. Gastroenterol. Hepatol."},{"key":"ref_3","first-page":"209","article-title":"Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries","volume":"71","author":"Sung","year":"2021","journal-title":"CA Cancer J. Clin."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1016\/j.livres.2024.05.004","article-title":"Hepatitis B virus as a risk factor for hepatocellular carcinoma: There is still much work to do","volume":"8","author":"Abdelhamed","year":"2024","journal-title":"Liver Res."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/0092-8674(92)90599-8","article-title":"The reverse transcriptase of hepatitis B virus acts as a protein primer for viral DNA synthesis","volume":"71","author":"Wang","year":"1992","journal-title":"Cell"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"127","DOI":"10.1099\/vir.0.004861-0","article-title":"Persistence of the hepatitis B virus covalently closed circular DNA in HepaRG human hepatocyte-like cells","volume":"90","author":"Hantz","year":"2009","journal-title":"J. Gen. Virol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"489","DOI":"10.1038\/317489a0","article-title":"The hepatitis B virus","volume":"317","author":"Tiollais","year":"1985","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1038\/s41564-020-0678-0","article-title":"Core components of DNA lagging strand synthesis machinery are essential for hepatitis B virus cccDNA formation","volume":"5","author":"Wei","year":"2020","journal-title":"Nat. Microbiol."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Knolle, P.A., Huang, L.-R., Kosinska, A., Wohlleber, D., and Protzer, U. (2021). Improving Therapeutic Vaccination against Hepatitis B\u2014Insights from Preclinical Models of Immune Therapy against Persistent Hepatitis B Virus Infection. Vaccines, 9.","DOI":"10.3390\/vaccines9111333"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"852","DOI":"10.1111\/jvh.13490","article-title":"Clinical effects of NTCP-inhibitor myrcludex B","volume":"28","author":"Cheng","year":"2021","journal-title":"J. Viral Hepat."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2180","DOI":"10.1053\/j.gastro.2020.02.058","article-title":"Safety and Efficacy of 48 Weeks REP 2139 or REP 2165, Tenofovir Disoproxil, and Pegylated Interferon Alfa-2a in Patients With Chronic HBV Infection Na\u00efve to Nucleos(t)ide Therapy","volume":"158","author":"Bazinet","year":"2020","journal-title":"Gastroenterology"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"189","DOI":"10.1002\/hep4.1633","article-title":"Persistent Control of Hepatitis B Virus and Hepatitis Delta Virus Infection Following REP 2139-Ca and Pegylated Interferon Therapy in Chronic Hepatitis B Virus\/Hepatitis Delta Virus Coinfection","volume":"5","author":"Bazinet","year":"2021","journal-title":"Hepatol. Commun."},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Weber, N.D., Stone, D., Sedlak, R.H., De Silva Feelixge, H.S., Roychoudhury, P., Schiffer, J.T., Aubert, M., and Jerome, K.R. (2014). AAV-mediated delivery of zinc finger nucleases targeting hepatitis B virus inhibits active replication. PLoS ONE, 9.","DOI":"10.1371\/journal.pone.0097579"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1889","DOI":"10.1038\/mt.2013.170","article-title":"Inactivation of Hepatitis B Virus Replication in Cultured Cells and In Vivo with Engineered Transcription Activator-Like Effector Nucleases","volume":"21","author":"Bloom","year":"2013","journal-title":"Mol. Ther."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/j.virol.2014.12.001","article-title":"Suppression of hepatitis B virus DNA accumulation in chronically infected cells using a bacterial CRISPR\/Cas RNA-guided DNA endonuclease","volume":"476","author":"Kennedy","year":"2015","journal-title":"Virology"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"947","DOI":"10.1038\/mt.2010.20","article-title":"Zinc-finger Nucleases as a Novel Therapeutic Strategy for Targeting Hepatitis B Virus DNAs","volume":"18","author":"Cradick","year":"2010","journal-title":"Mol. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1509","DOI":"10.1126\/science.1178811","article-title":"Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors","volume":"326","author":"Boch","year":"2009","journal-title":"Science"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"757","DOI":"10.1534\/genetics.110.120717","article-title":"Targeting DNA double-strand breaks with TAL effector nucleases","volume":"186","author":"Christian","year":"2010","journal-title":"Genetics"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"249","DOI":"10.1038\/s41591-018-0326-x","article-title":"Identification of preexisting adaptive immunity to Cas9 proteins in humans","volume":"25","author":"Charlesworth","year":"2019","journal-title":"Nat. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1389","DOI":"10.1016\/j.ymthe.2020.05.007","article-title":"Immunity to Cas9 as an Obstacle to Persistent Genome Editing","volume":"28","author":"Gough","year":"2020","journal-title":"Mol. Ther."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"347","DOI":"10.1038\/nbt.2842","article-title":"CRISPR-Cas systems for editing, regulating and targeting genomes","volume":"32","author":"Sander","year":"2014","journal-title":"Nat. Biotechnol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"606","DOI":"10.1038\/s41467-020-20672-5","article-title":"TALEN outperforms Cas9 in editing heterochromatin target sites","volume":"12","author":"Jain","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"303","DOI":"10.1038\/mt.2013.212","article-title":"An Efficient Antiviral Strategy for Targeting Hepatitis B Virus Genome Using Transcription Activator-Like Effector Nucleases","volume":"22","author":"Chen","year":"2014","journal-title":"Mol. Ther."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Smith, T., Singh, P., Chmielewski, K.O., Bloom, K., Cathomen, T., Arbuthnot, P., and Ely, A. (2021). Improved Specificity and Safety of Anti-Hepatitis B Virus TALENs Using Obligate Heterodimeric FokI Nuclease Domains. Viruses, 13.","DOI":"10.3390\/v13071344"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"134","DOI":"10.1016\/j.addr.2020.05.010","article-title":"In vitro transcribed mRNA for expression of designer nucleases: Advantages as a novel therapeutic for the management of chronic HBV infection","volume":"168","author":"Ely","year":"2021","journal-title":"Adv. Drug Deliv. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"eabf4398","DOI":"10.1126\/sciadv.abf4398","article-title":"Engineered ionizable lipid nanoparticles for targeted delivery of RNA therapeutics into different types of cells in the liver","volume":"7","author":"Kim","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"778","DOI":"10.1038\/nbt1319","article-title":"An improved zinc-finger nuclease architecture for highly specific genome editing","volume":"25","author":"Miller","year":"2007","journal-title":"Nat. Biotechnol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e39","DOI":"10.1002\/cpz1.39","article-title":"Cap 1 Messenger RNA Synthesis with Co-transcriptional CleanCap\u00ae Analog by In Vitro Transcription","volume":"1","author":"Henderson","year":"2021","journal-title":"Curr. Protoc."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"26","DOI":"10.1016\/j.omtn.2019.02.018","article-title":"A Facile Method for the Removal of dsRNA Contaminant from In Vitro-Transcribed mRNA","volume":"15","author":"Boros","year":"2019","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"808","DOI":"10.1016\/j.bbrc.2013.12.052","article-title":"Evaluation and identification of hepatitis B virus entry inhibitors using HepG2 cells overexpressing a membrane transporter NTCP","volume":"443","author":"Iwamoto","year":"2014","journal-title":"Biochem. Biophys. Res. Commun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1357","DOI":"10.1016\/0092-8674(90)90431-D","article-title":"Translational inactivation of RNA function: Discrimination against a subset of genomic transcripts during HBV nucleocapsid assembly","volume":"63","author":"Nassal","year":"1990","journal-title":"Cell"},{"key":"ref_32","doi-asserted-by":"crossref","unstructured":"Limani, S.W., Mnyandu, N., Ely, A., Wadee, R., Kramvis, A., Arbuthnot, P., and Maepa, M.B. (2021). In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors. Viruses, 13.","DOI":"10.3390\/v13112247"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"31715","DOI":"10.1074\/jbc.M113.501122","article-title":"Interleukin-1 and Tumor Necrosis Factor-\u03b1 Trigger Restriction of Hepatitis B Virus Infection via a Cytidine Deaminase Activation-induced Cytidine Deaminase (AID)","volume":"288","author":"Watashi","year":"2013","journal-title":"J. Biol. Chem."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1970","DOI":"10.1016\/j.ymthe.2021.04.001","article-title":"A single dose of self-transcribing and replicating RNA-based SARS-CoV-2 vaccine produces protective adaptive immunity in mice","volume":"29","author":"Gan","year":"2021","journal-title":"Mol. Ther."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1509","DOI":"10.1016\/j.ymthe.2018.03.010","article-title":"A Novel Amino Lipid Series for mRNA Delivery: Improved Endosomal Escape and Sustained Pharmacology and Safety in Non-human Primates","volume":"26","author":"Sabnis","year":"2018","journal-title":"Mol. Ther. J. Am. Soc. Gene Ther."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Marion, P.L., Salazar, F.H., Liittschwager, K., Bordier, B.B., Seeger, C., Winters, M.A., Cooper, A.D., and Cullen, J.M. (2003). A transgenic mouse lineage useful for testing antivirals targeting hepatitis B virus. Frontiers in Viral Hepatitis, Elsevier. Available online: https:\/\/linkinghub.elsevier.com\/retrieve\/pii\/B9780444509864500692.","DOI":"10.1016\/B978-044450986-4\/50069-2"},{"key":"ref_37","unstructured":"Guo, H., and Cuconati, A. (2017). A T7 Endonuclease I Assay to Detect Talen-Mediated Targeted Mutation of HBV cccDNA. Hepatitis B Virus, Springer. Available online: http:\/\/link.springer.com\/10.1007\/978-1-4939-6700-1_8."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"381","DOI":"10.2135\/cropsci2008.07.0413","article-title":"CEL I Nuclease Digestion for SNP Discovery and Marker Development in Common Bean (Phaseolus vulgaris L.)","volume":"49","author":"Galeano","year":"2009","journal-title":"Crop Sci."},{"key":"ref_39","unstructured":"Mackay, J.P., and Segal, D.J. (2010). A Rapid and General Assay for Monitoring Endogenous Gene Modification. Engineered Zinc Finger Proteins, Humana Press. Available online: http:\/\/link.springer.com\/10.1007\/978-1-60761-753-2_15."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"e91","DOI":"10.1093\/nar\/gkp446","article-title":"Efficient silencing of gene expression with modular trimeric Pol II expression cassettes comprising microRNA shuttles","volume":"37","author":"Ely","year":"2009","journal-title":"Nucleic Acids Res."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"51","DOI":"10.1128\/MMBR.64.1.51-68.2000","article-title":"Hepatitis B virus biology","volume":"64","author":"Seeger","year":"2000","journal-title":"Microbiol. Mol. Biol. Rev. MMBR"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"197","DOI":"10.1016\/j.yexcr.2004.02.031","article-title":"Monocyte chemoattractant protein-1 and macrophage inflammatory protein-2 are involved in both excitotoxin-induced neurodegeneration and regeneration","volume":"297","author":"Kalehua","year":"2004","journal-title":"Exp. Cell Res."},{"key":"ref_43","doi-asserted-by":"crossref","unstructured":"Patel, M.N., Tiwari, S., Wang, Y., O\u2019Neill, S., Wu, J., Omo-Lamai, S., Espy, C., Chase, L.S., Majumder, A., and Hoffman, E. (2025). Safer non-viral DNA delivery using lipid nanoparticles loaded with endogenous anti-inflammatory lipids. Nat. Biotechnol., 1\u201311.","DOI":"10.1038\/s41587-025-02556-5"},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"e02137-16","DOI":"10.1128\/JVI.02137-16","article-title":"High Prevalence of Infectious Adeno-associated Virus (AAV) in Human Peripheral Blood Mononuclear Cells Indicative of T Lymphocytes as Sites of AAV Persistence","volume":"91","author":"Khalid","year":"2017","journal-title":"J. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"107857","DOI":"10.1016\/j.heares.2019.107857","article-title":"Dual-AAV delivery of large gene sequences to the inner ear","volume":"394","author":"Reisinger","year":"2020","journal-title":"Hear. Res."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"1661","DOI":"10.1073\/pnas.97.4.1661","article-title":"Identification of hepatitis B virus indigenous to chimpanzees","volume":"97","author":"Hu","year":"2000","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"803","DOI":"10.3727\/096368908786516800","article-title":"Rescue of Fertility in Homozygous Mice for the Urokinase Plasminogen Activator Transgene by the Transplantation of Mouse Hepatocytes","volume":"17","author":"Brezillon","year":"2008","journal-title":"Cell Transplant."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"e60747","DOI":"10.7554\/eLife.60747","article-title":"Application of human liver organoids as a patient-derived primary model for HBV infection and related hepatocellular carcinoma","volume":"10","author":"Hossain","year":"2021","journal-title":"eLife"},{"key":"ref_49","doi-asserted-by":"crossref","unstructured":"Lim, C.K., Romeo, O., Chilver, A.P., Tran, B.M., Flanagan, D.J., Kirby, E.N., Breen, J., Vincan, E., Warner, N., and McCartney, E.M. (bioRxiv, 2022). Human Liver Organoids as a Patient-derived Model for HBV Infection and Cellular Response, bioRxiv, preprint.","DOI":"10.1101\/2022.10.20.513112"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"6158","DOI":"10.1128\/jvi.69.10.6158-6169.1995","article-title":"High-level hepatitis B virus replication in transgenic mice","volume":"69","author":"Guidotti","year":"1995","journal-title":"J. Virol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"955","DOI":"10.1089\/hum.2014.048","article-title":"Cellular immune response against firefly luciferase after sleeping beauty-mediated gene transfer in vivo","volume":"25","author":"Vezys","year":"2014","journal-title":"Hum. Gene Ther."}],"container-title":["Viruses"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/8\/1090\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,9]],"date-time":"2025-10-09T18:25:20Z","timestamp":1760034320000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1999-4915\/17\/8\/1090"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,8,7]]},"references-count":51,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2025,8]]}},"alternative-id":["v17081090"],"URL":"https:\/\/doi.org\/10.3390\/v17081090","relation":{},"ISSN":["1999-4915"],"issn-type":[{"value":"1999-4915","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,8,7]]}}}