Synthetic mRNA: Production, Introduction Into Cells, and Physiological Consequences (Methods in Molecular Biology, 1428)

Preface
Contents
Contributors
Part I: Introduction
	Chapter 1: Synthetic mRNA: Production, Introduction into Cells, and Physiological Consequences
		1 Introduction
		2 Basic Methodology for the In Vitro Synthesis of mRNA
			2.1 The RNA Chain
			2.2 The Cap
			2.3 The Poly(A) Tract
		3 Improving the Stability and Translational Efficiency of Synthetic mRNA
			3.1 The Cap
				3.1.1 ARCAs
				3.1.2 Caps that Are Resistant to Decapping Enzymes
				3.1.3 Other Modified Cap Analogs
			3.2 The 5- and 3-UTR
			3.3 The Coding Region
			3.4 The Poly(A) Tract
			3.5 The 3-End
		4 Delivery of Exogenous mRNA to the Cell
			4.1 Direct Uptake of Naked mRNA
			4.2 Cationic Liposome-Mediated RNA Transfection
			4.3 Protamine-Complexed mRNA
			4.4 Electroporation and Nucleoporation
			4.5 Other Techniques
		5 Exogenous mRNA and the Innate Immunity Response
		6 Applications for Synthetic mRNA
			6.1 Protein Replacement to Correct Diseases
			6.2 Vaccines Against Cancer
				6.2.1 Ex Vivo Introduction of Synthetic mRNA into DCs
				6.2.2 Electroporation of T Cells with Synthetic mRNA
			6.3 Vaccines Against Infectious Diseases
				6.3.1 Influenza
				6.3.2 HIV
			6.4 Engineering the Genome
			6.5 Generation of iPSCs
		7 Concluding Remarks
		References
Part II: Synthesis of mRNA
	Chapter 2: Synthetic Capped mRNAs for Cap-Specific Photo-Cross-Linking Experiments
		1 Introduction
		2 Materials
			2.1 Synthesis of m27,2-OGppp6SG
			2.2 Template for the Synthesis of H4-12 mRNA
			2.3 In Vitro Synthesis of Histone H4-12 mRNA
			2.4 Photo-cross-linking Experiment
		3 Methods
			3.1 Synthesis of m27,2-OGppp6SG
				3.1.1 Synthesis of 6SGMP-Im
				3.1.2 Coupling 6SGMP-Im and m27,2-OGDP
			3.2 Amplification of a PCR Template for H4-12 mRNA Transcript
			3.3 In Vitro Synthesis of Histone H4-12 mRNA Capped with m27,2-OGppp6SG
			3.4 In Vitro Synthesis of Histone H4-12 mRNA Capped with m7,6SGppp
			3.5 Photo-cross-linking Experiments
		4 Notes
		References
	Chapter 3: Enzymatic Modification of 5-Capped RNA and Subsequent Labeling by Click Chemistry
		1 Introduction
		2 Materials
			2.1 Synthesis of AdoMet Analogs
			2.2 Recombinant Production and Purification of Enzymes
			2.3 T7 In Vitro Transcription
			2.4 Capping of RNA
			2.5 Labeling of RNA
		3 Methods
			3.1 Synthesis of AdoViBe
			3.2 Synthesis of Ab-SAM
				3.2.1 (E)-1-Azido-4-Bromobut-2-Ene
				3.2.2 Synthesis and Characterization of Ab-SAM
			3.3 Synthesis of AdoEnYn
			3.4 Recombinant Production and Purification of Enzymes
				3.4.1 GlaTgs2-V34A
				3.4.2 MTAN
				3.4.3 LuxS
				3.4.4 Purification
			3.5 In Vitro T7 Transcription
			3.6 Capping of In Vitro Produced RNA
			3.7 Labeling of Azido-Modified RNA by SPAAC
			3.8 Chemoenzymatic Labeling of Capped RNA by IEDDA
			3.9 Chemoenzymatic Labeling of Capped RNA by CuAAC
		4 Notes
		References
	Chapter 4: Preparation of Functional, Fluorescently Labeled mRNA Capped with Anthraniloyl-m7GpppG
		1 Introduction
		2 Materials
			2.1 Thin-Layer Chromatography (TLC) Development Solvents
			2.2 Solutions and Buffers for the In Vitro Transcription of RNA
			2.3 Buffers for RNA Capping and Tailing
			2.4 Reagents for Determination of Capping Efficiency
		3 Methods
			3.1 Synthesis of a Fluorescent Cap Analog: Ant-GTP
			3.2 Synthesis of RNA Transcripts: In Vitro Transcription
			3.3 Incorporation of Ant-GTP Analog into mRNA: Capping of mRNA
			3.4 Measurement of RNA Quantity and Determination of Capping Efficiency
				3.4.1 RNA Quantification
				3.4.2 Determination of Capping Efficiency
				3.4.3 Decapping Reaction
				3.4.4 In Vitro Translation Assay
		4 Notes
		References
	Chapter 5: Intronless beta-Globin Reporter: A Tool for Studying Nuclear RNA Stability Elements
		1 Introduction
		2 Materials
			2.1 Construction of a betaDelta1,2-ENE Reporter Plasmid
			2.2 Transient Transfection of Mammalian Cells with Reporter Plasmid
			2.3 Measurement of beta-Globin mRNA Levels by Northern Blot Analysis
		3 Methods
			3.1 Construction of a betaDelta1,2-ENE Reporter Plasmid
			3.2 Transient Transfection of Mammalian Cells with betaDelta1,2 Reporter Plasmids
			3.3 Detection of beta-Globin mRNA Levels by Northern Blot Analysis
			3.4 Quantitation of beta-Globin mRNA Levels
		4 Notes
		References
	Chapter 6: Synthetic mRNA with Superior Properties that Mimics the Intracellular Fates of Natural Histone mRNA
		1 Introduction
		2 Materials
			2.1 Cap Analogs
			2.2 In Vitro Synthesis of Firefly Luciferase mRNA with Various 5- and 3-Ends
			2.3 Cell Culture, Synchronization, and Cell Cycle Analysis
			2.4 Introduction of Synthetic mRNAs into Cultured Mammalian Cells by Nucleoporation
			2.5 Analysis of Translational Efficiency in Cultured Cells
			2.6 Analysis of mRNA Stability in Cultured Cells
			2.7 Ligation-Coupled RT-PCR (LC-RT-PCR)
			2.8 Sequencing Gel Electrophoresis
			2.9 Deep Sequencing Analysis
		3 Methods
			3.1 In Vitro Synthesis of Firefly Luciferase mRNA with Various 5- and 3-Ends
			3.2 Cell Culture, Synchronization, and Cell Cycle Analysis
			3.3 Introduction of Synthetic mRNAs into Cultured Mammalian Cells
			3.4 Measurement of Translational Efficiency of Luc-SL and Luc-TL in HeLa Cells
			3.5 Measurement of Luc-SL and Luc-TL mRNA Stability in Cells
			3.6 Ligation-Coupled RT-PCR (LC-RT-PCR) and Sequencing Gel Electrophoresis
			3.7 Cloning and Sequencing of LC-RT-PCR Products
			3.8 Application of Deep Sequencing Technique to Analyze Histone mRNA Degradation Products
				3.8.1 Preadenylated Linker Ligation
				3.8.2 Linker Mediated Reverse Transcription
				3.8.3 PCR Round 1: Molecular Barcoding
				3.8.4 AMPure XP PCR Purification of First Round PCR
				3.8.5 PCR Round 2: Addition of Illumina Flow Cell Adapters
				3.8.6 Library Analysis and Quantitation
				3.8.7 Illumina Sequencing: 300 Cycle MiSeq Paired End Sequencing Run
		4 Notes
		References
	Chapter 7: Engineering WT1-Encoding mRNA to Increase Translational Efficiency in Dendritic Cells
		1 Introduction
		2 Materials
			2.1 Construction of Plasmid Template for Synthesis of WT1 mRNA
			2.2 In Vitro Transcription of mRNA
			2.3 Electroporation of Dendritic Cells
		3 Methods
			3.1 Construction of Plasmid Template for Synthesis of WT1 mRNA
				3.1.1 Cloning of the Genes of Interest into pGEM or pST1 Vector and Production of Plasmid DNA (Fig.1)
				3.1.2 Linearization of Plasmid DNA
			3.2 In Vitro Transcription of Capped mRNA
			3.3 Electroporation of Dendritic Cells (See Note 8)
		4 Notes
		References
Part III: Introduction of Synthetic mRNA into Cells
	Chapter 8: Electroporation of Alphavirus RNA Translational Reporters into Fibroblastic and Myeloid Cells as a Tool to Study th...
		1 Introduction
		2 Materials
			2.1 Media/Components for Cell Harvest
			2.2 Translation Reporter RNA
			2.3 Electroporation Components
			2.4 Luciferase Assay Components
			2.5 Alternate Neon Electroporation Components
		3 Methods
			3.1 Harvesting Cells
			3.2 Electroporation of Cells using a Bio-Rad Electroporator
			3.3 Alternate Cell Harvesting Protocol for using Neon Transfection System
			3.4 Electroporation of Cells Using Neon Transfection System
			3.5 Harvesting Cells for Dual-Luciferase Assay
			3.6 Dual-Luciferase Assay
		4 Notes
		References
	Chapter 9: GMP-Grade mRNA Electroporation of Dendritic Cells for Clinical Use
		1 Introduction
		2 Materials
			2.1 General Equipment
			2.2 In Vitro Generation of Human Monocyte-Derived DC
			2.3 mRNA Electroporation of Human Monocyte-Derived DC
			2.4 Cryopreservation and Thawing of Electroporated Human Monocyte-Derived DC
		3 Methods
			3.1 In Vitro Generation of Human Monocyte-Derived DC
			3.2 mRNA Electroporation of Human Monocyte-Derived DC
				3.2.1 Preparation Phase and Harvesting of Human Monocyte-Derived DC
				3.2.2 Electroporation of Human Monocyte-Derived DC
			3.3 Cryopreservation and Thawing of Electroporated Human Monocyte-Derived DC
		4 Notes
		References
	Chapter 10: Large-Scale mRNA Transfection of Dendritic Cells by Electroporation in Continuous Flow Systems
		1 Introduction
		2 Materials
			2.1 Fabrication of Electroporation Device
			2.2 Electroporation System
			2.3 Dendritic Cell  Preparation
			2.4 Electroporation
			2.5 Evaluation of Transfection Efficiency
		3 Methods
			3.1 Fabrication of Electroporation Device
			3.2 Electroporation System
			3.3 Dendritic Cell Preparation
			3.4 Electroporation
			3.5 Evaluation of Transfection Efficiency
		4 Notes
		References
	Chapter 11: FLT3 Ligand as a Molecular Adjuvant for Naked RNA Vaccines
		1 Introduction
		2 Materials
			2.1 Generation of FLT3 Ligand BMDCs
			2.2 mRNA Transfer into FLT3 Ligand BMDCs
			2.3 Effect of FLT3 Ligand on Cellularity and Activation of DCs
			2.4 Effect of FLT3 Ligand on Luciferase Expression
			2.5 Monitoring of Elicited Immune Responses
		3 Methods
			3.1 Generation and Characterization of FLT3 Ligand BMDCs
			3.2 mRNA Transfection into Murine FLT3 Ligand BMDCs
			3.3 Effect of FLT3 Ligand on Cellularity and Activation of DCs
				3.3.1 Injection of FLT3 Ligand
				3.3.2 Measuring In Vivo Effect of FLT3 Ligand on Cellularity and Expansion of DCs
			3.4 In Vivo Effect of RNA on Activation of DCs in Lymph Node After FLT3 Ligand Treatment
			3.5 Measuring In Vivo Luciferase Expression After FLT3 Ligand Treatment
			3.6 Monitoring of Elicited Cellular Immune Responses After RNA Vaccination
				3.6.1 Blood
				3.6.2 Spleen
		4 Notes
		References
	Chapter 12: Transfecting Human Monocytes with RNA
		1 Introduction
		2 Materials
			2.1 Solutions and Media
			2.2 In Vitro Transcribed RNA Preparation Components
			2.3 Denaturing Formaldehyde Agarose Gel Electrophoresis Components
			2.4 Magnetic Bead-Based Human Monocyte Isolation Supplies
			2.5 Counterflow Centrifugation Elutriation
			2.6 Supplies for Electroporation
			2.7 Antibodies and Reagents for Flow Cytometry (FACS) Analyses
			2.8 Cryopreservation of Monocytes
		3 Methods
			3.1 Generation of In Vitro Transcribed mRNA
			3.2 In Vitro Transcription Reaction mMESSAGEm MACHINE T7 kit
			3.3 Magnetic Bead-Based Isolation of Human Monocytes
			3.4 Electroporation of Monocytes with mRNA or siRNA
			3.5 Phenotype of Monocytes Using Flow Cytometry
			3.6 7-AAD Staining to Assess Cell Viability
			3.7 Cryopreservation of Monocytes
			3.8 Isolation of Human Monocytes by Elutriation
		4 Notes
		References
	Chapter 13: In Vitro Synthesis, Delivery, and Bioavailability of Exogenous mRNA in Gene Transfer Mediated by PiggyBac Transpos...
		1 Introduction
		2 Materials
			2.1 Plasmid Vectors
			2.2 Checking of DNA and RNA Quality
			2.3 Preparation of Plasmid DNA Templates
			2.4 In Vitro Synthesis of ARCA Capped and Poly(A) Tailed mRNA
			2.5 In Vitro Translation of In Vitro-Synthesized mRNA
			2.6 Cell Culture
			2.7 Nucleic Acid Transfection
			2.8 Protein Extraction and Western Blot
			2.9 Gel Retardation Assay of jetPEITM/mRNA Complexes
			2.10 Half-Life Calculation of mRNA Transcripts
				2.10.1 RNA Extraction
				2.10.2 RT-qPCR
			2.11 Uptake and Intracellular Trafficking of mRNA Polyplexes
				2.11.1 Cell Transfection, Fixation, and Permeabilization for Immunofluorescence
				2.11.2 mRNA/jetPEITM Co-Localization
				2.11.3 mRNA Clathrin-Dependent Endocytosis
				2.11.4 mRNA and SG or P Bodies Co-localizations
				2.11.5 Confocal Microscopy
			2.12 Transposition Assay
		3 Methods
			3.1 Checking DNA and RNA Quality
			3.2 Preparation of Plasmid DNA Templates
				3.2.1 Plasmid Linearization
				3.2.2 Proteinase K Treatment
				3.2.3 Phenol-Chloroform Extraction and Ethanol Precipitation
				3.2.4 Quality Control Electrophoresis
			3.3 In Vitro Synthesis of ARCA Capped and Poly(A) Tailed mRNA
				3.3.1 In Vitro Transcription Reaction
				3.3.2 Polyadenylation of in Vitro-Synthesized RNA
				3.3.3 mRNA Recovery by Lithium Chloride Precipitation
			3.4 Exogenous mRNA Transfection in Culture Cells
				3.4.1 Cells Seeding
				3.4.2 Transfection Using jetPEITM Reagent
				3.4.3 Transfection Using TransMessengerTM Reagent
				3.4.4 Transfection Using TransIT-mRNA Reagent
				3.4.5 Transfection Using Lipofectamine 2000 reagent
			3.5 Assaying mRNA Transfection Efficiency
				3.5.1 Total Protein Extraction
				3.5.2 SDS-PAGE and Western-Blot
			3.6 Gel Retardation Assay of jetPEITM/mRNA Complexes
			3.7 mRNA In Vitro Translation
				3.7.1 Preparation of the mRNA Complexes
				3.7.2 In Vitro Translation Reaction
			3.8 Half-Life Calculation of mRNA Transcripts
				3.8.1 Total RNA Extraction
				3.8.2 Retro-Transcription of Total RNA
				3.8.3 Quantitative PCR Analysis
			3.9 Uptake and Intracellular Trafficking of mRNA Polyplexes
				3.9.1 Cell Transfection, Fixation, and Permeabilization for Immunofluorescence
				3.9.2 mRNA Clathrin-Dependent Endocytosis
				3.9.3 mRNA/jetPEITM Co-Localization
				3.9.4 mRNA and SG or P Bodies Co-localizations
				3.9.5 Confocal Microscopy
			3.10 Transposition Assay in Hela Cells
			3.11 Transposition Assay in HS-27a and HS-5 Stromal Cells
				3.11.1 Cell Seeding
				3.11.2 DNA Complex Preparation
				3.11.3 RNA Complex Preparation
				3.11.4 DNA and RNA Complexes Co-transfection
		4 Notes
		References
	Chapter 14: Transfection of Human Keratinocytes with Nucleoside-Modified mRNA Encoding CPD-Photolyase to Repair DNA Damage
		1 Introduction
		2 Materials
			2.1 Transient Transfection
			2.2 UVB Treatment
			2.3 Photoreactivation
			2.4 CPD-Specific ELISA
		3 Methods
			3.1 Transfection of CPD-Photolyase mRNA into HaCaT Cells
			3.2 Exposing HaCaT Cells Transfected With CPD-Photolyase mRNA to UVB Irradiation
			3.3 Isolation of Genomic DNA
			3.4 DNA Sample Coating
			3.5 Measuring the Amount of UVB-Induced Cyclobutane Pyrimidine Dimers
		4 Notes
		References
Part IV: Alteration of Cellular Function with Synthetic mRNA
	Chapter 15: Delivery of Synthetic mRNA Encoding FOXP3 Antigen into Dendritic Cells for Inflammatory Breast Cancer Immunotherapy
		1 Introduction
		2 Materials
			2.1 IBC Cells and Culture Conditions
			2.2 Preparation of Synthetic FOXP3 mRNA [21]
			2.3 Generation of DCs from PBMCs
			2.4 Transfection of DCs with Antigen Encoding Synthetic mRNA
			2.5 Stimulation of T Cells
			2.6 CTL Assay
		3 Methods
			3.1 In Vitro Synthesis of FOXP3 mRNA
				3.1.1 Cloning of Human FOXP3 mRNA
				3.1.2 Plasmid Linearization
				3.1.3 In Vitro Transcription
				3.1.4 Transcript Purification
				3.1.5 Transcript Quantitation and Storage
			3.2 Generation of DCs from PBMCs
			3.3 Introduction of Synthetic FOXP3 mRNA into DCs
				3.3.1 Delivery by Electroporation
				3.3.2 Delivery by Lipofection [14]
				3.3.3 Delivery of Synthetic mRNA with Cationic Polymers [25]
			3.4 Application of FOXP3 mRNA-Transfected DCs for Anticancer Immunotherapy
				3.4.1 In Vitro Stimulation of T Cells
				3.4.2 Evaluation of CTL Response Against FOXP3 Expressing IBC Cells
		4 Notes
		References
	Chapter 16: Immune Monitoring Using mRNA-Transfected Dendritic Cells
		1 Introduction
		2 Materials
			2.1 Immune Monitoring, Day Minus 1
			2.2 Immune Monitoring, Day 0 (Prestimulation Culture)
			2.3 Immune Monitoring, Day 1
			2.4 Immune Monitoring, Day 6
			2.5 Immune Monitoring, Day 7
			2.6 Immune Monitoring, Day 8
			2.7 Immune Monitoring, Day 10
		3 Methods
			3.1 Immune Monitoring, Day Minus 1
			3.2 Immune Monitoring, Day 0 (Prestimulation Culture)
				3.2.1 Preparing PBMCs
				3.2.2 Preparing DCs
			3.3 Immune Monitoring, Day 1
				3.3.1 Adding IL-2 to Prestimulation Culture
				3.3.2 Analyzing Transfection Control
			3.4 Immune Monitoring, Day 6
				3.4.1 Coating ELISpot Plate
				3.4.2 Coating a 96-Well Round-Bottom Plate for Proliferation Assay
			3.5 Immune Monitoring, Day 7
				3.5.1 Preparing ELISpot Plate
				3.5.2 Preparing PBMCs
				3.5.3 Preparing DCs
				3.5.4 Baseline FACS on PKH-Stained PBMCs
			3.6 Immune Monitoring, Day 8
				3.6.1 Processing the ELISpot Plate
				3.6.2 Analyzing Transfection Control
			3.7 Immune Monitoring, Day 10
		4 Notes
		References
	Chapter 17: Transfection of Tumor-Infiltrating T Cells with mRNA Encoding CXCR2
		1 Introduction
		2 Materials
			2.1 In Vitro Synthesis of CXCR2 mRNA
			2.2 Isolation and Expansion of Human TILs
			2.3 Electroporation of TILs
			2.4 Expression and Signaling of CXCR2 by Flow Cytometry
			2.5 Specific Migration of CXCR2-Transfected TILs
		3 Methods
			3.1 In Vitro Synthesis of CXCR2 mRNA
			3.2 Isolation and Expansion of Human TILs
			3.3 Electroporation of TILs
			3.4 Expression and Signaling of CXCR2 by Flow Cytometry
			3.5 Specific Migration of CXCR2-Transfected TILs
		4 Notes
		References
	Chapter 18: mRNA Electroporation of Dendritic Cells with WT1, Survivin, and TriMix (a Mixture of caTLR4, CD40L, and CD70)
		1 Introduction
		2 Materials
			2.1 Reagents
			2.2 Equipment
		3 Methods
			3.1 mRNA Electroporation of Immature Dendritic Cells
			3.2 Preparation of Mature Dendritic Cells
		4 Notes
		References
	Chapter 19: Redirecting T Cell Specificity Using T Cell Receptor Messenger RNA Electroporation
		1 Introduction
		2 Materials
			2.1 Expansion of T Cells
			2.2 Electroporation of Expanded T Cells with mRNA
				2.2.1 Linearization of Plasmid
				2.2.2 In Vitro Transcription
				2.2.3 Synthesis of Capped and Tailed RNA
				2.2.4 Purification of Capped and Tailed mRNA
			2.3 Electroporation
			2.4 Assay for TCR Function
		3 Methods
			3.1 Expansion of T Cells
			3.2 Electroporation of Expanded T Cells with mRNA
				3.2.1 Linearization of Plasmid
				3.2.2 In Vitro Transcription
				3.2.3 Synthesis of Capped and Tailed RNA
				3.2.4 Purification of Capped and Tailed mRNA
			3.3 Electroporation Procedure
			3.4 Assay for TCR Function
		4 Notes
		References
	Chapter 20: Measuring Hematocrit in Mice Injected with In Vitro-Transcribed Erythropoietin mRNA
		1 Introduction
		2 Materials
			2.1 Treatment of Mice
			2.2 Blood Draw and Hematocrit Measurement
		3 Methods
			3.1 Experimental Planning
			3.2 Experimental Procedure
		4 Notes
		References
	Chapter 21: Genetic Modification of Human Pancreatic Progenitor Cells Through Modified mRNA
		1 Introduction
		2 Materials
			2.1 Tail PCR
			2.2 In Vitro Transcription
			2.3 modRNA Processing and Purification
			2.4 modRNA Transfection in hPPCs
			2.5 Analysis of Protein Expression and Cell Survival
			2.6 Analysis of Downstream Gene Expression
		3 Methods
			3.1 Tail PCR
			3.2 In Vitro Transcription
			3.3 ModRNA Purification
				3.3.1 DNase Treatment
				3.3.2 Dephosphorylation
				3.3.3 Column Purification
			3.4 Analysis of modRNA Transfection and Translational Efficiency
			3.5 Analysis of Posttransfection Cell Survival Rate
			3.6 Analysis of Innate Immunity and Downstream Gene Expression
		4 Notes
		References
Index