{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,12,6]],"date-time":"2025-12-06T09:58:38Z","timestamp":1765015118041,"version":"3.46.0"},"reference-count":25,"publisher":"IMR Press","issue":"11","license":[{"start":{"date-parts":[[2025,11,27]],"date-time":"2025-11-27T00:00:00Z","timestamp":1764201600000},"content-version":"unspecified","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"crossref","award":["82271640"],"award-info":[{"award-number":["82271640"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Front. Biosci. (Landmark Ed)"],"abstract":"<jats:p>Background: In contemporary gene therapy research, recombinant adeno-associated virus (rAAV) has emerged as a pivotal delivery vehicle due to its favorable safety profile and capacity for sustained transgene expression. The most commonly utilized rAAV variants are hybrid vectors constructed by packaging the AAV2 genome within the capsid proteins of alternative serotypes (e.g., AAV1, AAV5, AAV8, or AAV9). This chimeric design combines the stable genomic integration and long-term expression characteristics inherent to AAV2 with the distinct tissue tropisms conferred by different capsid serotypes. Consequently, these engineered rAAVs exhibit enhanced organ-targeting specificity, enabling more efficient and selective gene delivery to desired tissues while minimizing off-target effects. Methods: To identify the optimal hybrid vector for germ cell-directed gene delivery in mice, ten distinct chimeric AAV variants were generated by pseudotyping the AAV2 genome with diverse capsid proteins, followed by microinjection into seminiferous tubules via the efferent ducts. Transduction efficiency was comparatively evaluated at 4 weeks post-injection. Results: We demonstrated that AAV2\/9 mediated robust and widespread enhanced green fluorescent protein (EGFP) expression in the mouse testis. Through immunofluorescence assays, we demonstrated that AAV2\/9 demonstrated the capability to express in nearly all testicular cells, with remarkable efficiency and durability. Conclusions: AAV2\/9 demonstrated superior efficacy as a gene delivery vector for murine testicular germ cells without observable adverse effects on testicular development or spermatogenesis. This favorable safety profile, combined with high transduction efficiency establishes AAV2\/9 as a promising candidate for therapeutic gene transfer to testicular germ cells. Collectively, our study identifies AAV2\/9 as a premier vector for germ cell-directed gene therapy, providing crucial preclinical evidence to inform capsid selection for treating male infertility.<\/jats:p>","DOI":"10.31083\/fbl46354","type":"journal-article","created":{"date-parts":[[2025,12,6]],"date-time":"2025-12-06T09:55:11Z","timestamp":1765014911000},"source":"Crossref","is-referenced-by-count":0,"title":["AAV2\/9 Enables Superior Germ Cell Transduction in Mouse Testes"],"prefix":"10.31083","volume":"30","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7979-9198","authenticated-orcid":true,"given":"Bangguo","family":"Wu","sequence":"first","affiliation":[{"name":"NHC Key Lab of Reproduction Regulation, Shanghai Engineering Research Center of Reproductive Health Drugs and Devices, Shanghai Institute for Biomedical and Pharmaceutical Technologies, School of Pharmacy, Fudan University, 200237 Shanghai, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6684-7015","authenticated-orcid":true,"given":"Yadong","family":"Guo","sequence":"additional","affiliation":[{"name":"Department of Urology, Shanghai Tenth People\u2019s Hospital, Tongji University, 200040 Shanghai, China"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7943-0625","authenticated-orcid":true,"given":"Runsheng","family":"Li","sequence":"additional","affiliation":[{"name":"NHC Key Lab of Reproduction Regulation, Shanghai Engineering Research Center of Reproductive Health Drugs and Devices, Shanghai Institute for Biomedical and Pharmaceutical Technologies, School of Pharmacy, Fudan University, 200237 Shanghai, China"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"16242","published-online":{"date-parts":[[2025,11,27]]},"reference":[{"key":"ref1","doi-asserted-by":"publisher","unstructured":"Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X. Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduction and Targeted Therapy. 2020; 5: 1.","DOI":"10.1038\/s41392-019-0089-y"},{"key":"ref2","doi-asserted-by":"publisher","unstructured":"Chanda S, Lepikhov K, Dahlem C, Schymik HS, Hoppst\u00e4dter J, Geber AK, et al. Gene Editing and Small Molecule Inhibitors of the RNA Binding Protein IGF2BP2\/IMP2 Show its Potential as an Anti-Cancer Drug Target. Frontiers in Bioscience (Landmark edition). 2024; 29: 41.","DOI":"10.31083\/j.fbl2901041"},{"key":"ref3","doi-asserted-by":"publisher","unstructured":"Tamehri Zadeh SS, Shapiro MD. Therapeutic Gene Editing in Dyslipidemias. Reviews in Cardiovascular Medicine. 2024; 25: 286.","DOI":"10.31083\/j.rcm2508286"},{"key":"ref4","doi-asserted-by":"publisher","unstructured":"Raguram A, Banskota S, Liu DR. Therapeutic in vivo delivery of gene editing agents. Cell. 2022; 185: 2806\u20132827.","DOI":"10.1016\/j.cell.2022.03.045"},{"key":"ref5","doi-asserted-by":"publisher","unstructured":"Rossi A, Romano R, Fecarotta S, Dell\u2019Anno M, Pecorella V, Passeggio R, et al. Multi-year enzyme expression in patients with mucopolysaccharidosis type VI after liver-directed gene therapy. Med (New York, N.Y.). 2025; 6: 100544.","DOI":"10.1016\/j.medj.2024.10.021"},{"key":"ref6","doi-asserted-by":"publisher","unstructured":"Wang JH, Zhan W, Gallagher TL, Gao G. Recombinant adeno-associated virus as a delivery platform for ocular gene therapy: A comprehensive review. Molecular Therapy: the Journal of the American Society of Gene Therapy. 2024; 32: 4185\u20134207.","DOI":"10.1016\/j.ymthe.2024.10.017"},{"key":"ref7","doi-asserted-by":"publisher","unstructured":"Zai W, Yang M, Jiang K, Guan J, Wang H, Hu K, et al. Optimized RNA interference therapeutics combined with interleukin-2 mRNA for treating hepatitis B virus infection. Signal Transduction and Targeted Therapy. 2024; 9: 150.","DOI":"10.1038\/s41392-024-01871-8"},{"key":"ref8","doi-asserted-by":"publisher","unstructured":"Kon E, Ad-El N, Hazan-Halevy I, Stotsky-Oterin L, Peer D. Targeting cancer with mRNA-lipid nanoparticles: key considerations and future prospects. Nature Reviews. Clinical Oncology. 2023; 20: 739\u2013754.","DOI":"10.1038\/s41571-023-00811-9"},{"key":"ref9","doi-asserted-by":"publisher","unstructured":"Wang JH, Gessler DJ, Zhan W, Gallagher TL, Gao G. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Signal Transduction and Targeted Therapy. 2024; 9: 78.","DOI":"10.1038\/s41392-024-01780-w"},{"key":"ref10","doi-asserted-by":"publisher","unstructured":"Du S, Li W, Zhang Y, Xue Y, Hou X, Yan J, et al. Cholesterol-Amino-Phosphate (CAP) Derived Lipid Nanoparticles for Delivery of Self-Amplifying RNA and Restoration of Spermatogenesis in Infertile Mice. Advanced Science (Weinheim, Baden-Wurttemberg, Germany). 2023; 10: e2300188.","DOI":"10.1002\/advs.202300188"},{"key":"ref11","doi-asserted-by":"publisher","unstructured":"Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nature Reviews. Drug Discovery. 2019; 18: 358\u2013378.","DOI":"10.1038\/s41573-019-0012-9"},{"key":"ref12","doi-asserted-by":"publisher","unstructured":"Gao G, Vandenberghe LH, Alvira MR, Lu Y, Calcedo R, Zhou X, et al. Clades of Adeno-associated viruses are widely disseminated in human tissues. Journal of Virology. 2004; 78: 6381\u20136388.","DOI":"10.1128\/JVI.78.12.6381-6388.2004"},{"key":"ref13","doi-asserted-by":"publisher","unstructured":"Lin A, Balazs AB. Adeno-associated virus gene delivery of broadly neutralizing antibodies as prevention and therapy against HIV-1. Retrovirology. 2018; 15: 66.","DOI":"10.1186\/s12977-018-0449-7"},{"key":"ref14","doi-asserted-by":"publisher","unstructured":"Beloukhova MI, Lukashev AN, Volchkov PY, Zamyatnin AA, Jr, Deviatkin AA. Robust AAV Genotyping Based on Genetic Distances in Rep Gene That Are Maintained by Ubiquitous Recombination. Viruses. 2022; 14: 1038.","DOI":"10.3390\/v14051038"},{"key":"ref15","doi-asserted-by":"publisher","unstructured":"Nisanov AM, Rivera de Jes\u00fas JA, Schaffer DV. Advances in AAV capsid engineering: Integrating rational design, directed evolution and machine learning. Molecular Therapy: the Journal of the American Society of Gene Therapy. 2025; 33: 1937\u20131945.","DOI":"10.1016\/j.ymthe.2025.03.056"},{"key":"ref16","doi-asserted-by":"publisher","unstructured":"Zhou Y, Sach T, Ong JY, Lim TA, Berecz Z, Deniston C, et al. Adeno-associated virus serotype 2 capsids with proteolytic cuts by trypsin remain intact and potent. Gene Therapy. 2025; 32: 121\u2013131.","DOI":"10.1038\/s41434-024-00507-4"},{"key":"ref17","doi-asserted-by":"publisher","unstructured":"Zhu Q, Sun J, An C, Li X, Xu S, He Y, et al. Mechanism of LncRNA Gm2044 in germ cell development. Frontiers in Cell and Developmental Biology. 2024; 12: 1410914.","DOI":"10.3389\/fcell.2024.1410914"},{"key":"ref18","doi-asserted-by":"publisher","unstructured":"Xia K, Wang F, Lai X, Dong L, Luo P, Zhang S, et al. AAV-mediated gene therapy produces fertile offspring in the Lhcgr-deficient mouse model of Leydig cell failure. Cell Reports Medicine. 2022; 3: 100792.","DOI":"10.1016\/j.xcrm.2022.100792"},{"key":"ref19","doi-asserted-by":"publisher","unstructured":"Watanabe S, Kanatsu-Shinohara M, Ogonuki N, Matoba S, Ogura A, Shinohara T. In Vivo Genetic Manipulation of Spermatogonial Stem Cells and Their Microenvironment by Adeno-Associated Viruses. Stem Cell Reports. 2018; 10: 1551\u20131564.","DOI":"10.1016\/j.stemcr.2018.03.005"},{"key":"ref20","doi-asserted-by":"publisher","unstructured":"Kaiser VM, Gonzalez-Cordero A. Organoids - the future of pre-clinical development of AAV gene therapy for CNS disorders. Gene Therapy. 2025.  (online ahead of print)","DOI":"10.1038\/s41434-025-00527-8"},{"key":"ref21","doi-asserted-by":"publisher","unstructured":"Mingozzi F, High KA. Immune responses to AAV vectors: overcoming barriers to successful gene therapy. Blood. 2013; 122: 23\u201336.","DOI":"10.1182\/blood-2013-01-306647"},{"key":"ref22","doi-asserted-by":"publisher","unstructured":"Rajasekaran S, Thatte J, Periasamy J, Javali A, Jayaram M, Sen D, et al. Infectivity of adeno-associated virus serotypes in mouse testis. BMC Biotechnology. 2018; 18: 70.","DOI":"10.1186\/s12896-018-0479-1"},{"key":"ref23","doi-asserted-by":"publisher","unstructured":"Lester Suarez-Amaran , Liujiang Song, Anna P. Tretiakova, Sheila A. Mikhail, Richard Jude Samulski. AAV vector development, back to the future. Molecular Therapy. 2025; 33: 1903-1936.","DOI":"10.1016\/j.ymthe.2025.03.064"},{"key":"ref24","doi-asserted-by":"publisher","unstructured":"Vassalli G, B\u00fceler H, Dudler J, von Segesser LK, Kappenberger L. Adeno-associated virus (AAV) vectors achieve prolonged transgene expression in mouse myocardium and arteries in vivo: a comparative study with adenovirus vectors. International Journal of Cardiology. 2003; 90: 229\u2013238.","DOI":"10.1016\/s0167-5273(02)00554-5"},{"key":"ref25","doi-asserted-by":"publisher","unstructured":"Oziolor EM, Kumpf SW, Qian J, Gosink M, Sheehan M, Rubitski DM, et al. Comparing molecular and computational approaches for detecting viral integration of AAV gene therapy constructs. Molecular Therapy. Methods & Clinical Development. 2023; 29: 395\u2013405.","DOI":"10.1016\/j.omtm.2023.04.009"}],"container-title":["Frontiers in Bioscience-Landmark"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.imrpress.com\/journal\/FBL\/30\/11\/10.31083\/FBL46354","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,12,6]],"date-time":"2025-12-06T09:55:43Z","timestamp":1765014943000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.imrpress.com\/journal\/FBL\/30\/11\/10.31083\/FBL46354"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,27]]},"references-count":25,"journal-issue":{"issue":"11","published-print":{"date-parts":[[2025]]}},"alternative-id":["S2768-6701(25)01910-0"],"URL":"https:\/\/doi.org\/10.31083\/fbl46354","relation":{},"ISSN":["2768-6701","2768-6698"],"issn-type":[{"value":"2768-6701","type":"print"},{"value":"2768-6698","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,27]]},"article-number":"46354"}}