{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T15:02:18Z","timestamp":1774278138499,"version":"3.50.1"},"reference-count":45,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T00:00:00Z","timestamp":1774224000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000005","name":"DOD","doi-asserted-by":"publisher","award":["W81XWH-22-1-0065"],"award-info":[{"award-number":["W81XWH-22-1-0065"]}],"id":[{"id":"10.13039\/100000005","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biomolecules"],"abstract":"<jats:p>Studies have shown that IL-10 has therapeutic potential for inflammatory diseases. However, it is challenging to use IL-10 as a therapeutic drug because it also possesses pro-inflammatory functions. To reduce these pro-inflammatory effects of IL-10, we have designed three IL-10 mutants using structure-based computational design technology. We demonstrated that these mutants exhibited significantly lower activity in IL-10-responsive cell lines than wild-type IL-10. Using recombinant adeno-associated virus (rAAV8) vectors expressing wild-type or mutant IL-10 molecules, we performed gene therapy experiments in IL-10 KO mice. The results showed that our vectors mediated high levels of transgene expression. Importantly, IL-10 gene therapy increased body weight gain, reduced colon injury, and prevented the development of inflammatory bowel disease (IBD). Moreover, IL-10 mutant gene therapy elicited significantly lower stimulation of CD8 T and NK cells compared with the wild-type IL-10 group. In summary, our IL-10 mutants provide a protective effect comparable to wild-type IL-10 in the IL-10 KO mouse model, suggesting that they may potentially have reduced pro-inflammatory function. While rigorous investigations of safety and efficacy in different disease models will be required, these results indicate the therapeutic potential of IL-10 mutant gene therapy for inflammatory diseases such as IBD.<\/jats:p>","DOI":"10.3390\/biom16030482","type":"journal-article","created":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T13:53:34Z","timestamp":1774274014000},"page":"482","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional Evaluation of Computationally Designed IL-10 in IL-10 KO Mice"],"prefix":"10.3390","volume":"16","author":[{"given":"Jordan","family":"Stokes","sequence":"first","affiliation":[{"name":"Departments of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iram","family":"Hyder","sequence":"additional","affiliation":[{"name":"Departments of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-2505-1680","authenticated-orcid":false,"given":"Zhihang","family":"Shen","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2027-1591","authenticated-orcid":false,"given":"Peter","family":"Ramdhan","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Allison","family":"Bayer","sequence":"additional","affiliation":[{"name":"Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Clive","family":"Wasserfall","sequence":"additional","affiliation":[{"name":"Department of Pathology, Immunology and Laboratory Medicine, University of Florida College of Medicine, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chenglong","family":"Li","sequence":"additional","affiliation":[{"name":"Department of Medicinal Chemistry, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6523-165X","authenticated-orcid":false,"given":"Sihong","family":"Song","sequence":"additional","affiliation":[{"name":"Departments of Pharmaceutics, University of Florida College of Pharmacy, Gainesville, FL 32610, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,3,23]]},"reference":[{"key":"ref_1","first-page":"619","article-title":"Prevalence of Inflammatory Bowel Disease in Pediatric and Adult Populations: Recent Estimates From Large National Databases in the United States, 2007\u20132016","volume":"26","author":"Ye","year":"2020","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Cai, Z., Wang, S., and Li, J. (2021). Treatment of Inflammatory Bowel Disease: A Comprehensive Review. Front. Med., 8.","DOI":"10.3389\/fmed.2021.765474"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"629","DOI":"10.1016\/S1734-1140(11)70575-8","article-title":"Treatment of inflammatory bowel disease (IBD)","volume":"63","author":"Pithadia","year":"2011","journal-title":"Pharmacol. Rep."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"185","DOI":"10.2147\/DDDT.S11290","article-title":"Current and emerging drugs for the treatment of inflammatory bowel disease","volume":"5","author":"Triantafillidis","year":"2011","journal-title":"Drug Des. Dev. Ther."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1739","DOI":"10.1136\/gut.2009.199679","article-title":"New IBD genetics: Common pathways with other diseases","volume":"60","author":"Lees","year":"2011","journal-title":"Gut"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Fillatreau, S., and O\u2019Garra, A. (2014). IL-10 in Humans: Lessons from the Gut, IL-10\/IL-10 Receptor Deficiencies, and IL-10 Polymorphisms. Interleukin-10 in Health and Disease, Springer.","DOI":"10.1007\/978-3-662-43492-5"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"e20190418","DOI":"10.1084\/jem.20190418","article-title":"Biology and therapeutic potential of interleukin-10","volume":"217","author":"Saraiva","year":"2020","journal-title":"J. Exp. Med."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"948","DOI":"10.1089\/jir.2014.0221","article-title":"The Potentiation of IFN-gamma and Induction of Cytotoxic Proteins by Pegylated IL-10 in Human CD8 T Cells","volume":"35","author":"Chan","year":"2015","journal-title":"J. Interferon Cytokine Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"eabc8433","DOI":"10.1126\/science.abc8433","article-title":"Structure-based decoupling of the pro- and anti-inflammatory functions of interleukin-10","volume":"371","author":"Saxton","year":"2021","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"17158","DOI":"10.1073\/pnas.0909520106","article-title":"Distinct immune responses to transgene products from rAAV1 and rAAV8 vectors","volume":"106","author":"Lu","year":"2009","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2436","DOI":"10.1002\/ibd.21673","article-title":"Neutralizing antibodies against adeno-associated viruses in inflammatory bowel disease patients: Implications for gene therapy","volume":"17","author":"Comijn","year":"2011","journal-title":"Inflamm. Bowel Dis."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"78","DOI":"10.1038\/s41392-024-01780-w","article-title":"Adeno-associated virus as a delivery vector for gene therapy of human diseases","volume":"9","author":"Wang","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_13","unstructured":"(2025). Schr\u00f6dinger Release 2025-4: Maestro, Schr\u00f6dinger LLC."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3381","DOI":"10.1093\/nar\/gkg520","article-title":"SWISS-MODEL: An automated protein homology-modeling server","volume":"31","author":"Schwede","year":"2003","journal-title":"Nucleic Acids Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1542","DOI":"10.1021\/ct200909j","article-title":"Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 1. Generalized Born","volume":"8","author":"Williamson","year":"2012","journal-title":"J. Chem. Theory Comput."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"3878","DOI":"10.1021\/ct400314y","article-title":"Routine Microsecond Molecular Dynamics Simulations with AMBER on GPUs. 2. Explicit Solvent Particle Mesh Ewald","volume":"9","author":"Poole","year":"2013","journal-title":"J. Chem. Theory Comput."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1668","DOI":"10.1002\/jcc.20290","article-title":"The Amber biomolecular simulation programs","volume":"26","author":"Case","year":"2005","journal-title":"J. Comput. Chem."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"528","DOI":"10.1021\/acs.jctc.9b00591","article-title":"ff19SB: Amino-Acid-Specific Protein Backbone Parameters Trained against Quantum Mechanics Energy Surfaces in Solution","volume":"16","author":"Tian","year":"2020","journal-title":"J. Chem. Theory Comput."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"3863","DOI":"10.1021\/jz501780a","article-title":"Building water models: A different approach","volume":"5","author":"Izadi","year":"2014","journal-title":"J. Phys. Chem. Lett."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1002\/jcc.26798","article-title":"Taking the Monte-Carlo gamble: How not to buckle under the pressure!","volume":"43","author":"Gomez","year":"2022","journal-title":"J. Comput. Chem."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1016\/0021-9991(77)90098-5","article-title":"Numerical integration of the cartesian equations of motion of a system with constraints: Molecular dynamics of n-alkanes","volume":"23","author":"Ryckaert","year":"1977","journal-title":"J. Comput. Phys."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"952","DOI":"10.1002\/jcc.540130805","article-title":"Settle: An analytical version of the SHAKE and RATTLE algorithm for rigid water models","volume":"13","author":"Miyamoto","year":"1992","journal-title":"J. Comput. Chem."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"10089","DOI":"10.1063\/1.464397","article-title":"Particle mesh Ewald: An N\u22c5log(N) method for Ewald sums in large systems","volume":"98","author":"Darden","year":"1993","journal-title":"J. Chem. Phys."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"523","DOI":"10.1002\/bip.360320508","article-title":"Langevin dynamics of peptides: The frictional dependence of isomerization rates of N-acetylalanyl-N\u2019-methylamide","volume":"32","author":"Loncharich","year":"1992","journal-title":"Biopolymers"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"3084","DOI":"10.1021\/ct400341p","article-title":"PTRAJ and CPPTRAJ: Software for processing and analysis of molecular dynamics trajectory data","volume":"9","author":"Roe","year":"2013","journal-title":"J. Chem. Theory Comput."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"3314","DOI":"10.1021\/ct300418h","article-title":"MMPBSA.py: An Efficient Program for End-State Free Energy Calculations","volume":"8","author":"Miller","year":"2012","journal-title":"J. Chem. Theory Comput."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"260","DOI":"10.15430\/JCP.2015.20.4.260","article-title":"Adequate Dextran Sodium Sulfate-induced Colitis Model in Mice and Effective Outcome Measurement Method","volume":"20","author":"Park","year":"2015","journal-title":"J. Cancer Prev."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"1299","DOI":"10.1038\/sj.gt.3301422","article-title":"Stable therapeutic serum levels of human alpha-1 antitrypsin (AAT) after portal vein injection of recombinant adeno-associated virus (rAAV) vectors","volume":"8","author":"Song","year":"2001","journal-title":"Gene Ther."},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"794","DOI":"10.1093\/ecco-jcc\/jjy035","article-title":"Development of Reliable, Valid and Responsive Scoring Systems for Endoscopy and Histology in Animal Models for Inflammatory Bowel Disease","volume":"12","author":"Koelink","year":"2018","journal-title":"J. Crohn\u2019s Colitis"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"16878","DOI":"10.1038\/s41598-017-17204-5","article-title":"QuPath: Open source software for digital pathology image analysis","volume":"7","author":"Bankhead","year":"2017","journal-title":"Sci. Rep."},{"key":"ref_31","doi-asserted-by":"crossref","unstructured":"Terrabuio, E., Zenaro, E., and Constantin, G. (2023). The role of the CD8+ T cell compartment in ageing and neurodegenerative disorders. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1233870"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"371","DOI":"10.1016\/j.jaci.2022.09.022","article-title":"Human natural killer cells: Form, function, and development","volume":"151","author":"Mace","year":"2023","journal-title":"J. Allergy Clin. Immunol."},{"key":"ref_33","doi-asserted-by":"crossref","unstructured":"Luo, M., Zhao, F., Cheng, H., Su, M., and Wang, Y. (2024). Macrophage polarization: An important role in inflammatory diseases. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1352946"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"706","DOI":"10.1016\/j.immuni.2014.03.011","article-title":"Interleukin-10 receptor signaling in innate immune cells regulates mucosal immune tolerance and anti-inflammatory macrophage function","volume":"40","author":"Shouval","year":"2014","journal-title":"Immunity"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"3815","DOI":"10.4049\/jimmunol.147.11.3815","article-title":"IL-10 inhibits cytokine production by activated macrophages","volume":"147","author":"Fiorentino","year":"1991","journal-title":"J. Immunol."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1146\/annurev-immunol-031210-101312","article-title":"Regulation and functions of the IL-10 family of cytokines in inflammation and disease","volume":"29","author":"Ouyang","year":"2011","journal-title":"Annu. Rev. Immunol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"e20220686","DOI":"10.1084\/jem.20220686","article-title":"STAT3 regulates CD8+ T cell differentiation and functions in cancer and acute infection","volume":"220","author":"Sun","year":"2023","journal-title":"J. Exp. Med."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1073\/pnas.88.4.1172","article-title":"Isolation and expression of human cytokine synthesis inhibitory factor cDNA clones: Homology to Epstein-Barr virus open reading frame BCRFI","volume":"88","author":"Vieira","year":"1991","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"21053","DOI":"10.1016\/S0021-9258(19)36892-9","article-title":"Characterization of interleukin-10 receptors on human and mouse cells","volume":"268","author":"Tan","year":"1993","journal-title":"J. Biol. Chem."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"13913","DOI":"10.1073\/pnas.251532298","article-title":"Adeno-associated virus vector-mediated IL-10 gene delivery prevents type 1 diabetes in NOD mice","volume":"98","author":"Goudy","year":"2001","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"2270","DOI":"10.4049\/jimmunol.171.5.2270","article-title":"Systemic overexpression of IL-10 induces CD4+CD25+ cell populations in vivo and ameliorates type 1 diabetes in nonobese diabetic mice in a dose-dependent fashion","volume":"171","author":"Goudy","year":"2003","journal-title":"J. Immunol."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"708","DOI":"10.2337\/diabetes.52.3.708","article-title":"Adeno-associated virus-mediated IL-10 gene therapy inhibits diabetes recurrence in syngeneic islet cell transplantation of NOD mice","volume":"52","author":"Zhang","year":"2003","journal-title":"Diabetes"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1186\/1479-5876-9-21","article-title":"Alpha-1 antitrypsin protein and gene therapies decrease autoimmunity and delay arthritis development in mouse model","volume":"9","author":"Grimstein","year":"2011","journal-title":"J. Transl. Med."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"131","DOI":"10.1016\/j.omtm.2018.10.007","article-title":"Alpha 1 Antitrypsin Gene Therapy Extends the Lifespan of Lupus-Prone Mice","volume":"11","author":"Elshikha","year":"2018","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"679","DOI":"10.1089\/hum.2016.029","article-title":"Alpha-1 Antitrypsin Gene Therapy Ameliorates Bone Loss in Ovariectomy-Induced Osteoporosis Mouse Model","volume":"27","author":"Akbar","year":"2016","journal-title":"Hum. Gene Ther."}],"container-title":["Biomolecules"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/3\/482\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,3,23]],"date-time":"2026-03-23T14:13:36Z","timestamp":1774275216000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2218-273X\/16\/3\/482"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,3,23]]},"references-count":45,"journal-issue":{"issue":"3","published-online":{"date-parts":[[2026,3]]}},"alternative-id":["biom16030482"],"URL":"https:\/\/doi.org\/10.3390\/biom16030482","relation":{},"ISSN":["2218-273X"],"issn-type":[{"value":"2218-273X","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,3,23]]}}}