南太湖新工科产业学报 3136-2249 求真学术出版 04330a89-37c4-417f-9eca-70003bc10e36 10.68249/jstnei.2026.0006 0006 CD5-targeted LNP-mRNA generates CD19 CAR-T cells in the body to achieve lupus remission without clearing lymph nodes Liu GangBelarus State University Alena Mikalaeuna VasiukevichBelarus State University 2026729 11 2438

Conventional methods that rely on viral vectors, ex vivo cell manufacturing, and lymphocyte-depleting preconditioning significantly limit the use of chimeric antigen receptor T-cell (CAR-T) therapy, despite its potential in treating systemic lupus erythematosus (SLE). This study aimed to design an in vivo method for generating CD19 CAR-T cells utilising lipid nanoparticle (LNP)-mRNA and validate the feasibility of disease remission in lupus models without lymphodepletion. We created a CD5/LNP-CAR19 nanoformulation that is precisely delivered to T cells by encasing the mRNA encoding a second-generation CAR that targets human CD19 in LNPs coupled with CD5 antibodies using microfluidic technology.A single intravenous injection was given to MRL/lpr lupus mice as a model, and T-cell transfection efficiency, CAR expression dynamics, B-cell depletion, autoantibody levels, proteinuria, kidney pathology improvements, and safety indicators such as cytokine release were systematically evaluated using flow cytometry, in vivo imaging, ELISA, and histopathology. In peripheral T cells, CD5/LNP-CAR19 effectively and selectively produced temporary CAR expression, which lasted more than five days and reached 32.4% ± 5.1% CAR⁺ T cells 24 hours after injection. Deep and long-lasting B-cell depletion was caused by a single dose; over 95% of B cells in peripheral blood and lymphoid tissues were destroyed, and the effects persisted for more than eight weeks. Urine protein/creatinine ratios recovered to normal, kidney immune complex deposits and pathology scores were drastically reversed, and anti-dsDNA antibody titers dramatically decreased in the therapy group. In the medicated group, survival rose from 40% in the untreated mice to 90%. Throughout, no lymphocyte depletion was carried out, and there was no discernible cytokine release syndrome, neurotoxicity, or weight loss. In a lupus model without lymphodepletion, this study first demonstrated that CD5-targeted LNP-mRNA may instruct T cells in vivo to temporarily produce enough functional CD19 CAR-T cells, allowing for safe long-term remission. With substantial clinical translational potential, this "in vivo, ready-to-go" CAR-T approach provides a very accessible and safe new therapy option for B-cell-mediated autoimmune disorders.

 CAR-T cellsLipid nanoparticlesmRNAIn vivo generationCD19Systemic lupus erythematosusLymphodepletion-freeCD5 targeting CC BY-NC-ND 4.0 [1] Tsokos GC. Systemic lupus erythematosus. N Engl J Med. 2011;365(22):2110-2121. [2] Furie R, Petri M, Zamani O, et al. A phase III, randomized, placebo-controlled study of belimumab, a monoclonal antibody that inhibits B lymphocyte stimulator, in patients with systemic lupus erythematosus. Arthritis Rheum. 2011;63(12):3918-3930. [3] Merrill JT, Neuwelt CM, Wallace DJ, et al. Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, double-blind, phase II/III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum. 2010;62(1):222-233. [4] Rovin BH, Furie R, Latinis K, et al. Efficacy and safety of rituximab in patients with active proliferative lupus nephritis: the Lupus Nephritis Assessment with Rituximab study. Arthritis Rheum. 2012;64(4):1215-1226. [5] Maude SL, Laetsch TW, Buechner J, et al. Tisagenlecleucel in children and young adults with B-cell lymphoblastic leukemia. N Engl J Med. 2018;378(5):439-448. [6] Neelapu SS, Locke FL, Bartlett NL, et al. Axicabtagene ciloleucel CAR T-cell therapy in refractory large B-cell lymphoma. N Engl J Med. 2017;377(26):2531-2544. [7] Cappell KM, Sherry RM, Yang JC, et al. Long-term follow-up of anti-CD19 chimeric antigen receptor T-cell therapy. J Clin Oncol. 2020;38(32):3805-3815. [8] Mougiakakos D, Krönke G, Völkl S, et al. CD19-targeted CAR T cells in refractory systemic lupus erythematosus. N Engl J Med. 2021;385(6):567-569. [9] Mackensen A, Müller F, Mougiakakos D, et al. Anti-CD19 CAR T cell therapy for refractory systemic lupus erythematosus. Nat Med. 2022;28(10):2124-2132. [10] Müller F, Boeltz S, Knitza J, et al. CD19-targeted CAR T cells in refractory antisynthetase syndrome. Lancet. 2023;401(10379):815-818. [11] Haghikia A, Hegelmaier T, Wolleschak D, et al. Anti-CD19 CAR T cells for refractory myasthenia gravis. Lancet Neurol. 2023;22(12):1104-1105. [12] Levine BL, Miskin J, Wonnacott K, Keir C. Global manufacturing of CAR T cell therapy. Mol Ther Methods Clin Dev. 2017;4:92-101. [13] Amini L, Silbert SK, Maude SL, et al. Preparing for CAR T cell therapy: patient selection, bridging therapies and lymphodepletion. Nat Rev Clin Oncol. 2022;19(5):342-355. [14] Shah NN, Qin H, Yates B, et al. Clonal expansion of CAR T cells harboring lentivector integration in the CBL gene following anti-CD22 CAR T-cell therapy. Blood Adv. 2019;3(15):2317-2322. [15] Fiorenza S, Ritchie DS, Ramsey SD, Turtle CJ. Value and affordability of CAR T-cell therapy in the United States. Bone Marrow Transplant. 2020;55(9):1706-1715. [16] Polack FP, Thomas SJ, Kitchin N, et al. Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine. N Engl J Med. 2020;383(27):2603-2615. [17] Parayath NN, Stephan SB, Koehne AL, Nelson PS, Stephan MT. In vitro-transcribed antigen receptor mRNA nanocarriers for transient expression in circulating T cells in vivo. Nat Commun. 2020;11(1):6080. [18] Billingsley MM, Singh N, Ravikumar P, Zhang R, June CH, Mitchell MJ. Ionizable lipid nanoparticle-mediated mRNA delivery for human CAR T cell engineering. Nano Lett. 2020;20(3):1578-1589. [19] Tombácz I, Laczkó D, Shahnawaz H. Highly efficient CD4+ T cell targeting and genetic recombination using engineered CD4+ cell-homing mRNA-LNPs. Molecular Therapy, 2021; 29, 3293-3304. [20] Jing-e Zhou, Lei Sun, Yujie Jia, Zhehao Wang, Tengshuo Luo, Jingwen Tan, Xiaoyan Fang, Hongjia Zhu, Jing Wang, Lei Yu, Zhiqiang Yan,Lipid nanoparticles produce chimeric antigen receptor T cells with interleukin-6 knockdown in vivo,Journal of Controlled Release,Volume 350,2022,Pages 298-307. [21] Rurik JG, Tombácz I, Yadegari A, et al. CAR T cells produced in vivo to treat cardiac injury. Science. 2022;375(6576):91-96. [22] Katsuyama T, Tsokos GC, Moulton VR. Aberrant T cell signaling and subsets in systemic lupus erythematosus. Front Immunol. 2018;9:1088. [23] Kansal R, Richardson N, Neeli I, et al. Sustained B cell depletion by CD19-targeted CAR T cells is a highly effective treatment for murine lupus. Sci Transl Med. 2019;11(482):eaav1648.