Revisiting the Need for mRNA Nucleoside Modification in CAR T Cell Engineering

Revisiting the Need for mRNA Nucleoside Modification in CAR T Cell Engineering | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var 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Profile Anna Luisa Hiller , View ORCID Profile Charlotte Maeve Dunne , View ORCID Profile Michael Launspach , View ORCID Profile Arnd Kleyer , View ORCID Profile David Nils Simon , View ORCID Profile Dimitrios Laurin Wagner , View ORCID Profile Chantal Pichon , View ORCID Profile Gerhard Krönke , View ORCID Profile Michael Schmueck-Henneresse , View ORCID Profile Hans-Dieter Volk , View ORCID Profile Manfred Gossen , View ORCID Profile Norman Michael Drzeniek doi: https://doi.org/10.1101/2025.01.21.630650 Nourhan Kahwaji 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 2 Pantherna Therapeutics GmbH , 16761 Hennigsdorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Nourhan Kahwaji Niklas Kotzian 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 3 Freie Universität Berlin, Institute of Chemistry and Biochemistry , Arnimallee 20, 14195 Berlin : niklas Find this author on Google Scholar Find this author on PubMed Search for this author on this site Jasmin Melissa Prinz 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Yaolin Pu 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 4 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and HumboldtUniversität zu Berlin, Berlin Center for Advanced Therapies (BeCAT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Yaolin Pu Jonas Kath 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 4 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and HumboldtUniversität zu Berlin, Berlin Center for Advanced Therapies (BeCAT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Jonas Kath Samira Picht 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Samira Picht Anna Luisa Hiller 5 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Infectious Diseases and Critical Care Medicine , Charitéplatz 1, 10117 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Anna Luisa Hiller Charlotte Maeve Dunne 6 Pantherna Therapeutics GmbH , 16761 Hennigsdorf, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Charlotte Maeve Dunne Michael Launspach 7 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Pediatric Oncology and Hematology , Charité, Augustenburger Platz 1, 13353 Berlin 8 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin , Anna-Louisa-Karsch-Straße 2, 10178 Berlin, Germany 9 German Cancer Consortium (DKTK), Partner Site Berlin , Invalidenstr. 80, 10115 Berlin, Germany 10 German Cancer Research Center (DKFZ) , Im Neuenheimer Feld 280, 69120 Heidelberg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Launspach Arnd Kleyer 11 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology , Charitéplatz 1, 10117 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Arnd Kleyer David Nils Simon 11 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology , Charitéplatz 1, 10117 Berlin, Germany 12 Deutsches Rheuma-Forschungszentrum , Charitéplatz 1, 10117 Berlin, Germany 13 Department of Internal Medicine 3, University of Erlangen-Nuremberg and Universitätsklinikum Erlangen , Ulmenweg 18, 91054 Erlangen, Germany 14 Deutsches Zentrum für Immuntherapie (DZI), University of Erlangen-Nuremberg and Universitätsklinikum Erlangen , Erlangen, Ulmenweg 18, 91054 Erlangen, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for David Nils Simon Dimitrios Laurin Wagner 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 4 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and HumboldtUniversität zu Berlin, Berlin Center for Advanced Therapies (BeCAT) , Augustenburger Platz 1, 13353 Berlin, Germany 15 Center for Cell and Gene Therapy, Baylor College of Medicine, Texas Children’s Hospital, and Houston Methodist Hospital , One Baylor Plaza, Houston, TX 77030, USA 16 Department of Molecular and Cellular Biology, Baylor College of Medicine, Texas Children’s Hospital, and Houston Methodist Hospital , One Baylor Plaza, Houston, TX 77030, USA 17 Dan L Duncan Comprehensive Cancer Center , Houston, 1919 Old Spanish Trl, Houston, TX 77054, USA Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Dimitrios Laurin Wagner Chantal Pichon 18 ART ARNm US 55 Inserm, LI2RSO University of Orléans , F-45100 Orléans, France 19 Institut Universitaire de France , 1 rue Descartes, F-75035 Paris, France Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Chantal Pichon Gerhard Krönke 11 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology , Charitéplatz 1, 10117 Berlin, Germany 12 Deutsches Rheuma-Forschungszentrum , Charitéplatz 1, 10117 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gerhard Krönke Michael Schmueck-Henneresse 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Michael Schmueck-Henneresse Hans-Dieter Volk 20 Charité – Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt Universität zu Berlin, Institute of Medical Immunology , Augustenburger Platz 1, 13353 Berlin, Germany 21 tcBalance Biopharmaceuticals GmbH , Marienstr. 7, 10117 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Hans-Dieter Volk Manfred Gossen 22 Institute of Active Polymers, Helmholtz-Zentrum Hereon , Kantstraße 55, 14513 Teltow, Germany 23 Berlin-Brandenburg Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Manfred Gossen Norman Michael Drzeniek 1 Berlin Institute of Health (BIH) at Charité–Universitätsmedizin Berlin, BIH Center for Regenerative Therapies (BCRT) , Augustenburger Platz 1, 13353 Berlin, Germany 11 Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology , Charitéplatz 1, 10117 Berlin, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Norman Michael Drzeniek For correspondence: norman.drzeniek{at}charite.de Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract mRNA-based chimeric antigen receptor (CAR)-T cells offer the promise of enhanced safety and simplified manufacturing. However, in vitro-transcribed (IVT) mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To address these challenges and enable efficient IVT-mRNA expression, modified nucleosides, such as N1-methyl-pseudouridine (m1Ψ), have become the gold standard for CAR-T cell production, albeit at increased cost. In this study, immune responses to IVT-mRNA were evaluated across five primary human cell types, including T-cells. Unexpectedly, T-cells, unlike other immune and non-immune cell types tested, exhibited no immune activation in response to unmodified mRNA. T-cell viability and cytokine secretion patterns remained unaffected, regardless of whether unmodified mRNA was delivered via lipid nanoparticles (LNPs) or electroporation. Furthermore, CAR expression levels in T-cells were not influenced by mRNA modification with m1Ψ or 5-methoxy-uridine (5moU) nucleosides. The absence of nucleoside modifications did not compromise CAR-T cell cytotoxic potency, demonstrating that such modifications are not required for producing functional CAR-T cells. These findings eliminate the need for nucleoside modification in T-cell mRNA, simplifying and reducing the cost of CAR-T cell manufacturing while positioning IVT-mRNA as a highly efficient and minimally invasive tool for CAR-T cell engineering. Introduction Virus-transduced CAR-T-cell therapies are often associated with high cost, burdensome logistics and lingering safety concerns [ 1 ]. Novel non-malignant applications of CAR-T-cells, such as breakthrough treatment of autoimmune disease [ 2 ] and promising elimination of fibrosis and senescence [ 3 , 4 ], have sparked an acute interest in mRNA-based transient CAR-T-cell products with improved safety and reduced manufacturing complexity, that facilitates scalability[ 5 ]. mRNA-CAR-T-cells are being developed against autoimmunity [ 6 ] and cancer [ 7 ] alike. Nevertheless, mRNA production remains expensive, largely due to the use of chemically modified nucleosides. Excessive immune activation upon recognition of IVT-mRNA by pattern recognition receptors (PRR), a major roadblock in the development of mRNA therapeutics [ 8 ], has been largely overcome by mRNA nucleoside modifications, such as N1-methyl-pseudouridine (m1Ψ) in Comirnaty and Spikevax SARS-CoV2 vaccines [ 9 ], or 5-methoxy-uridine (5moU), which has shown even greater reduction of mRNA immune activation in our previous studies [ 10 , 11 ]. m1Ψ has become a gold standard in the mRNA field and to the best of our knowledge, all reports of mRNA-based CAR-T-cell engineering use m1Ψ-modified mRNA [ 12 , 13 ]. However, modified nucleosides amount to ∼35% of mRNA manufacturing cost at large-scale. Surprisingly little is known about the different sensitivity of different cell types toward mRNA-triggered immune activation and thus about cell type-dependent requirements for mRNA modification. In our previous work, we extensively characterized the immune response toward mRNA with different chemical modifications in bone marrow stromal cells [ 10 , 11 ]. Asking to what extent those insights could be transferred to other cell types, here we screened responses toward unmodified and nucleoside-modified (m1Ψ, 5moU) mRNA in 5 primary human cell types, including T-cells, macrophages, endothelial, epithelial and stromal cells. Surprisingly, and in contrast to all other cell types tested, T-cells exhibited no immune response to unmodified mRNA, regardless of the transfection method. mRNAs with different nucleoside chemistries resulted in comparable expression of CD19-CAR in primary human T-cells. To confirm that the use of unmodified mRNA does not functionally impair T-cells, we demonstrated that mRNA-CAR-T cells transfected with unmodified mRNA exhibited equivalent cytotoxic activity compared to those transfected with industry-standard m1Ψ-modified CAR-mRNA. Methods Cell Culture Blood was drawn from n≥3 consenting adult donors (Charité ethics committee approval EA4/091/19). From the mononuclear fraction obtained by density gradient centrifugation, primary T-cells and monocytes were isolated using magnetic cell sorting with beads against CD3 and CD14 respectively (Miltenyi Biotec, Germany)[ 11 ]. T-cells were cultured and activated in ImmunoCult-XF TCell Expansion Medium with recombinant IL-2 and ImmunoCult HumanCD3/CD28/CD2 TCell-Activator (Stemcell Technologies, Canada). Macrophages were differentiated within 7 days of culture in RPMI1640 containing 10% FCS,1% Penicillin/Streptomycin and 50ng/mL M-CSF (Miltenyi) [ 11 , 14 ]. Bone marrow stromal cells were supplied by the BIH Center for Regenerative Therapies (BCRT) core facility “Cell Harvesting” [ 15 ]. Written informed consent was given, and approval was obtained from the Charité IRB/local ethics committee (EA2/089/20). Human umbilical vein endothelial cells were purchased from Promocell, Germany. Tubular epithelial cells were a kind gift from Prof. Babel’s group at BCRT. Non-immune cells were cultured in low-glucose DMEM containing 10% FCS, 1% Glutamax and 1% Penicillin/Streptomycin. mRNA Synthesis and Transfection DNA sequences encoding enhanced green fluorescent protein (GFP) or chimeric antigen receptor (CAR; Supplemental Table 1) were cloned into the pRNA2-(A)128 vector, amplified by PCR and transcribed using the TranscriptAidT7 kit (Thermo Fischer, USA) into mRNA containing uridine, m1Ψ or 5moU (Jena Bioscience, Germany). mRNA was purified using lithium chloride precipitation, washed in 70% ethanol and stored in aqueous solution, as previously described [ 11 ]. mRNA was complexed with Lipofectamine MessengerMax (LMM; ThermoFischer) at a ratio of 1ug:2ul [ 10 ] and added to cells at 2ug mRNA per 1*10 6 cells. Because LMM did not transfect T-cells (Figure S1), mRNA was microfluidically formulated with Genvoy T-cell lipid mix (Cytiva, USA) using setting #3 on a NanoAssemblr Spark and added to cells after 48h of activation. T-cells or macrophages were electroporated using a 4D-Nucleofector (Lonza, Switzerland) in P3 buffer using the EH-115 or DP-148 programs respectively. Quantification of mRNA Expression and Cell Death All readouts were performed 24h post-transfection. GFP fluorescent imaging was performed on a Nikon ECLIPSE Ti (Nikon Instruments, Japan). GFP- and CAR-expression were quantified using using MACSQuantVYB (Miltenyi) or CytoflexLX (Beckmann Coulter, USA) flowcytometers. CD19-CARs were stained using anti-Myc-Tag (CAR1[ 16 ] 1:50; 9B11; CellSignalingTechnology, USA) or anti-IgG-Fcγ (CAR2[ 17 ]; 1:50; 109-605-098; Jackson ImmunoResearch, USA) antibodies. DAPI was used (1 µg/mL) for live/dead discrimination. Data was analysed in FlowJov10; a representative gating strategy is shown in Figure S2. Caspase activity was quantified as previously described [ 10 ]. Cytokine and Interferon Analysis Concentration of interferons α and β were measured by ELISA and T-cell secretomes were measured using ProteomeProfilerXL Human Cytokine array (all R&D Systems, USA) from supernatants 24h post-transfection, according to the manufacturer’s instructions. Cytotoxicity Assay CAR-dependent cytotoxicity was assessed in a VITAL assay, as previously described [ 16 , 18 ]. Briefly, CAR-T-cells were incubated at decreasing ratios with Nalm6 (CD19+ GFP+; target) and Nalm6 (CD19-RFP+; control) cells for 8h, stained with DAPI, and ratios of surviving target and control cells were analyzed by flow cytometry. Cytotoxicity was normalized against mock-transfected, CAR-negative T-cells using the formula: Data Analysis Statistical analysis was carried out in Prism10 (GraphPad Software., USA). A one-way or two-way ANOVA with Tukey’s or Dunnet’s post-hoc test was used to test for significant differences between groups. Heatmaps were generated in R and Python v3.11.0. Results and Discussion Primary human T-cells, monocyte-derived macrophages, and three non-hematopoietic primary cell types representative of different tissues (human umbilical vein endothelial cells; bone marrow stromal cells; renal tubular epithelial cells) were screened for antiviral responses toward IVT-mRNA, which can reduce cell viability and impair mRNA expression [ 11 ]. Non-hematopoietic cells showed very similar interferon response, caspase activation and cell death in response to unmodified mRNA, which was greatly reduced by uridine substitution with m1Ψ and further reduced by 5moU. Macrophages showed the strongest immune response and apoptosis to unmodified mRNA ( Figure1a-d ; Figure S3; Figure S4). This was also reflected in the relatively large impact of uridine modification on mRNA expression in macrophages ( Figure 1e-f ; Figure S5). Download figure Open in new tab Figure 1. Responses toward mRNA with different nucleoside chemistry vary between cell types. Renal tubular epithelial cells, umbilical vein endothelial cells, bone marrow stromal cells, macrophages and T-cells (all primary human cells) were transfected with unmodified (U) or m1Ψ-modified or 5moU-modified GFP-encoding mRNA and cellular responses were measured 24h post-transfection: a, b) Secretion of type-I interferons β and α in supernatants, which are hallmarks of cellular immune responses against RNA viruses. c) Increase in activity of caspases 3 and 7, as an indicator of apoptosis induction. d) Cell viability measured using flow cytometry. The fraction of mRNA-expressing cells (e) and expression level (f) measured by quantifying GFP fluorescence using flow cytometry. n= 3-12, detailed graphs and statistical information can be found in Supplemental Figures S3-5. Surprisingly and in contrast to all other cell types, T-cells did not show any adverse response to unmodified mRNA in terms of interferon secretion, caspase activation or cell death. Also, all three mRNAs were expressed equally, suggesting no beneficial impact of uridine modification in T-cells ( Figure 1 ; Figure S3-5). Except for T-cells requiring mRNA to be encapsulated in lipid nanoparticles (LNPs; Figure S1), all other cells could be transfected with lipofectamine-complexed mRNA. We therefore asked whether the lack of a recognizable immune reaction toward unmodified mRNA in T-cells depended on the LNP-based delivery. This was not the case, as shown in side-by-side comparison of mRNA delivery using LNP versus electroporation (EP): T-cells displayed no increased cell death in response to unmodified mRNA for either transfection method ( Figure 2a ; Figure S6). Electroporated T-cells exhibited low viability loss in response to EP-related stress (mock-EP), which did not increase when mRNA was added. Download figure Open in new tab Figure 2. Human T-cells show no immune activation by unmodified IVT-mRNA. Unmodified mRNA was introduced into primary human T-cells using lipofection (LNP) and electroporation (EP) to exclude dependency of immune activation on the delivery mechanism. a) 24h following either transfection method, T-cells showed strong expression of mRNA-encoded GFP and no reduction in cell viability was observed compared to untreated or mock-electroporated cells (EP without mRNA). n=1 representative set of flow cytometry plots is shown out of n=12 from 4 biological donors (Figure S6). b) Interferon β secretion from T-cells was quantified using ELISA and revealed no interferon induction after transfection with unmodified mRNA through either EP or LNP. Monocyte-derived macrophages (MΦ) were used as a positive control. n=3 donors, median is shown. c) EP led to more T cell death compared to LNP, but in contrast to macrophages, cell death was not increased by mRNA treatment. n=4 donors. Mean is shown. d) A protein array measuring 105 cytokines in T cell supernatants 24h post-transfection did not reveal any secretion pattern shift upon transfection with unmodified mRNA. n=1. e) Publicly available bulk RNA sequencing data [ 19 ] was interrogated for transcripts of pattern recognition receptors relevant for intracellular recognition of mRNA (NOD2, RIGI, TLR7, TLR8) and related adapter proteins (MyD88, TRIF, MAVS). The analysis revealed that the two uridine-recognizing RNA receptors (TLR7, TLR8) were not expressed in any T lymphocyte subsets and their adapters MyD88 and TRIF were expressed at lower levels than in other leukocytes. TPM: transcripts per million. n=4. * p<0,05; ** p<0,01; *** p<0,001; ns= not significant. In direct comparison with macrophages, T-cells did not show any interferon-β induction or apoptosis following transfection. Macrophages displayed both responses, although the same LNP-formulation was used to transfect both cell types ( Figure 2b-c ). To assess whether mRNA transfection influenced the production of cytokines beyond type-I interferons, a protein array profiling 105 cytokines was performed on T-cell supernatants. In contrast to bespoke secretome shifts previously described in stromal cells [ 11 ], T-cells transfected with unmodified mRNA via either EP or LNP exhibited indistinguishable cytokine secretion patterns ( Figure 2d ). Notably, the production of key T-cell cytokines, including IFN-γ, IL-2 or the inflammation marker TNFα, remained unaffected by mRNA treatment. In conclusion, multidimensional profiling of primary human T-cells revealed no evidence of immune activation or downstream side effects in response to unmodified mRNA. Interrogations of RNA sequencing data from two independent sources [ 19 , 20 ] revealed that among RNA-sensing PRRs, TLRs 7 and 8, which recognize unmodified uridine were expressed in T-cells at lower levels than in other leukocytes, suggesting that T-cells might not be equipped to recognize uridine-containing mRNA as a pathogen-associated pattern ( Figure 2e , Figure S7). While an in-depth characterization of RNA-sensing pathways in T-cells was outside the scope of this study, our aim was to determine whether the costly nucleoside modifications traditionally used in mRNA-based CAR-T cell production are necessary for generating potent CAR-T cells ( Figure 3a ). Unmodified, m1Ψ-modified, and 5moU-modified CAR-mRNA resulted in equally high CD19-CAR expression rates (>85%; Figure 3b ), high T-cell viability (>90%; Fig. 3b ) and equally potent cytotoxicity against B-cells ( Figure 3c ). These results demonstrate that in applications of mRNA to T-cells, uridine modifications could be omitted in favour of unmodified mRNA. Download figure Open in new tab Figure 3. CAR-T cells generated with unmodified versus nucleotide-modified mRNA are functionally equivalent. a) Schematic: mRNA with or without nucleoside modifications encoding two different anti-CD19 chimeric antigen receptors (designated by magenta and blue respectively) was used to generate CAR-T cells directed against a target B cell line (NALM-6 cells). > 85% of T-cells showed CAR expression (b) and high viability (c) , irrespective of the mRNA nucleoside modification used. n= 7 donors. d) A VITAL assay to quantify antigen-specific cytotoxicity of CAR-T cells against NALM-6 B cells [ 16 ] was performed at an increasing ratio (E:T:C) between effector CAR-T cells (E), CD19+ target cells (T) and CD19-control cells (C). It revealed no difference in killing functionality between CAR-T cells generated with unmodified versus m1Ψ-modified or 5moU-modified mRNA. n=6: n=3 donors and n=2 CAR constructs. ns= not significant. Compared to virus-transduction of CAR-T-cells, mRNA-based manufacturing could reduce cost, time and complexity[ 5 ]. Nevertheless, mRNA is commonly limited by antiviral immune responses. Uridine modification used to mitigate this immune activation account for ∼35% of IVT-mRNA manufacturing costs. However, human T-cells appear to lack receptors required to recognize uridine on exogenous mRNA. This study demonstrates that unmodified mRNA does not elicit immune reactions in T-cells, highlighting it as a safe, effective, and cost-efficient alternative for CAR-T cell manufacturing. Authorship contributions NoKa: Investigation, Formal analysis, Visualization. NiKo: Investigation, Formal analysis. JMP: Investigation, Visualization. YP: Investigation, Formal analysis. JK: Validation, Methodology. SP: Methodology. ALH: Software. CMD: Methodology. ML: Methodology, Writing – review & editing. AK: Writing – review & editing. DNS: Writing – review & editing. DLW: Methodology, Resources. CP: Writing – review & editing. GK: Writing – review & editing. MSH: Methodology, Resources, Funding acquisition, Writing – review & editing. HDV: Conceptualization, Funding acquisition, Writing – review & editing. MG: Conceptualization, Supervision, Resources, Writing – review & editing. NMD: Conceptualization, Project administration, Supervision, Methodology, Investigation, Formal analysis, Visualization, Writing – original draft, Funding acquisition. Conflict of interest disclosures No.Ka. and C.M.D. are currently employees of Pantherna Therapeutics GmbH, focused on RNA therapeutics. The opinions expressed in this article are those of the authors and not necessarily those of Pantherna. Pantherna was neither financially involved in the creation nor in the publication of this article. H.-D.V. and D.L.W. are co-founders of TCBalance Biopharmaceuticals GmbH focused on regulatory T cell therapy. The opinions expressed in this article are those of the authors and not necessarily those of TCBalance. TCBalance was neither financially involved in the creation nor in the publication of this article. The other authors declare no conflicts of interest. Data availability All underlying data of this manuscript can be obtained from the corresponding author upon request. The pRNA2-(A)128 vector is available on Addgene (ID: 174006). The plasmid containing the CD19-CAR 2 sequence can be obtained from Addgene (ID: 183473). Acknowledgements This project had received funding from the Einstein Center for Regenerative Therapies (ECRT) Advanced Scientist Kickbox. 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