The Emerging Role of In Vitro-Transcribed mRNA in Adoptive T Cell Immunotherapy
Authors: Jessica B. Foster, David M. Barrett, Katalin Karikó
Journal: Molecular Therapy (2019)

Adoptive T cell therapy is a form of cellular therapy that utilizes human immune cells, often empowered by the expression of recombinant proteins, to attack selected targets present on tumor or infected cells. T cell-based immunotherapy has been progressing over the past several decades, and reached a milestone with the recent US Food and Drug Administration (FDA) approval of chimeric antigen receptor T cell therapy for relapsed and refractory leukemia and lymphoma. Although most studies have used viral vectors, a growing number of researchers have come to appreciate in vitro-transcribed (IVT) mRNA for the development, testing, and application of T cell-based immunotherapeutics. IVT mRNA offers inherent safety features, highly efficient recombinant protein translation, and the ability to control pharmacokinetic properties of the therapy. In this review, we discuss the history of IVT mRNA in adoptive T cell therapy, from tumor-infiltrating lymphocytes and T cell receptor-based therapies to chimeric antigen receptor therapy and gene-editing techniques, as well as prior and ongoing clinical trials. Adoptive T cell therapy is a form of cellular therapy that utilizes human immune cells, often empowered by the expression of recombinant proteins, to attack selected targets present on tumor or infected cells. T cell-based immunotherapy has been progressing over the past several decades, and reached a milestone with the recent US Food and Drug Administration (FDA) approval of chimeric antigen receptor T cell therapy for relapsed and refractory leukemia and lymphoma. Although most studies have used viral vectors, a growing number of researchers have come to appreciate in vitro-transcribed (IVT) mRNA for the development, testing, and application of T cell-based immunotherapeutics. IVT mRNA offers inherent safety features, highly efficient recombinant protein translation, and the ability to control pharmacokinetic properties of the therapy. In this review, we discuss the history of IVT mRNA in adoptive T cell therapy, from tumor-infiltrating lymphocytes and T cell receptor-based therapies to chimeric antigen receptor therapy and gene-editing techniques, as well as prior and ongoing clinical trials. The existence of mRNA, a labile molecule that carries information of the encoded protein from the chromosome to the site of translation, was discovered in the early 1960s. However, technologies to generate in vitro-transcribed (IVT) mRNA only became available in 1984, and ever since, IVT mRNA has been utilized for its therapeutic potential.1Sahin U. Karikó K. Türeci Ö. mRNA-based therapeutics—developing a new class of drugs.Nat. Rev. Drug Discov. 2014; 13: 759-780Google Scholar Within cells, mRNA is the blueprint for protein production. IVT mRNA can be translated into any protein when introduced into cells, thus providing an opportunity to manipulate and augment cells in any imaginable way. In addition, IVT mRNA has become an attractive tool because of its rapid and facile production, as well as its safety profile. Unlike DNA, IVT mRNA has no risk of causing insertional mutagenesis and no long-term concern for side effects because of its labile nature. The affordability and ease-of-use allow for iterative trials in the development of novel therapies. Immunotherapy is the use of the immune system to attack specific targets, such as malignant cells or viruses. Immunotherapy encompasses many different types of therapy that utilize different components of the immune system. Adoptive T cell therapy is one form of immunotherapy that focuses on ex vivo expansion of T cells and manipulation of cells for targeted cytotoxicity, prior to returning the T cells into the patient (Figure 1). In this review, we discuss the recent use of IVT mRNA in adoptive T cell therapy, ranging from tumor-directed immunotherapy to infectious disease applications. We highlight the strengths and weaknesses of using IVT mRNA for this approach, as well as the recent clinical trials applying this technology. Non-viral gene transfer into primary T lymphocytes has long been problematic because of the poor efficiency of delivery. However, initial preclinical and clinical studies confirmed that dendritic cells (DCs) electroporated ex vivo with antigen-encoding IVT mRNA generate potent immune responses.2Van Tendeloo V.F.I. Ponsaerts P. Lardon F. Nijs G. Lenjou M. Van Broeckhoven C. Van Bockstaele D.R. Berneman Z.N. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.Blood. 2001; 98: 49-56Google Scholar, 3Zhao Y. Boczkowski D. Nair S.K. Gilboa E. Inhibition of invariant chain expression in dendritic cells presenting endogenous antigens stimulates CD4+ T-cell responses and tumor immunity.Blood. 2003; 102: 4137-4142Google Scholar, 4Su Z. Dannull J. Yang B.K. Dahm P. Coleman D. Yancey D. Sichi S. Niedzwiecki D. Boczkowski D. Gilboa E. Vieweg J. Telomerase mRNA-transfected dendritic cells stimulate antigen-specific CD8+ and CD4+ T cell responses in patients with metastatic prostate cancer.J. Immunol. 2005; 174: 3798-3807Google Scholar It was also noted that electroporation of IVT mRNA not only increased efficiency of transgene expression, but also increased DC viability compared with when DNA plasmids were delivered.2Van Tendeloo V.F.I. Ponsaerts P. Lardon F. Nijs G. Lenjou M. Van Broeckhoven C. Van Bockstaele D.R. Berneman Z.N. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells.Blood. 2001; 98: 49-56Google Scholar Similar results were also observed in macrophages5Van De Parre T.J. Martinet W. Schrijvers D.M. Herman A.G. De Meyer G.R. mRNA but not plasmid DNA is efficiently transfected in murine J774A.1 macrophages.Biochem. Biophys. Res. Commun. 2005; 327: 356-360Google Scholar and CD40-activated B cells electroporated with IVT mRNA.6Van den Bosch G.A. Ponsaerts P. Nijs G. Lenjou M. Vanham G. Van Bockstaele D.R. Berneman Z.N. Van Tendeloo V.F. Ex vivo induction of viral antigen-specific CD8 T cell responses using mRNA-electroporated CD40-activated B cells.Clin. Exp. Immunol. 2005; 139: 458-467Google Scholar, 7Van den Bosch G.A. Van Gulck E. Ponsaerts P. Nijs G. Lenjou M. Apers L. Kint I. Heyndrickx L. Vanham G. Van Bockstaele D.R. et al.Simultaneous activation of viral antigen-specific memory CD4+ and CD8+ T-cells using mRNA-electroporated CD40-activated autologous B-cells.J. Immunother. 2006; 29: 512-523Google Scholar Smits et al.8Smits E. Ponsaerts P. Lenjou M. Nijs G. Van Bockstaele D.R. Berneman Z.N. Van Tendeloo V.F. RNA-based gene transfer for adult stem cells and T cells.Leukemia. 2004; 18: 1898-1902Google Scholar in Belgium were the first to electroporate IVT mRNA into T lymphocytes. They discovered that only stimulated T cells translated the electroporated mRNA, whereas the non-stimulated ones did not.8Smits E. Ponsaerts P. Lenjou M. Nijs G. Van Bockstaele D.R. Berneman Z.N. Van Tendeloo V.F. RNA-based gene transfer for adult stem cells and T cells.Leukemia. 2004; 18: 1898-1902Google Scholar Shortly afterward, investigators at the National Cancer Institute (NCI) applied electroporation to transduce peripheral blood mononuclear cells (PBMCs) as well. After investigating a variety of electroporation conditions, they could achieve >90% efficiency and >80% viability.9Zhao Y. Zheng Z. Cohen C.J. Gattinoni L. Palmer D.C. Restifo N.P. Rosenberg S.A. Morgan R.A. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation.Mol. Ther. 2006; 13: 151-159Google Scholar They also electroporated T cells with IVT mRNA encoding the α and β chains of T cell receptor (TCR) directed against NY-ESO-1, MART-1, and p53. These T cells transduced with TCR mRNA produced interferon (IFN) gamma when exposed to T2 cells pulsed with the corresponding peptides or specific melanoma cell lines expressing NY-ESO-1.9Zhao Y. Zheng Z. Cohen C.J. Gattinoni L. Palmer D.C. Restifo N.P. Rosenberg S.A. Morgan R.A. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation.Mol. Ther. 2006; 13: 151-159Google Scholar That same year, Schaft et al.10Schaft N. Dörrie J. Müller I. Beck V. Baumann S. Schunder T. Kämpgen E. Schuler G. A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.Cancer Immunol. Immunother. 2006; 55: 1132-1141Google Scholar also reported successful introduction of glycoprotein 100 (gp100)-specific TCR into primary T cells using IVT mRNA, again with excellent cytotoxicity in peptide-loaded T2 cells and melanoma cell lines. In 2006, Rabinovich et al.11Rabinovich P.M. Komarovskaya M.E. Ye Z.J. Imai C. Campana D. Bahceci E. Weissman S.M. Synthetic messenger RNA as a tool for gene therapy.Hum. Gene Ther. 2006; 17: 1027-1035Google Scholar were the first to transduce T cells with IVT mRNA encoding chimeric antigen receptors (CARs) directed against CD19 and demonstrated functionality of those T cells in vitro. By 2009, several studies reported using IVT mRNA to create hematopoietic cells with CARs. Birkholz et al.12Birkholz K. Hombach A. Krug C. Reuter S. Kershaw M. Kämpgen E. Schuler G. Abken H. Schaft N. Dörrie J. Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer.Gene Ther. 2009; 16: 596-604Google Scholar reported the efficacy of ErbB2 and carcinoembryonic antigen (CEA)-directed CAR T cells created with IVT mRNA, with modest cytotoxicity against a variety of cancer cell lines in vitro. Yoon et al.13Yoon S.H. Lee J.M. Cho H.I. Kim E.K. Kim H.S. Park M.Y. Kim T.G. Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model.Cancer Gene Ther. 2009; 16: 489-497Google Scholar applied the electroporation method described by Zhao et al.9Zhao Y. Zheng Z. Cohen C.J. Gattinoni L. Palmer D.C. Restifo N.P. Rosenberg S.A. Morgan R.A. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation.Mol. Ther. 2006; 13: 151-159Google Scholar on stimulated peripheral blood lymphocytes as the effector cells. Using T cells transduced with IVT mRNA encoding Her2/neu-directed CAR and containing signaling domains of CD28 and CD3 zeta, antitumor activity could be demonstrated in both in vitro and in vivo ovarian tumor models.13Yoon S.H. Lee J.M. Cho H.I. Kim E.K. Kim H.S. Park M.Y. Kim T.G. Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model.Cancer Gene Ther. 2009; 16: 489-497Google Scholar As a follow-up, they investigated cytokine-induced killer cells electroporated with IVT mRNA encoding the same Her2/neu CAR.14Yoon S.H. Lee J.M. Woo S.J. Park M.J. Park J.S. Kim H.S. Park M.Y. Sohn H.J. Kim T.G. Transfer of Her-2/neu specificity into cytokine-induced killer (CIK) cells with RNA encoding chimeric immune receptor (CIR).J. Clin. Immunol. 2009; 29: 806-814Google Scholar Again, they showed significant antitumor effects against in vitro and in vivo tumor models. While in both studies lymphocytes transduced with CAR mRNA inhibited tumor growth better than Herceptin, a monoclonal antibody (mAb) specific for Her2/neu, in both studies tumor growth was slowed only without any tumor regression.13Yoon S.H. Lee J.M. Cho H.I. Kim E.K. Kim H.S. Park M.Y. Kim T.G. Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model.Cancer Gene Ther. 2009; 16: 489-497Google Scholar, 14Yoon S.H. Lee J.M. Woo S.J. Park M.J. Park J.S. Kim H.S. Park M.Y. Sohn H.J. Kim T.G. Transfer of Her-2/neu specificity into cytokine-induced killer (CIK) cells with RNA encoding chimeric immune receptor (CIR).J. Clin. Immunol. 2009; 29: 806-814Google Scholar In 2009, Rabinovich et al.15Rabinovich P.M. Komarovskaya M.E. Wrzesinski S.H. Alderman J.L. Budak-Alpdogan T. Karpikov A. Guo H. Flavell R.A. Cheung N.K. Weissman S.M. Bahceci E. Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.Hum. Gene Ther. 2009; 20: 51-61Google Scholar separated T cells and natural killer (NK) cells from PBMCs for transfection with CAR mRNA. The IVT CAR mRNA was introduced into CD3+/CD4+ T cells and CD3+/CD8+ T cells, as well as NK cells. Electroporation of all these cell populations resulted in high levels of surface expression of CAR.15Rabinovich P.M. Komarovskaya M.E. Wrzesinski S.H. Alderman J.L. Budak-Alpdogan T. Karpikov A. Guo H. Flavell R.A. Cheung N.K. Weissman S.M. Bahceci E. Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.Hum. Gene Ther. 2009; 20: 51-61Google Scholar In addition, all cell groups were capable of producing target-specific cytotoxicity; however, CD8+ cytotoxic T lymphocytes (CTLs) showed the most robust tumor killing and were successful in treating a murine lymphoma model.15Rabinovich P.M. Komarovskaya M.E. Wrzesinski S.H. Alderman J.L. Budak-Alpdogan T. Karpikov A. Guo H. Flavell R.A. Cheung N.K. Weissman S.M. Bahceci E. Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.Hum. Gene Ther. 2009; 20: 51-61Google Scholar All together, these studies shaped the subsequent 10 years of research, where focus honed on T cells for adoptive cellular therapy (Table 1).Table 1List of Published Works Utilizing IVT mRNA for Adoptive T Cell ImmunotherapyTargetDisease or ModelReferencesmRNA Encoding CAR T CellsCD19B cell lymphoma, leukemia11Rabinovich P.M. Komarovskaya M.E. Ye Z.J. Imai C. Campana D. Bahceci E. Weissman S.M. Synthetic messenger RNA as a tool for gene therapy.Hum. Gene Ther. 2006; 17: 1027-1035Google Scholar, 15Rabinovich P.M. Komarovskaya M.E. Wrzesinski S.H. Alderman J.L. Budak-Alpdogan T. Karpikov A. Guo H. Flavell R.A. Cheung N.K. Weissman S.M. Bahceci E. Chimeric receptor mRNA transfection as a tool to generate antineoplastic lymphocytes.Hum. Gene Ther. 2009; 20: 51-61Google Scholar, 47Almåsbak H. Rian E. Hoel H.J. Pulè M. Wälchli S. Kvalheim G. Gaudernack G. Rasmussen A.M. Transiently redirected T cells for adoptive transfer.Cytotherapy. 2011; 13: 629-640Google ScholarALL48Barrett D.M. Zhao Y. Liu X. Jiang S. Carpenito C. Kalos M. Carroll R.G. June C.H. Grupp S.A. Treatment of advanced leukemia in mice with mRNA engineered T cells.Hum. Gene Ther. 2011; 22: 1575-1586Google Scholar, 49Barrett D.M. Liu X. Jiang S. June C.H. Grupp S.A. Zhao Y. Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia.Hum. Gene Ther. 2013; 24: 717-727Google Scholar, 50Almåsbak H. Walseng E. Kristian A. Myhre M.R. Suso E.M. Munthe L.A. Andersen J.T. Wang M.Y. Kvalheim G. Gaudernack G. Kyte J.A. Inclusion of an IgG1-Fc spacer abrogates efficacy of CD19 CAR T cells in a xenograft mouse model.Gene Ther. 2015; 22: 391-403Google Scholar, 51Foster J.B. Choudhari N. Perazzelli J. Storm J. Hofmann T.J. Jain P. Storm P.B. Pardi N. Weissman D. Waanders A.J. et al.Purification of mRNA encoding chimeric antigen receptor is critical for generation of a robust T-cell response.Hum. Gene Ther. 2018; (Published online October 2, 2018)https://doi.org/10.1089/hum.2018.145Google Scholar, 65Liu X. Barrett D.M. Jiang S. Fang C. Kalos M. Grupp S.A. June C.H. Zhao Y. Improved anti-leukemia activities of adoptively transferred T cells expressing bispecific T-cell engager in mice.Blood Cancer J. 2016; 6: e430Google ScholarHodgkin's lymphoma52Svoboda J. Rheingold S.R. Gill S.I. Grupp S.A. Lacey S.F. Kulikovskaya I. Suhoski M.M. Melenhorst J.J. Loudon B. Mato A.R. et al.Nonviral RNA chimeric antigen receptor-modified T cells in patients with Hodgkin lymphoma.Blood. 2018; 132: 1022-1026Google ScholarMesothelinmesothelioma33Zhao Y. Moon E. Carpenito C. Paulos C.M. Liu X. Brennan A.L. Chew A. Carroll R.G. Scholler J. Levine B.L. et al.Multiple injections of electroporated autologous T cells expressing a chimeric antigen receptor mediate regression of human disseminated tumor.Cancer Res. 2010; 70: 9053-9061Google Scholar, 34Maus M.V. Haas A.R. Beatty G.L. Albelda S.M. Levine B.L. Liu X. Zhao Y. Kalos M. June C.H. T cells expressing chimeric antigen receptors can cause anaphylaxis in humans.Cancer Immunol. Res. 2013; 1: 26-31Google Scholar, 49Barrett D.M. Liu X. Jiang S. June C.H. Grupp S.A. Zhao Y. Regimen-specific effects of RNA-modified chimeric antigen receptor T cells in mice with advanced leukemia.Hum. Gene Ther. 2013; 24: 717-727Google Scholarmesothelioma, pancreatic cancer35Beatty G.L. Haas A.R. Maus M.V. Torigian D.A. Soulen M.C. Plesa G. Chew A. Zhao Y. Levine B.L. Albelda S.M. et al.Mesothelin-specific chimeric antigen receptor mRNA-engineered T cells induce anti-tumor activity in solid malignancies.Cancer Immunol. Res. 2014; 2: 112-120Google Scholarpancreatic cancer36Beatty G.L. O'Hara M.H. Lacey S.F. Torigian D.A. Nazimuddin F. Chen F. Kulikovskaya I.M. Soulen M.C. McGarvey M. Nelson A.M. et al.Activity of mesothelin-specific chimeric antigen receptor T cells against pancreatic carcinoma metastases in a phase 1 trial.Gastroenterology. 2018; 155: 29-32Google Scholarovarian cancer37Hung C.F. Xu X. Li L. Ma Y. Jin Q. Viley A. Allen C. Natarajan P. Shivakumar R. Peshwa M.V. Emens L.A. Development of anti-human mesothelin-targeted chimeric antigen receptor messenger RNA-transfected peripheral blood lymphocytes for ovarian cancer therapy.Hum. Gene Ther. 2018; 29: 614-625Google ScholarMCSPmelanoma40Krug C. Birkholz K. Paulus A. Schwenkert M. Schmidt P. Hoffmann N. Hombach A. Fey G. Abken H. Schuler G. et al.Stability and activity of MCSP-specific chimeric antigen receptors (CARs) depend on the scFv antigen-binding domain and the protein backbone.Cancer Immunol. Immunother. 2015; 64: 1623-1635Google Scholar, 41Harrer D.C. Simon B. Fujii S.I. Shimizu K. Uslu U. Schuler G. Gerer K.F. Hoyer S. Dörrie J. Schaft N. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma.BMC Cancer. 2017; 17: 551Google ScholarHer-2/neuovarian cancer13Yoon S.H. Lee J.M. Cho H.I. Kim E.K. Kim H.S. Park M.Y. Kim T.G. Adoptive immunotherapy using human peripheral blood lymphocytes transferred with RNA encoding Her-2/neu-specific chimeric immune receptor in ovarian cancer xenograft model.Cancer Gene Ther. 2009; 16: 489-497Google Scholar, 14Yoon S.H. Lee J.M. Woo S.J. Park M.J. Park J.S. Kim H.S. Park M.Y. Sohn H.J. Kim T.G. Transfer of Her-2/neu specificity into cytokine-induced killer (CIK) cells with RNA encoding chimeric immune receptor (CIR).J. Clin. Immunol. 2009; 29: 806-814Google ScholarErbB2, CEAgastric, colorectal, ovarian, and breast cancers12Birkholz K. Hombach A. Krug C. Reuter S. Kershaw M. Kämpgen E. Schuler G. Abken H. Schaft N. Dörrie J. Transfer of mRNA encoding recombinant immunoreceptors reprograms CD4+ and CD8+ T cells for use in the adoptive immunotherapy of cancer.Gene Ther. 2009; 16: 596-604Google ScholarFolate receptorovarian cancer39Schutsky K. Song D.G. Lynn R. Smith J.B. Poussin M. Figini M. Zhao Y. Powell Jr., D.J. Rigorous optimization and validation of potent RNA CAR T cell therapy for the treatment of common epithelial cancers expressing folate receptor.Oncotarget. 2015; 6: 28911-28928Google ScholarCD33AML54Kenderian S.S. Ruella M. Shestova O. Klichinsky M. Aikawa V. Morrissette J.J. Scholler J. Song D. Porter D.L. Carroll M. et al.CD33-specific chimeric antigen receptor T cells exhibit potent preclinical activity against human acute myeloid leukemia.Leukemia. 2015; 29: 1637-1647Google ScholarCD123AML55Tasian S.K. Kenderian S.S. Shen F. Ruella M. Shestova O. Kozlowski M. Li Y. Schrank-Hacker A. Morrissette J.J.D. Carroll M. et al.Optimized depletion of chimeric antigen receptor T cells in murine xenograft models of human acute myeloid leukemia.Blood. 2017; 129: 2395-2407Google Scholar, 56Cummins K.D. Frey N. Nelson A.M. Schmidt A. Luger S. Isaacs R.E. Lacey S.F. Hexner E. Melenhorse J.J. June C.H. et al.Treating relapsed / refractory (RR) AML with biodegradable anti-CD123 CAR modified T cells.Blood. 2017; 130: 1359Google ScholarLigands of NKGD2(CD314)Ewing's sarcoma45Lehner M. Götz G. Proff J. Schaft N. Dörrie J. Full F. Ensser A. Muller Y.A. Cerwenka A. Abken H. et al.Redirecting T cells to Ewing's sarcoma family of tumors by a chimeric NKG2D receptor expressed by lentiviral transduction or mRNA transfection.PLoS ONE. 2012; 7: e31210Google ScholarGD2neuroblastoma38Singh N. Liu X. Hulitt J. Jiang S. June C.H. Grupp S.A. Barrett D.M. Zhao Y. Nature of tumor control by permanently and transiently modified GD2 chimeric antigen receptor T cells in xenograft models of neuroblastoma.Cancer Immunol. Res. 2014; 2: 1059-1070Google ScholarEGFRglioblastoma44Caruso H.G. Torikai H. Zhang L. Maiti S. Dai J. Do K.A. Singh H. Huls H. Lee D.A. Champlin R.E. et al.Redirecting T-cell specificity to EGFR using mRNA to self-limit expression of chimeric antigen receptor.J. Immunother. 2016; 39: 205-217Google ScholarVEGFR2melanoma42Inoo K. Inagaki R. Fujiwara K. Sasawatari S. Kamigaki T. Nakagawa S. Okada N. Immunological quality and performance of tumor vessel-targeting CAR-T cells prepared by mRNA-EP for clinical research.Mol. Ther. Oncolytics. 2016; 3: 16024Google ScholarCD20canine lymphoma53Panjwani M.K. Smith J.B. Schutsky K. Gnanandarajah J. O'Connor C.M. Powell Jr., D.J. Mason N.J. Feasibility and safety of RNA-transfected CD20-specific chimeric antigen receptor T cells in dogs with spontaneous B cell lymphoma.Mol. Ther. 2016; 24: 1602-1614Google ScholarEpCAMgastric cancer, ovarian cancer43Ang W.X. Li Z. Chi Z. Du S.H. Chen C. Tay J.C. Toh H.C. Connolly J.E. Xu X.H. Wang S. Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis.Oncotarget. 2017; 8: 13545-13559Google ScholarChimeric CD3 binder-CD28-4-1BBexpansion protocol for T cell manufacture64Liu X. Jiang S. Fang C. Li H. Zhang X. Zhang F. June C.H. Zhao Y. Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.Protein Cell. 2017; 8: 514-526Google Scholarc-Metbreast cancer46Tchou J. Zhao Y. Levine B.L. Zhang P.J. Davis M.M. Melenhorst J.J. Kulikovskaya I. Brennan A.L. Liu X. Lacey S.F. et al.Safety and efficacy of intratumoral injections of chimeric antigen receptor (CAR) T cells in metastatic breast cancer.Cancer Immunol. Res. 2017; 5: 1152-1161Google ScholarmRNA Encoding T Cell Enhancers or TCRs Targeting Cancer and Viral AntigensNY-ESO1, MART1, P53melanoma9Zhao Y. Zheng Z. Cohen C.J. Gattinoni L. Palmer D.C. Restifo N.P. Rosenberg S.A. Morgan R.A. High-efficiency transfection of primary human and mouse T lymphocytes using RNA electroporation.Mol. Ther. 2006; 13: 151-159Google Scholargp100melanoma10Schaft N. Dörrie J. Müller I. Beck V. Baumann S. Schunder T. Kämpgen E. Schuler G. A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.Cancer Immunol. Immunother. 2006; 55: 1132-1141Google Scholar, 41Harrer D.C. Simon B. Fujii S.I. Shimizu K. Uslu U. Schuler G. Gerer K.F. Hoyer S. Dörrie J. Schaft N. RNA-transfection of γ/δ T cells with a chimeric antigen receptor or an α/β T-cell receptor: a safer alternative to genetically engineered α/β T cells for the immunotherapy of melanoma.BMC Cancer. 2017; 17: 551Google ScholarcaTLR4melanoma25Pato A. Eisenberg G. Machlenkin A. Margalit A. Cafri G. Frankenburg S. Merims S. Peretz T. Lotem M. Gross G. Messenger RNA encoding constitutively active Toll-like receptor 4 enhances effector functions of human T cells.Clin. Exp. Immunol. 2015; 182: 220-229Google ScholarIL-2, IL-12, and IL-15melanoma26Weinstein-Marom H. Pato A. Levin N. Susid K. Itzhaki O. Besser M.J. Peretz T. Margalit A. Lotem M. Gross G. Membrane-attached cytokines expressed by mRNA electroporation act as potent T-cell adjuvants.J. Immunother. 2016; 39: 60-70Google ScholarcaCD40melanoma27Levin N. Weinstein-Marom H. Pato A. Itzhaki O. Besser M.J. Eisenberg G. Peretz T. Lotem M. Gross G. Potent activation of human T cells by mRNA encoding constitutively active CD40.J. Immunol. 2018; 201: 2959-2968Google ScholarGM-CSF in OT1 T cellsintracranial melanoma, astrocytoma28Pohl-Guimarães F. Yang C. Dyson K.A. Wildes T.J. Drake J. Huang J. Flores C. Sayour E.J. Mitchell D.A. RNA-modified T cells mediate effective delivery of immunomodulatory cytokines to brain tumors.Mol. Ther. 2018; (Published online October 17, 2018)https://doi.org/10.1016/j.ymthe.2018.10.007Scopus (16) Google ScholarCXCR2CMV20Mitchell D.A. Karikari I. Cui X. Xie W. Schmittling R. Sampson J.H. Selective modification of antigen-specific T cells by RNA electroporation.Hum. Gene Ther. 2008; 19: 511-521Google Scholargag, nefHIV22Hofmann C. Höfflin S. Hückelhoven A. Bergmann S. Harrer E. Schuler G. Dörrie J. Schaft N. Harrer T. Human T cells expressing two additional receptors (TETARs) specific for HIV-1 recognize both epitopes.Blood. 2011; 118: 5174-5177Google ScholarCD86, 4-1BBLimproved activity64Liu X. Jiang S. Fang C. Li H. Zhang X. Zhang F. June C.H. Zhao Y. Novel T cells with improved in vivo anti-tumor activity generated by RNA electroporation.Protein Cell. 2017; 8: 514-526Google ScholarHBV envelope and corehepatitis B21Kah J. Koh S. Volz T. Ceccarello E. Allweiss L. Lütgehetmann M. Bertoletti A. Dandri M. Lymphocytes transiently expressing virus-specific T cell receptors reduce hepatitis B virus infection.J. Clin. Invest. 2017; 127: 3177-3188Google ScholarWT1-specific TCR (with inhibition of endogenous TCR)AML23Campillo-Davo D. Fujiki F. Van den Bergh J.M.J. De Reu H. Smits E.L.J.M. Goossens H. Sugiyama H. Lion E. Berneman Z.N. Van Tendeloo V. Efficient and non-genotoxic RNA-based engineering of human T cells using tumor-specific T cell receptors with minimal TCR mispairing.Front. Immunol. 2018; 9: 2503Google ScholarNuclease mRNA for Gene Editing of T CellsDisruption of TCR and CD52 with TALEN; CD19 CARlymphoma57Poirot L. Philip B. Schiffer-Mannioui C. Le Clerre D. Chion-Sotinel I. Derniame S. Potrel P. Bas C. Lemaire L. Galetto R. et al.Multiplex genome-edited T-cell manufacturing platform for "off-the-shelf" adoptive T-cell immunotherapies.Cancer Res. 2015; 75: 3853-3864Google ScholarDisruption of TCR and CD52 with TALEN; CD19 CARinfant ALL58Qasim W. Zhan H. Samarasinghe S. Adams S. Amrolia P. Stafford S. Butler K. Rivat C. Wright G. Somana K. et al.Molecular remission of infant B-ALL after infusion of universal TALEN gene-edited CAR T cells.Sci. Transl. Med. 2017; 9 (eaaj2013)Google ScholarDisruption of CCR5 with megaTAL; HIV CARHIV61Sather B.D. Romano Ibarra G.S. Sommer K. Curinga G. Hale M. Khan I.F. Singh S. Song Y. Gwiazda K. Sahni J. et al.Efficient modification of CCR5 in primary human hematopoietic cells using a megaTAL nuclease and AAV donor template.Sci. Transl. Med. 2015; 7 (307ra156)Google ScholarDisruption of CCR5 with zinc-finger nucleaseplatform for gene editing60Wang J. DeClercq J.J. Hayward S.B. Li P.W. Shivak D.A. Gregory P.D. Lee G. Holmes M.C. Highly efficient homology-driven genome editing in human T cells by combining zinc-finger nuclease mRNA and AAV6 donor delivery.Nucleic Acids Res. 2016; 44: e30Google ScholarInsertion of CD19 CAR into TRAC with CRISPR/Cas9leukemia59Eyquem J. Mansilla-Soto J. Giavridis T. van der Stegen S.J. Hamieh M. Cunanan K.M. Odak A. Gönen M. Sadelain M. Targeting a CAR to the TRAC locus with CRISPR/Cas9 enhances tumour rejection.Nature. 2017; 543: 113-117Google Scholar Open table in a new tab Synthetically created TCRs mimic the structure of endogenous α and β chains, enabling optimization to increase affinity if desired. The first report of effective transfer of a TCR into T lymphocytes using a retroviral vector was published in 1999 by Clay et al.16Clay T.M. Custer M.C. Sachs J. Hwu P. Rosenberg S.A. Nishimura M.I. Efficient transfer of a tumor antigen-reactive TCR to human peripheral blood lymphocytes confers anti-tumor reactivity.J. Immunol. 1999; 163: 507-513Google Scholar Based on these promising results, several clinical trials were initiated using TCRs genetically designed with increased affinity for tumor antigens. However, a few of these trials resulted in patient deaths when the increased affinity TCR bound not only to the intended target antigen but cross-reacted with normal tissues.17Morgan R.A. Chinnasamy N. Abate-Daga D. Gros A. Robbins P.F. Zheng Z. Dudley M.E. Feldman S.A. Yang J.C. Sherry R.M. et al.Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.J. Immunother. 2013; 36: 133-151Google Scholar, 18Linette G.P. Stadtmauer E.A. Maus M.V. Rapoport A.P. Levine B.L. Emery L. Litzky L. Bagg A. Carreno B.M. Cimino P.J. et al.Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced T cells in myeloma and melanoma.Blood. 2013; 122: 863-871Google Scholar, 19Cameron B.J. Gerry A.B. Dukes J. Harper J.V. Kannan V. Bianchi F.C. Grand F. Brewer J.E. Gupta M. Plesa G. et al.Identification of a Titin-derived HLA-A1-presented peptide as a cross-reactive target for engineered MAGE A3-directed T cells.Sci. Transl. Med. 2013; 5: 197ra103Google Scholar These unfortunate events highlighted the importance of safety issues when using permanently altered T cells and increased the interest in safe alternatives such as IVT mRNA. As discussed above, Zhao e