{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,3,9]],"date-time":"2026-03-09T15:46:03Z","timestamp":1773071163392,"version":"3.50.1"},"reference-count":25,"publisher":"MDPI AG","issue":"17","license":[{"start":{"date-parts":[[2023,8,31]],"date-time":"2023-08-31T00:00:00Z","timestamp":1693440000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100012190","name":"Ministry of Science and Higher Education of the Russian Federation","doi-asserted-by":"publisher","award":["075-15-2021-1346"],"award-info":[{"award-number":["075-15-2021-1346"]}],"id":[{"id":"10.13039\/501100012190","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Dysferlinopathy treatment is an active area of investigation. Gene therapy is one potential approach. We studied muscle regeneration and inflammatory response after injection of an AAV-9 with a codon-optimized DYSF gene. A dual-vector system AAV.DYSF.OVERLAP with overlapping DYSF cDNA sequences was generated. Two AAV vectors were separately assembled by a standard triple-transfection protocol from plasmids carrying parts of the DYSF gene. Artificial myoblasts from dysferlin-deficient fibroblasts were obtained by MyoD overexpression. RT-PCR and Western blot were used for RNA and protein detection in vitro. A dysferlinopathy murine model (Bla\/J) was used for in vivo studies. Histological assay, morphometry, and IHC were used for the muscle tissue analysis. Dysferlin was detected in vitro and in vivo at subphysiological levels. RT-PCR and Western Blot detected dysferlin mRNA and protein in AAV.DYSF.OVERLAP-transduced cells, and mRNA reached a 7-fold elevated level compared to the reference gene (GAPDH). In vivo, the experimental group showed intermediate median values for the proportion of necrotic muscle fibers, muscle fibers with internalized nuclei, and cross-sectional area of muscle fibers compared to the same parameters in the control groups of WT and Bla\/J mice, although the differences were not statistically significant. The inverse relationship between the dosage and the severity of inflammatory changes in the muscles may be attributed to the decrease in the number of necrotic fibers. The share of transduced myofibers reached almost 35% in the group with the highest dose. The use of two-vector systems based on AAV is justified in terms of therapeutic efficacy. The expression of dysferlin at a subphysiological level, within a short observation period, is capable of inducing the restoration of muscle tissue structure, reducing inflammatory activity, and mitigating necrotic processes. Further research is needed to provide a more detailed assessment of the impact of the transgene and viral vector on the inflammatory component, including longer observation periods.<\/jats:p>","DOI":"10.3390\/ijms241713551","type":"journal-article","created":{"date-parts":[[2023,9,1]],"date-time":"2023-09-01T08:19:39Z","timestamp":1693556379000},"page":"13551","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Dual Adeno-Associated Virus 9 with Codon-Optimized DYSF Gene Promotes In Vivo Muscle Regeneration and May Decrease Inflammatory Response in Limb Girdle Muscular Dystrophy Type R2"],"prefix":"10.3390","volume":"24","author":[{"given":"Ivan A.","family":"Yakovlev","sequence":"first","affiliation":[{"name":"Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia"},{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Aleksei M.","family":"Emelin","sequence":"additional","affiliation":[{"name":"Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Yana S.","family":"Slesarenko","sequence":"additional","affiliation":[{"name":"Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Igor S.","family":"Limaev","sequence":"additional","affiliation":[{"name":"Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iuliia A.","family":"Vetrova","sequence":"additional","affiliation":[{"name":"Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Liliya D.","family":"Belikova","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia"},{"name":"Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119992 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8957-6142","authenticated-orcid":false,"given":"Ekaterina N.","family":"Grafskaia","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pavel A.","family":"Bobrovsky","sequence":"additional","affiliation":[{"name":"Lopukhin Federal Research and Clinical Center of Physical-Chemical Medicine of Federal Medical Biological Agency, 119435 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mikhail V.","family":"Pokrovsky","sequence":"additional","affiliation":[{"name":"Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elena V.","family":"Kuzubova","sequence":"additional","affiliation":[{"name":"Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Vladimir M.","family":"Pokrovsky","sequence":"additional","affiliation":[{"name":"Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pyotr A.","family":"Lebedev","sequence":"additional","affiliation":[{"name":"Laboratory for Modeling and Gene Therapy of Human Diseases, Belgorod State National Research University, 308015 Belgorod, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3804-6245","authenticated-orcid":false,"given":"Sergei N.","family":"Bardakov","sequence":"additional","affiliation":[{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Artur A.","family":"Isaev","sequence":"additional","affiliation":[{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Roman V.","family":"Deev","sequence":"additional","affiliation":[{"name":"Genotarget LLC, Skolkovo Innovation Center, 121205 Moscow, Russia"},{"name":"PJSC Human Stem Cells Institute, 129110 Moscow, Russia"},{"name":"Department of Pathological Anatomy, I. I. Mechnikov North-West State Medical University, Ministry of Health of the Russian Federation, 191036 St. Petersburg, Russia"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,8,31]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"289","DOI":"10.4103\/0028-3886.43447","article-title":"Dysferlinopathies","volume":"56","author":"Urtizberea","year":"2008","journal-title":"Neurol. India"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"31","DOI":"10.1038\/1682","article-title":"Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy","volume":"20","author":"Liu","year":"1998","journal-title":"Nat Genet."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"2891","DOI":"10.2353\/ajpath.2010.090058","article-title":"Inflammasome Up-Regulation and Activation in Dysferlin-Deficient Skeletal Muscle","volume":"176","author":"Rawat","year":"2010","journal-title":"Am. J. Pathol."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1016\/0165-5728(95)00122-0","article-title":"Analysis of cytokine expression in muscle in inflammatory myopathies, Duchenne dystrophy, and non-weak controls","volume":"63","author":"Lundberg","year":"1995","journal-title":"J. Neuroimmunol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"865","DOI":"10.1002\/art.1780400514","article-title":"Cytokine production in muscle tissue of patients with idiopathic inflammatory myopathies","volume":"40","author":"Lundberg","year":"1997","journal-title":"Arthritis Rheum."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"841","DOI":"10.1242\/jcs.198861","article-title":"Dysferlin mediates membrane tubulation and links T-tubule biogenesis to muscular dystrophy","volume":"130","author":"Hofhuis","year":"2017","journal-title":"J. Cell Sci."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"37","DOI":"10.23868\/201707014","article-title":"Gingiva as a source of stromal cells with high differentiating and reparative potential","volume":"12","author":"Zorin","year":"2017","journal-title":"Genes Cells"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"15009","DOI":"10.1038\/mtm.2015.9","article-title":"A comparison of AAV strategies distinguishes overlapping vectors for efficient systemic delivery of the 6.2 kb Dysferlin coding sequence","volume":"2","author":"Pryadkina","year":"2015","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"2150","DOI":"10.1016\/j.ymthe.2017.05.013","article-title":"Structure-Based Designed Nano-Dysferlin Significantly Improves Dysferlinopathy in BLA\/J Mice","volume":"25","author":"Llanga","year":"2017","journal-title":"Mol. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Grose, W.E., Clark, K.R., Griffin, D., Malik, V., Shontz, K.M., Montgomery, C.L., Lewis, S., Brown, R.H., Janssen, P.M., and Mendell, J.R. (2012). Homologous recombination mediates functional recovery of dysferlin deficiency following AAV5 gene transfer. PLoS ONE, 7.","DOI":"10.1371\/journal.pone.0039233"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"1897","DOI":"10.1093\/hmg\/ddq065","article-title":"Efficient recovery of dysferlin deficiency by dual adeno-associated vector-mediated gene transfer","volume":"19","author":"Lostal","year":"2010","journal-title":"Hum. Mol. Genet."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"560629","DOI":"10.1155\/2014\/560629","article-title":"Macrophage plasticity in skeletal muscle repair","volume":"2014","author":"Rigamonti","year":"2014","journal-title":"BioMed Res. Int."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"648","DOI":"10.1016\/j.nmd.2012.03.002","article-title":"Myogenesis in dysferlin-deficient myoblasts is inhibited by an intrinsic inflammatory response","volume":"22","author":"Cohen","year":"2012","journal-title":"Neuromuscul. Disord."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"1313","DOI":"10.1002\/cphy.c170052","article-title":"Immunobiology of Inherited Muscular Dystrophies","volume":"8","author":"Tidball","year":"2018","journal-title":"Compr. Physiol."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"166","DOI":"10.1002\/mus.21166","article-title":"Dysferlin associates with the developing T-tubule system in rodent and human skeletal muscle","volume":"41","author":"Klinge","year":"2010","journal-title":"Muscle Nerve"},{"key":"ref_16","first-page":"134","article-title":"Intracellular localization of dysferlin and its association with the dihydropyridine receptor","volume":"24","author":"Ampong","year":"2005","journal-title":"Acta Myol."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"15865","DOI":"10.1038\/s41598-021-95378-9","article-title":"Loss of dysferlin or myoferlin results in differential defects in excitation-contraction coupling in mouse skeletal muscle","volume":"11","author":"Barefield","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"20831","DOI":"10.1073\/pnas.1307960110","article-title":"Dysferlin stabilizes stress-induced Ca2+ signaling in the transverse tubule membrane","volume":"110","author":"Kerr","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"206","DOI":"10.1016\/j.tcb.2004.03.001","article-title":"Dysferlin and the plasma membrane repair in muscular dystrophy","volume":"14","author":"Bansal","year":"2004","journal-title":"Trends Cell Biol."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1104","DOI":"10.1093\/jnen\/63.10.1104","article-title":"In Vivo and In Vitro Dysferlin Expression in Human Muscle Satellite Cells","volume":"63","author":"Gallardo","year":"2004","journal-title":"J. Neuropathol. Exp. Neurol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"902","DOI":"10.1002\/1531-8249(200012)48:6<902::AID-ANA11>3.0.CO;2-Z","article-title":"Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb\u2013girdle muscular dystrophies","volume":"48","author":"Piccolo","year":"2000","journal-title":"Ann. Neurol."},{"key":"ref_22","first-page":"25","article-title":"Formation of the recombinant adenovirus encoding codon-optimized dysferlin gene and analysis of the recombinant protein expression in cell culture in vitro","volume":"7","author":"Starostina","year":"2012","journal-title":"Genes Cells"},{"key":"ref_23","unstructured":"Mavlikeev, M.O., Kiyasov, A.P., and Deev, R.V. (2023). Histological technique in the pathomorphological laboratory: Educational and methodological manual (Gistologicheskaya Tekhnika v Patomorfologicheskoj Laboratorii: Uchebno-Metodicheskoe Posobie), Prakticheskaya Medicina."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"359","DOI":"10.1080\/01913123.2022.2105464","article-title":"Structural and ultrastructural changes in the skeletal muscles of dysferlin-deficient mice during postnatal ontogenesis","volume":"46","author":"Chernova","year":"2022","journal-title":"Ultrastruct. Pathol."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1002\/acn3.172","article-title":"AAV.Dysferlin Overlap Vectors Restore Function in Dysferlinopathy Animal Models","volume":"2","author":"Sondergaard","year":"2015","journal-title":"Ann. Clin. Transl. Neurol."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/17\/13551\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T20:44:25Z","timestamp":1760129065000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/17\/13551"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,8,31]]},"references-count":25,"journal-issue":{"issue":"17","published-online":{"date-parts":[[2023,9]]}},"alternative-id":["ijms241713551"],"URL":"https:\/\/doi.org\/10.3390\/ijms241713551","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,8,31]]}}}