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However, the potential genotoxicity of viral\u2010based CAR\u2010T therapy, their considerable manufacturing cycle and high costs, prompts questions about its safety and leads to limitations in clinical application. Aiming to explore a safe, efficient, and low\u2010cost mRNA\u2010based CAR\u2010T therapy, we characterize a nonviral, mRNA\u2010based approach utilizing a current good manufacturing practice (cGMP)\u2010compatible electroporation (EP) platform to generate anti\u2010CD19, anti\u2010CD22, and tandem anti\u2010CD19\/CD22 CAR\u2010T products with a turn\u2010around time of less than 48\u2009h. The protocol yields a more than 90% cellular viability with a CAR expression of exceeding 60% within the 24\u2009h of transfection. The mRNA\u2010based CAR\u2010T products have a significantly enhanced T cell activation profile with CD69 upregulation, production of tumor necrosis factor\u2010alpha (TNF\u2010\u03b1), interleukin\u20102 (IL\u20102), and Granzyme B, and higher Ki67 expression level. Robust in vitro tumor neutralization by the mRNA\u2010based CAR\u2010T was observed compared to control. Interestingly, we have observed that the short\u2010term cell cryopreservation after EP resulted in a 50% reduction of cellular expansion. However, the cryopreservation and thaw did not have an observed negative impact on the in vitro tumor neutralization. Lastly, we have evaluated the metabolic activity of the mRNA\u2010based CAR\u2010T products, which has a clearly inducible and significant increase in basal respiration (by 60%) and a 2\u2010fold spare respiratory capacity (SRC) upon exposure with the antigen positive target cells. We believe this approach holds promise as a viable alternative that addresses the limitations of current CAR\u2010T therapies, offering a potential solution for future nonviral CAR\u2010T clinical application.<\/jats:p>","DOI":"10.1155\/jimr\/9452254","type":"journal-article","created":{"date-parts":[[2026,7,31]],"date-time":"2026-07-31T05:52:57Z","timestamp":1785477177000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Characterization of Short Production Cycle and Nongenotoxic mRNA\u2010Based CAR\u2010T Cells Targeting CD19\u2010Positive and CD22\u2010Positive 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