TY - JOUR AU - Anderson, Bart R. AU - Muramatsu, Hiromi AU - Nallagatla, Subba R. AU - Bevilacqua, Philip C. AU - Sansing, Lauren H. AU - Weissman, Drew AU - Karikó, Katalin TI - Incorporation of pseudouridine into mRNA enhances translation by diminishing PKR activation JF - NUCLEIC ACIDS RESEARCH J2 - NUCLEIC ACIDS RES VL - 38 PY - 2010 IS - 17 SP - 5884 EP - 5892 PG - 9 SN - 0305-1048 DO - 10.1093/nar/gkq347 UR - https://m2.mtmt.hu/api/publication/32039148 ID - 32039148 N1 - Funding Agency and Grant Number: National Institutes of HealthUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USA [R01AI50484, R21DE019059, T32GM07229, T32DK07748, T32RR007063, R42HL87688, R01GM058709]; NATIONAL CENTER FOR RESEARCH RESOURCESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Center for Research Resources (NCRR) [T32RR007063] Funding Source: NIH RePORTER; NATIONAL HEART, LUNG, AND BLOOD INSTITUTEUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Heart Lung & Blood Institute (NHLBI) [R42HL087688] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Allergy & Infectious Diseases (NIAID) [R01AI050484] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF DENTAL &CRANIOFACIAL RESEARCHUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Dental & Craniofacial Research (NIDCR) [R21DE019059] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of Diabetes & Digestive & Kidney Diseases (NIDDK) [T32DK007748] Funding Source: NIH RePORTER; NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCESUnited States Department of Health & Human ServicesNational Institutes of Health (NIH) - USANIH National Institute of General Medical Sciences (NIGMS) [T32GM007229, R01GM058709] Funding Source: NIH RePORTER Funding text: The National Institutes of Health (R01AI50484, R21DE019059 to D.W., T32GM07229, T32DK07748, T32RR007063 to B.R.A., R42HL87688 to K.K. and R01GM058709 to P.C.B.). Funding for open access charge: National Institutes of Health grant R42HL87688. Department of Medicine, University of Pennsylvania, Philadelphia, PA, United States Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, United States Department of Chemistry, Pennsylvania State University, University Park, PA, United States Department of Neurology, University of Pennsylvania, Philadelphia, PA, United States Cited By :156 Export Date: 28 May 2021 CODEN: NARHA Correspondence Address: Karikó, K.; Department of Neurosurgery, University of Pennsylvania, Philadelphia, PA, United States; email: kariko@mail.med.upenn.edu AB - Previous studies have shown that the translation level of in vitro transcribed messenger RNA (mRNA) is enhanced when its uridines are replaced with pseudouridines; however, the reason for this enhancement has not been identified. Here, we demonstrate that in vitro transcripts containing uridine activate RNA-dependent protein kinase (PKR), which then phosphorylates translation initiation factor 2-alpha (eIF-2 alpha), and inhibits translation. In contrast, in vitro transcribed mRNAs containing pseudouridine activate PKR to a lesser degree, and translation of pseudouridine-containing mRNAs is not repressed. RNA pull-down assays demonstrate that mRNA containing uridine is bound by PKR more efficiently than mRNA with pseudouridine. Finally, the role of PKR is validated by showing that pseudouridine- and uridine-containing RNAs were translated equally in PKR knockout cells. These results indicate that the enhanced translation of mRNAs containing pseudouridine, compared to those containing uridine, is mediated by decreased activation of PKR. LA - English DB - MTMT ER - TY - JOUR AU - Karikó, Katalin AU - Muramatsu, Hiromi AU - Welsh, Frank A. AU - Ludwig, Janos AU - Kato, Hiroki AU - Akira, Shizuo AU - Weissman, Drew TI - Incorporation of Pseudouridine Into mRNA Yields Superior Nonimmunogenic Vector With Increased Translational Capacity and Biological Stability JF - MOLECULAR THERAPY J2 - MOL THER VL - 16 PY - 2008 IS - 11 SP - 1833 EP - 1840 PG - 8 SN - 1525-0016 DO - 10.1038/mt.2008.200 UR - https://m2.mtmt.hu/api/publication/32039151 ID - 32039151 N1 - Funding Agency and Grant Number: National Institutes of Health (NIH) [NIAID AI-050484, NHLBI HL87688, NINDS NS-29331]; Ruth L. Kirschstein National Research Service Awards Funding text: This work was supported by the National Institutes of Health (NIH) grants NIAID AI-050484, NHLBI HL87688, and NINDS NS-29331. H. M. was supported by Ruth L. Kirschstein National Research Service Awards postdoctoral fellowship. We thank Houping Ni for technical assistance. K. K. and D. W. have formed a small biotech company that receives funding from the NIH to explore the use of nucleoside-modified mRNA for gene therapy. AB - In vitro-transcribed mRNAs encoding physiologically important proteins have considerable potential for therapeutic applications. However, in its present form, mRNA is unfeasible for clinical use because of its labile and immunogenic nature. Here, we investigated whether incorporation of naturally modified nucleotides into transcripts would confer enhanced biological properties to mRNA. We found that mRNAs containing pseudouridines have a higher translational capacity than unmodified mRNAs when tested in mammalian cells and lysates or administered intravenously into mice at 0.015-0.15 mg/kg doses. The delivered mRNA and the encoded protein could be detected in the spleen at 1, 4, and 24 hours after the injection, where both products were at significantly higher levels when pseudouridine-containing mRNA was administered. Even at higher doses, only the unmodified mRNA was immunogenic, inducing high serum levels of interferon-alpha ( IFN-alpha). These findings indicate that nucleoside modification is an effective approach to enhance stability and translational capacity of mRNA while diminishing its immunogenicity in vivo. Improved properties conferred by pseudouridine make such mRNA a promising tool for both gene replacement and vaccination. LA - English DB - MTMT ER - TY - JOUR AU - Karikó, Katalin AU - Buckstein, M. AU - Ni, H. AU - Weissman, D. TI - Suppression of RNA recognition by Toll-like receptors: The impact of nucleoside modification and the evolutionary origin of RNA JF - IMMUNITY J2 - IMMUNITY VL - 23 PY - 2005 IS - 2 SP - 165 EP - 175 PG - 11 SN - 1074-7613 DO - 10.1016/j.immuni.2005.06.008 UR - https://m2.mtmt.hu/api/publication/32039262 ID - 32039262 AB - DNA and RNA stimulate the mammalian innate immune system through activation of Toll-like receptors (TLRs). DNA containing methylated CpG motifs, however, is not stimulatory. Selected nucleosides in naturally occurring RNA are also methylated or otherwise modified, but the immunomodulatory effects of these alterations remain untested. We show that RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. We conclude that nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue. Copyright ©2005 by Elsevier Inc. LA - English DB - MTMT ER - TY - JOUR AU - Karikó, Katalin AU - Ni, H. AU - Capodici, J. AU - Lamphier, M. AU - Weissman, D. TI - mRNA Is an Endogenous Ligand for Toll-like Receptor 3 JF - JOURNAL OF BIOLOGICAL CHEMISTRY J2 - J BIOL CHEM VL - 279 PY - 2004 IS - 13 SP - 12542 EP - 12550 PG - 9 SN - 0021-9258 DO - 10.1074/jbc.M310175200 UR - https://m2.mtmt.hu/api/publication/32039266 ID - 32039266 AB - Toll-like receptors (TLRs) are the basic signaling receptors of the innate immune system. They are activated by molecules associated with pathogens or injured host cells and tissue. TLR3 has been shown to respond to double stranded (ds) RNA, a replication intermediary for many viruses. Here we present evidence that heterologous RNA released from or associated with necrotic cells or generated by in vitro transcription also stimulates TLR3 and induces immune activation. To assess RNA-mediated TLR3 activation, human embryonic kidney 293 cells stably expressing TLR3 and containing a nuclear factor-κB-dependent luciferase reporter were generated. Exposing these cells to in vitro transcribed RNA resulted in a TLR3-dependent induction of luciferase activity and interleukin-8 secretion. Treatment with in vitro transcribed mRNA activated nuclear factor-κB via TLR3 through a process that was dose-dependent and involved tyrosine phosphorylation. Furthermore, in vitro transcribed natural or 2′-fluoro-substituted mRNA induced the expression of TLR3, interferon regulatory factor-1, tumor necrosis factor-α, and interleukin-1 receptor-associated kinase-M mRNA in human dendritic cells (DCs). DCs responded to mRNA treatment by expressing activation markers, and this maturation was inhibited by antagonistic TLR3-specific antibody. Endogenous RNA released from or associated with necrotic cells also stimulated DCs, leading to interferon-α secretion, which could be abolished by pretreatment of necrotic cells with RNase. These results demonstrate that RNA, likely through secondary structure, is a potent host-derived activator of TLR3. This finding has potential physiologic relevance because RNA escaping from damaged tissue or contained within endocytosed cells could serve as an endogenous ligand for TLR3 that induces or otherwise modulates immune responses. LA - English DB - MTMT ER -