Synthetic mRNA

This volume presents detailed laboratory protocols for in vitro synthesis of mRNA with favorable properties, its introduction into cells by a variety of techniques, and the measurement of physiological and clinical consequences such as protein replacement and cancer immunotherapy. Synthetic techniques are described for structural features in mRNA that provide investigational tools such as fluorescence emission, click chemistry, photo-chemical crosslinking, and that produce mRNA with increased stability in the cell, increased translational efficiency, and reduced activation of the innate immune response. Protocols are described for clinical applications such as large-scale transfection of dendritic cells, production of GMP-grade mRNA, redirecting T cell specificity, and use of molecular adjuvants for RNA vaccines. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessarymaterials and reagents, step-by-step laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

• Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences* is a valuable and cutting-edge resource for both laboratory investigators and clinicians interested in this powerful and rapidly evolving technology.

Includes cutting-edge methods and protocols Provides step-by-step detail essential for reproducible results Contains key notes and implementation advice from the experts Includes supplementary material: sn.pub/extras

Inhalt
Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences.- Synthetic Capped mRNAs for Cap-Specific Photo-Crosslinking Experiments.- Enzymatic Modification of 5'-Capped RNA and Subsequent Labeling by Click Chemistry.- Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m 7 GpppG.- Intronless b-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements.- Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA.- Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells.- Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study the Innate Immune System.- GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical Use.- Large Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems.- FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines.- Transfecting Human Monocytes with RNA.- In Vitro Synthesis, Delivery and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transposition.- Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage.- Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy.- Immune Monitoring using mRNA-Transfected Dendritic Cells.- Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2.- mRNA Electroporation of Dendritic Cells with WT1, Survivin and TriMix (A Mixture of caTLR4, CD40L, and CD70).- Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation.- Measuring Hematocrit in Mice Injected with In Vitro-Transcribed Erythropoietin mRNA.- Genetic Modification of Human Pancreatic Progenitor Cells through Modified mRNA.