{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,7]],"date-time":"2026-08-07T14:30:57Z","timestamp":1786113057186,"version":"build-2736575974"},"reference-count":34,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,6,30]],"date-time":"2026-06-30T00:00:00Z","timestamp":1782777600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Silesian Centre for Entrepreneurship","award":["UDA-RPSL.01.02.00-24-0129\/21-00"],"award-info":[{"award-number":["UDA-RPSL.01.02.00-24-0129\/21-00"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Curr. Issues Mol. Biol.","CIMB"],"abstract":"<jats:p>Efficient and reproducible lentiviral vector production and T-cell transduction remain important technical challenges in CAR-T (Chimeric Antigen Receptor T-cell) cell manufacturing. In this study, we optimized HEK293T transfection and primary T-cell transduction parameters for lentiviral CAR constructs targeting BCMA (B-cell maturation antigen) and GPRC5D (G-protein coupled receptor family C group 5 member D). Lipofectamine 3000 and TurboFectin 8.0 were compared across different seeding densities and reagent-to-DNA ratios, with vector yields quantified by qPCR (Quantitative Polymerase Chain Reaction) and p24 ELISA (Enzyme-linked Immunosorbent Assay). Lipofectamine 3000 consistently generated higher viral titers and transduction efficiencies, as reflected by a greater proportion of GFP-positive (Green Fluorescent Protein) cells than TurboFectin 8.0, reaching peak titers of 9.65 \u00d7 108 copies\/mL for the anti-GPRC5D and 5.33 \u00d7 108 copies\/mL for the anti-BCMA vectors. Under optimized conditions, transduction efficiencies reached 43.8% GFP+ cells for BCMA-CAR and approximately 13\u201314% GFP-positive transduced cells for the GPRC5D construct within the tested TU\/mL range. Co-transduction experiments yielded approximately 62\u201366% GFP+ cells with detectable BCMA-binding and presumptive GPRC5D-CAR-expressing subpopulations identified based on GFP reporter expression. Immunophenotypic analysis demonstrated a relatively stable CD4\/CD8 distribution (~65\/35), enrichment of effector memory CD8+ cells, and expression of activation-associated markers. Collectively, these findings describe an optimized lentiviral transfection and transduction workflow that may support the further development of dual-targeting BCMA\/GPRC5D CAR-T manufacturing strategies in research and early translational settings.<\/jats:p>","DOI":"10.3390\/cimb48070679","type":"journal-article","created":{"date-parts":[[2026,7,1]],"date-time":"2026-07-01T00:50:37Z","timestamp":1782867037000},"page":"679","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["A Scalable Lentiviral Workflow for Laboratory-Scale Generation of BCMA\/GPRC5D Co-Transduced CAR-T Cells in Multiple Myeloma"],"prefix":"10.3390","volume":"48","author":[{"given":"Ewa","family":"Nowak","sequence":"first","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8360-359X","authenticated-orcid":false,"given":"Emilia","family":"Morawiec","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"},{"name":"Department of Microbiology, Academy of Silesia, 40-555 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4558-8437","authenticated-orcid":false,"given":"Adam","family":"Pude\u0142ko","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"},{"name":"Department of Immunology, Academy of Silesia, 40-555 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6436-8451","authenticated-orcid":false,"given":"Agnieszka","family":"Polak","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"},{"name":"Department of Internal Medicine and Clinical Pharmacology, Faculty of Medical Science in Katowice, Medical University of Silesia, 40-055 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mateusz","family":"Broncel","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Daria","family":"Matczy\u0144ska","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-2427-2680","authenticated-orcid":false,"given":"Dawid","family":"Zamojski","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"},{"name":"Department of Data Science and Engineering, Silesian University of Technology, 44-100 Gliwice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Micha\u0142","family":"Czerwinski","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Bednarska-Czerwi\u0144ska","sequence":"additional","affiliation":[{"name":"Gyncentrum Sp. z o.o. Medical Diagnostic Laboratory, Genetics and Molecular Biology in Sosnowiec, 40-851 Katowice, Poland"},{"name":"American Medical Clinic, 40-851 Katowice, Poland"},{"name":"Department of Gynecology and Obstetrics, Faculty of Medicine in Zabrze, Academy of Silesia in Katowice, 40-555 Katowice, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,30]]},"reference":[{"key":"ref_1","unstructured":"Kojima, R. (2021). Generation of CAR-T Cells by Lentiviral Transduction. Mammalian Cell Engineering: Methods and Protocols, Springer."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Mitra, A., Barua, A., Huang, L., Ganguly, S., Feng, Q., and He, B. (2023). From bench to bedside: The history and progress of CAR T cell therapy. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1188049"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Zhang, X., Zhang, H., Lan, H., Wu, J., and Xiao, Y. (2023). CAR-T cell therapy in multiple myeloma: Current limitations and potential strategies. Front. Immunol., 14.","DOI":"10.3389\/fimmu.2023.1101495"},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Labb\u00e9, R.P., Vessillier, S., and Rafiq, Q.A. (2021). Lentiviral Vectors for T Cell Engineering: Clinical Applications, Bioprocessing and Future Perspectives. Viruses, 13.","DOI":"10.3390\/v13081528"},{"key":"ref_5","doi-asserted-by":"crossref","unstructured":"Gutierrez-Guerrero, A., Cosset, F.-L., and Verhoeyen, E. (2020). Lentiviral Vector Pseudotypes: Precious Tools to Improve Gene Modification of Hematopoietic Cells for Research and Gene Therapy. Viruses, 12.","DOI":"10.3390\/v12091016"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"101315","DOI":"10.1016\/j.omtm.2024.101315","article-title":"Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process","volume":"32","author":"Tridgett","year":"2024","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"146","DOI":"10.1158\/2643-3230.BCD-20-0020","article-title":"Defining an Optimal Dual-Targeted CAR T-cell Therapy Approach Simultaneously Targeting BCMA and GPRC5D to Prevent BCMA Escape\u2013Driven Relapse in Multiple Myeloma","volume":"1","author":"Staehr","year":"2020","journal-title":"Blood Cancer Discov."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"88","DOI":"10.1186\/s13045-024-01611-z","article-title":"Targeting GPRC5D for multiple myeloma therapy","volume":"17","author":"Zhou","year":"2024","journal-title":"J. Hematol. Oncol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"210","DOI":"10.1038\/s41392-025-02269-w","article-title":"CAR-T cell therapy for cancer: Current challenges and future directions","volume":"10","author":"Zugasti","year":"2025","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1186\/s40364-022-00417-w","article-title":"CAR-T cell potency: From structural elements to vector backbone components","volume":"10","author":"Mazinani","year":"2022","journal-title":"Biomark. Res."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Sch\u00e4fer, D., Henze, J., Pfeifer, R., Schleicher, A., Brauner, J., Mockel-Tenbrinck, N., Barth, C., Gudert, D., Al Rawashdeh, W., and Johnston, I.C.D. (2020). A Novel Siglec-4 Derived Spacer Improves the Functionality of CAR T Cells Against Membrane-Proximal Epitopes. Front. Immunol., 11.","DOI":"10.3389\/fimmu.2020.01704"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Fujiwara, K., Tsunei, A., Kusabuka, H., Ogaki, E., Tachibana, M., and Okada, N. (2020). Hinge and Transmembrane Domains of Chimeric Antigen Receptor Regulate Receptor Expression and Signaling Threshold. Cells, 9.","DOI":"10.3390\/cells9051182"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Moreno-Cortes, E., Franco-Fuquen, P., Garcia-Robledo, J.E., Forero, J., Booth, N., and Castro, J.E. (2023). ICOS and OX40 tandem co-stimulation enhances CAR T-cell cytotoxicity and promotes T-cell persistence phenotype. Front. Oncol., 13.","DOI":"10.3389\/fonc.2023.1200914"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1007\/s11899-023-00687-7","article-title":"CAR-T Cell Therapy: The Efficacy and Toxicity Balance","volume":"18","author":"Chohan","year":"2023","journal-title":"Curr. Hematol. Malig. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3146","DOI":"10.1016\/j.ymthe.2023.09.021","article-title":"Arming CAR-T cells with cytokines and more: Innovations in the fourth-generation CAR-T development","volume":"31","author":"Tang","year":"2023","journal-title":"Mol. Ther."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"169","DOI":"10.20517\/cdr.2022.138","article-title":"Anti-BCMA novel therapies for multiple myeloma","volume":"6","author":"Sammartano","year":"2023","journal-title":"Cancer Drug Resist."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"985","DOI":"10.1038\/s41375-020-0734-z","article-title":"B-cell maturation antigen (BCMA) in multiple myeloma: Rationale for targeting and current therapeutic approaches","volume":"34","author":"Shah","year":"2020","journal-title":"Leukemia"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"e125","DOI":"10.1002\/cpmb.125","article-title":"Optimized Transgene Delivery Using Third-Generation Lentiviruses","volume":"133","author":"Gill","year":"2020","journal-title":"Curr. Protoc. Mol. Biol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"987","DOI":"10.3324\/haematol.2019.231183","article-title":"CD28.OX40 co-stimulatory combination is associated with long in vivo persistence and high activity of CAR.CD30 T-cells","volume":"106","author":"Guercio","year":"2020","journal-title":"Haematologica"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"715","DOI":"10.1016\/j.jcyt.2021.02.117","article-title":"Combined 4-1BB and ICOS co-stimulation improves anti-tumor efficacy and persistence of dual anti-CD19\/CD20 chimeric antigen receptor T cells","volume":"23","author":"Wang","year":"2021","journal-title":"Cytotherapy"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"92","DOI":"10.1016\/j.omtm.2016.12.006","article-title":"Global Manufacturing of CAR T Cell Therapy","volume":"4","author":"Levine","year":"2017","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"701","DOI":"10.4149\/BLL_2018_125","article-title":"Comparison of transfection efficiency of polymer-based and lipid-based transfection reagents","volume":"119","author":"Rahimi","year":"2018","journal-title":"Bratisl. Med. J."},{"key":"ref_23","first-page":"95","article-title":"An improved method for increasing the efficiency of gene transfection and transduction","volume":"10","author":"Shi","year":"2018","journal-title":"Int. J. Physiol. Pathophysiol. Pharmacol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1016\/j.exphem.2015.10.003","article-title":"Kinetics of lentiviral vector transduction in human CD34+ cells","volume":"44","author":"Uchida","year":"2016","journal-title":"Exp. Hematol."},{"key":"ref_25","first-page":"73","article-title":"Critical factors for lentivirus-mediated PRDX4 gene transfer in the HepG2 cell line","volume":"16","author":"Aznan","year":"2018","journal-title":"Oncol. Lett."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1016\/j.omtm.2021.03.015","article-title":"Efficient lentiviral transduction method to gene modify cord blood CD8+ T cells for cancer therapy applications","volume":"21","author":"Cornel","year":"2021","journal-title":"Mol. Ther. Methods Clin. Dev."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"497","DOI":"10.1182\/blood-2003-01-0297","article-title":"Human T lymphocytes transduced by lentiviral vectors in the absence of TCR activation maintain an intact immune competence","volume":"102","author":"Cavalieri","year":"2003","journal-title":"Blood"},{"key":"ref_28","doi-asserted-by":"crossref","unstructured":"Leyva, F.J., Anzinger, J.J., McCoy, J.P., and Kruth, H.S. (2011). Evaluation of transduction efficiency in macrophage colony-stimulating factor differentiated human macrophages using HIV-1 based lentiviral vectors. BMC Biotechnol., 11.","DOI":"10.1186\/1472-6750-11-13"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"1529","DOI":"10.1038\/s41375-018-0106-0","article-title":"Clinical use of lentiviral vectors","volume":"32","author":"Milone","year":"2018","journal-title":"Leukemia"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e1049","DOI":"10.1002\/cti2.1049","article-title":"Selecting costimulatory domains for chimeric antigen receptors: Functional and clinical considerations","volume":"8","author":"Weinkove","year":"2019","journal-title":"Clin. Transl. Immunol."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"2449373","DOI":"10.1155\/2022\/2449373","article-title":"An In Vitro Comparison of Costimulatory Domains in Chimeric Antigen Receptor T Cell for Breast Cancer Treatment","volume":"2022","author":"Khuisangeam","year":"2022","journal-title":"J. Immunol. Res."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"39","DOI":"10.1186\/s13045-022-01244-0","article-title":"Chimeric antigen receptors containing the OX40 signalling domain enhance the persistence of T cells even under repeated stimulation with multiple myeloma target cells","volume":"15","author":"Tan","year":"2022","journal-title":"J. Hematol. Oncol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3750","DOI":"10.1182\/blood-2014-01-552174","article-title":"Closely related T-memory stem cells correlate with in vivo expansion of CAR.CD19-T cells and are preserved by IL-7 and IL-15","volume":"123","author":"Xu","year":"2014","journal-title":"Blood"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"482","DOI":"10.1186\/s12967-024-05254-z","article-title":"Unraveling resistance mechanisms in anti-CD19 chimeric antigen receptor-T therapy for B-ALL: A novel in vitro model and insights into target antigen dynamics","volume":"22","author":"Li","year":"2024","journal-title":"J. Transl. Med."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/7\/679\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,5]],"date-time":"2026-08-05T17:02:06Z","timestamp":1785949326000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/7\/679"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,30]]},"references-count":34,"journal-issue":{"issue":"7","published-online":{"date-parts":[[2026,7]]}},"alternative-id":["cimb48070679"],"URL":"https:\/\/doi.org\/10.3390\/cimb48070679","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,30]]}}}