RNA Vaccines: Methods and Protocols (Methods in Molecular Biology, 2786)

Preface
Contents
Contributors
Chapter 1: Introduction to RNA Vaccines Post COVID-19
	1 Introduction
	2 Messenger RNA and Other RNA Vaccine Concepts
		2.1 Historical Background
		2.2 Vector Design
			2.2.1 Optimizing Translation
			2.2.2 Optimizing Vector Persistence
		2.3 Production
		2.4 Adjuvantation
		2.5 Delivery
		2.6 Immunomonitoring
		2.7 Regulatory Aspects and Validated Benchmarks
	3 Contents of This Book
		3.1 Overview
		3.2 Part I: Replicating RNA Vectors
		3.3 Part II: Non-replicating mRNA Vectors
		3.4 Part III: Formulation and Delivery of RNA Vaccines
		3.5 Part IV: Nonclinical and Clinical Development of RNA Vaccines
	4 Conclusion
	References
Part I: Replicating RNA Vectors
	Chapter 2: Self-Replicating RNA Derived from the Genomes of Positive-Strand RNA Viruses
		1 Introduction
		2 Methods for Establishment of Self-Replicating RNAs
			2.1 Basic Strategies: A Historical Review
			2.2 Road Map to Recovery of Self-Replicating RNA
		3 Use of Self-Replicating RNA for Vaccine Purposes
			3.1 Vaccines Based on Full-Length Viral RNA
			3.2 Replicons as Vaccines
			3.3 Self-Replicating RNAs as Vectors for Expression of Foreign Genes
		References
	Chapter 3: Negative-Strand RNA Virus-Vectored Vaccines
		1 Introduction: Negative-Strand RNA Viruses (Order Mononegavirales)
			1.1 Virion Structure and Genome Organization of Negative-Strand RNA Viruses
			1.2 Replication Cycle of Negative-Strand RNA Viruses
		2 Generating Negative-Stranded RNA Virus-Based Vector Vaccines
			2.1 Advantages and Disadvantages of Negative-Stranded RNA Virus Vectors
			2.2 Reverse Genetic Systems of Negative-Stranded RNA Viruses
			2.3 Designing a Recombinant Negative-Stranded RNA Viruses Vector Vaccine: Requirements Concerning the Vector
			2.4 Generating a Recombinant Newcastle Disease Virus-Vectored RNA Vaccine
		3 Scope of Recombinant Negative-Strand RNA Virus Vector Vaccines
			3.1 Newcastle Disease Virus (NDV)
			3.2 Measles Virus (MeV)
			3.3 Parainfluenza Viruses (PIV)
			3.4 Borna Disease Virus (BoDV)
			3.5 Rabies Virus (RABV) and Vesicular Stomatitis Virus (VSV)
		4 Negative-Strand RNA Virus Vaccines Against SARS-CoV-2
		5 Notes
		References
	Chapter 4: New Generation Self-Replicating RNA Vaccines Derived from Pestivirus Genome
		1 Introduction
		2 Cell Preparations and Cultures
			2.1 Chemicals and Solutions
			2.2 Main Equipment and Other Materials
			2.3 Dendritic Cells (DCs) and Monocytes for RepRNA Delivery Assessment
				2.3.1 Porcine Peripheral Blood Mononuclear Cell (PBMC) Preparation
				2.3.2 Isolation of Porcine DCs and Monocytes
				2.3.3 Culture of Monocytes to Differentiate Monocyte-Derived DCs
				2.3.4 Human Peripheral Blood Mononuclear Cell Preparation
				2.3.5 Isolation of Human DCs and Monocytes
				2.3.6 Culture of Monocytes to Differentiate Human Monocyte-Derived DCs
			2.4 Other Cells
		3 RepRNA Generation
			3.1 Characteristics of the RepRNA
				3.1.1 Overview of CSFV-Derived RepRNA
				3.1.2 Important Genetic Considerations for the RepRNA
				3.1.3 RepRNA Constructs for Various Vaccine or Protein Delivery (Table 4)
			3.2 In Vitro Transcription of RepRNA from Plasmid DNA
				3.2.1 Preparation and Linearization of the Plasmid DNA
				3.2.2 Phenol Extraction of Linearized DNA
				3.2.3 In Vitro Transcription
				3.2.4 RNA Cleaning Step
				3.2.5 Evaluation of the Physical Properties of the RepRNA
			3.3 Functional Assay of the Freshly Produced RepRNA
				3.3.1 Electroporation of RepRNA
				3.3.2 Quantifying the Specific Infectivity of RepRNA by Immunoperoxidase Staining
				3.3.3 Quantifying the Specific Infectivity of RepRNA by Confocal Microscopy
			3.4 RepRNA Association with Synthetic Delivery Vehicles
				3.4.1 PEI-Based Polyplexes
					Polyplex Formulation
					Polyplex Formulation with Incorporated Cell Penetrating Peptides
					RepRNA Protection from RNases Offered by Polyplexes
					Polyplex Internalization by Porcine and Human Cells
					RepRNA Translation/Replication Offered by Polyplexes in Transfected Cells
					Polyplex Formulation to Be Injected in Mice
					Polyplex Formulation to Be Injected in Pigs
				3.4.2 Coatsome-Replicon Vehicles
					Coatsome-Replicon Vehicle Formulation
					RepRNA Protection from RNases Offered by Coatsome-Replicon Vehicles
					Coatsome-Replicon Vehicle Internalization by Porcine and Human Cells
					RepRNA Translation/Replication Offered by Coatsome-Replicon Vehicles in Transfected Cells
					Coatsome-Replicon Vehicle Formulation to Be Injected in Mice
				3.4.3 The c-di-AMP Adjuvant
			3.5 Experimental Readouts
				3.5.1 In Vitro Readouts
				3.5.2 In Vivo Readouts
		4 Application of Synthetic RepRNA Delivery
		5 Conclusions
		6 Notes
		References
	Chapter 5: Trans-Amplifying RNA Vaccines Against Infectious Diseases: A Comparison with Non-Replicating and Self-Amplifying RNA
		1 Introduction
		2 Unmodified and Nucleoside-Modified Non-Replicating mRNA
		3 Self-Amplifying RNA
		4 Trans-Amplifying RNA
		5 Conceptual Advantages of Transreplicons
		6 Conclusions
		References
Part II: Non-Replicating RNA Vectors
	Chapter 6: Generation and Characterization of In Vitro Transcribed mRNA
		1 Introduction
		2 Materials
			2.1 Media
			2.2 Reagents
			2.3 Cell Lines
			2.4 Equipment
		3 Methods
			3.1 Plasmid Linearization
			3.2 Two-Step In Vitro Transcription and Capping mRNA
				3.2.1 Step 1: In Vitro Transcription
				3.2.2 Step 2: mRNA Capping
			3.3 One-Step Protocol for In Vitro Transcription, RNA Labeling, and mRNA Capping
				3.3.1 RNA Labeling Probes by In Vitro Transcription with UTP-X-CY3
			3.4 mRNA Integrity and Quality Controls
				3.4.1 Ultraviolet (UV) Spectrophotometry
				3.4.2 Fluorescence Spectrophotometry
				3.4.3 Electrophoresis
				3.4.4 Bioanalyzer
				3.4.5 Qubit RNA IQ Assay  Kits
			3.5 In Vitro Testing Using Primary Cell Cultures and Established Cell Lines
				3.5.1 Cell Culture Media Preparation
				3.5.2 Cell Seeding for mRNA Transfection
				3.5.3 Transfection to Test mRNA Constructs Using Lipofectamine MessengerMAX
				3.5.4 Transfection to Test LNP-Formulated mRNA Constructs
			3.6 Confirming Translation by Flow Cytometry or Microscopy
				3.6.1 Sample Preparation
				3.6.2 Intracellular Staining for Flow Cytometry
				3.6.3 Staining for Immunofluorescence Microscopy
			3.7 Confirming Translation by Western Blotting
				3.7.1 Sample Preparation
				3.7.2 Electrophoresis and Transfer
				3.7.3 Blocking and Antibody Incubation
		4 Notes
		References
	Chapter 7: Production and Evaluation of Nucleoside-Modified mRNA Vaccines for Infectious Diseases
		1 Introduction
		2 Materials
			2.1 mRNA Production and Purification
			2.2 iLNP Complexing of mRNA
			2.3 Intramuscular mRNA-iLNP Administration to  Mice
			2.4 Retro-Orbital Blood Collection from  Mice
			2.5 Spleen Isolation
			2.6 Stimulation and Staining of Mouse Splenocytes
			2.7 Preparation of Compensation Controls
			2.8 Flow Cytometry and T Cell Data Analysis
			2.9 Enzyme-Linked Immunosorbent Assay (ELISA)
			2.10 Virus Neutralization, Antibody-Dependent Cellular Cytotoxicity, Bacterial Protection and Killing Assays
		3 Methods
			3.1 Nucleoside-Modified mRNA Production and Purification
			3.2 iLNP Complexing of mRNA
			3.3 Intramuscular mRNA-iLNP Administration to  Mice
			3.4 Retro-Orbital Blood Collection from  Mice
			3.5 Spleen Isolation
			3.6 Stimulation and Staining of Mouse Splenocytes
			3.7 Preparation of Compensation Controls
			3.8 Flow Cytometry and T Cell Data Analysis
			3.9 Enzyme-Linked Immunosorbent Assay (ELISA)
			3.10 Virus Neutralization, Antibody-Dependent Cellular Cytotoxicity, Bacterial Protection and Killing Assays
		4 Notes
		References
	Chapter 8: Sequence-Optimized mRNA Vaccines Against Infectious Disease
		1 Introduction
		2 Materials
			2.1 Media
			2.2 Reagents
			2.3 Equipment
		3 Methods
			3.1 mRNA Synthesis
			3.2 mRNA Purification
			3.3 mRNA LNP Formulation
			3.4 Transfection
				3.4.1 Transfection of mRNA
				3.4.2 Transfection of mRNA-LNP
			3.5 Intradermal and Intramuscular Injections
				3.5.1 Intraperitoneal Anesthesia
				3.5.2 Intradermal Injection in the Back
				3.5.3 Intramuscular Injection
			3.6 Detection of Luciferase Expression After Intradermal Injection of Luciferase mRNA by In Vivo Imaging
			3.7 Detection of Luciferase Expression in Tissue Lysates
				3.7.1 Preparation of PpLuc Measuring Buffer and PpLuc Standard
				3.7.2 Preparation of Tissue Samples
				3.7.3 Preparation of Tissue Lysates
				3.7.4 Preparation of LIA Plate with Samples and PpLuc Standard
			3.8 Assessing the Immunogenicity of mRNA Vaccines
			3.9 Prophylactic Vaccination of Mice
				3.9.1 Humoral Immune Responses by ELISA
				3.9.2 Cellular Immune Responses by Intracellular Cytokine Staining (ICS)
			3.10 Assessing the Efficacy of mRNA Vaccines
		4 Notes
		References
	Chapter 9: Design and Synthesis of Circular RNA Expression Vectors
		1 Introduction
		2 Materials
			2.1 circRNA Production and Purification
			2.2 Agarose Gel Electrophoresis
			2.3 Gaussia Luciferase Reporter Assay
		3 Methods
			3.1 circRNA Production and Purification
			3.2 Agarose Gel Electrophoresis
			3.3 Gaussia Luciferase Reporter Assay
		4 Notes
		References
Part III: Formulation and Delivery of RNA Vaccines
	Chapter 10: Electroporation of mRNA as a Universal Technology Platform to Transfect a Variety of Primary Cells with Antigens a...
		1 Introduction
		2 Materials
			2.1 Equipment
			2.2 Consumables
		3 Methods
			3.1 General Remarks
			3.2 Preparatory Work
			3.3 Protocol
		4 Notes
		References
	Chapter 11: A Basic Method for Formulating mRNA-Lipid Nanoparticle Vaccines in the Lab
		1 Introduction
		2 Materials
			2.1 mRNA Production and Purification
			2.2 Microfluidic Mixer
			2.3 LNP Particles Formulation
			2.4 LNP Particle Characterization
			2.5 In Vitro Transfection
			2.6 Intramuscular and Subcutaneous mRNA Administration of mRNA-LNPs in  Mice
			2.7 In Vivo Expression Detection of Protein Expressed from mRNA-LNPs
			2.8 Sublingual Vein Blood Collection from  Mice
			2.9 Detection of Humoral Response (ELISA)
			2.10 Detection of Cytotoxic T-Cell Responses
		3 Methods
			3.1 mRNA Production and Purification
			3.2 Microfluidic Mixer Fabrication
			3.3 Particle Formulation
			3.4 LNP Encapsulation Efficiency
			3.5 Transfection
			3.6 mRNA Administration
			3.7 In Vivo Expression Detection
			3.8 Sublingual Vein Blood Collection and Serum Collection
			3.9 Detection of the Humoral Response (by ELISA)
			3.10 Detection of the Cellular Response by  FACS
		4 Notes
		References
	Chapter 12: Messenger RNA Lipid-Based Nanoparticles: Optimization of Formulations in the Lab
		1 Introduction
		2 Materials
			2.1 mRNA Synthesis
				2.1.1 Preparation and Setup
				2.1.2 Plasmid DNA Linearization and mRNA Production
				2.1.3 Post-transcriptional mRNA Treatments
				2.1.4 DNA and mRNA Purity Check
			2.2 mRNA Labeling
			2.3 Microfluidic Preparation of Liposomes and LNPs
			2.4 Thin-Film Method for Production of Liposomes
			2.5 Formulation of Lipoplexes, Lipopolyplexes, and Complexes with Lipofectamine MessengerMAX
			2.6 Characterization of the Formulations
			2.7 Cells and In Vitro mRNA Transfection
			2.8 Activation of mRNA Sensors by Western Blot
			2.9 Cellular Uptake Quantification of mRNA Formulations
			2.10 Confocal Microscopy
		3 Methods
			3.1 In Vitro mRNA Transcription
				3.1.1 DNA Linearization
				3.1.2 Synthesis of Capped mRNA
				3.1.3 Poly(A) Tailing
				3.1.4 Recovery of mRNA
			3.2 Post-transcriptional Purification
				3.2.1 Cellulose Purification
				3.2.2 Phosphatase Purification
			3.3 DNA and mRNA Quality Check
				3.3.1 Gel Electrophoresis with Sybrsafe for DNA
				3.3.2 Gel Electrophoresis with Ribogreen for mRNA
			3.4 Agilent 2100 Bioanalyzer
				3.4.1 Preparation of Gel and Sample
				3.4.2 Setting Up Bioanalyzer Equipment
				3.4.3 Preparation of the RNA Chip
			3.5 mRNA Labeling
			3.6 Microfluidic Production of Liposomes (Fig. 4)
			3.7 Thin-Film Method for Production of Liposomes
			3.8 Complexation of EGFP-mRNA and Liposomes into Lipoplexes (LRs) (for 1 Well of 24-Well Plate)
			3.9 Complexation of EGFP-mRNA, Polymer PTG1, and Liposomes into Lipopolyplexes (LPRs) (for 1 Well of 24-Well Plate, 0.5 μg of ...
			3.10 Microfluidic Production of mRNA Lipid Nanoparticles (LNPs) (Fig. 5)
			3.11 Preparation of Complexes with Lipofectamine MessengerMAX (LFM)
			3.12 Size, Polydispersity, and Zeta Potential Measurements of Prepared Formulations with SZ-100 Nanoparticle Analyzer (Horiba,...
			3.13 Encapsulation Efficiency with Quant-iT Ribogreen Assay Kit for RNA-LNPs
			3.14 In Vitro EGFP mRNA Transfection (Adherent Cells)
			3.15 Uptake Assay
			3.16 Evaluation of Endosomal Escape by Confocal Microscopy Analysis (Figs. 7 and 8)
			3.17 Assessment of Activation of Phosphorylation of PKR by Western Blot (Fig. 9)
				3.17.1 Isolation of Proteins (All Steps on Ice)
				3.17.2 Protein Quantification by BCA Assay
				3.17.3 SDS-PAGE (Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis)
		4 Notes
		References
	Chapter 13: Plant Expression of Trans-Encapsidated Chimeric Viral Vaccines with Animal RNA Replicons: An Update
		1 Introduction
		2 Materials
			2.1 Agro-Inoculation
			2.2 Chimeric Viral Vaccine Collection and Purification
		3 Methods
			3.1 Agro-Coinoculation
			3.2 Viral Vaccine Extraction and Purification
		4 Notes
		References
Part IV: Nonclinical and Clinical Development of RNA Vaccines
	Chapter 14: Producing Plasmid DNA Template for Clinical Grade RNA Vaccine Manufacture
		1 Introduction
		2 Plasmid DNA as a Biopharmaceutical Agent
			2.1 DNA Vaccines
			2.2 mRNA-Based Vaccines
		3 Regulatory Aspects
			3.1 High-Quality Grade
			3.2 GMP Grade
				3.2.1 Typical Process  Flow
				3.2.2 Quality Controls
				3.2.3 Plasmid DNA Storage
		References
	Chapter 15: Regulatory Considerations for Producing mRNA Vaccines for Clinical Trials
		1 Introduction
		2 Investigational Medicinal Product Dossier
			2.1 S - Active Substance
			2.2 P - Investigational Medicinal Product Under  Test
		3 Control of the Active Substance/IMP
		4 Notes
		References
	Chapter 16: Quality by Digital Design for Developing Platform RNA Vaccine and Therapeutic Manufacturing Processes
		1 Introduction
		2 mRNA Production Processes
			2.1 Template DNA Synthesis
			2.2 In Vitro Transcription
			2.3 Tangential Flow Filtration
			2.4 Chromatography
			2.5 Lipid Nanoparticle (LNP) Formulation
		3 Summary
		References
	Chapter 17: Nonclinical Safety Testing of RNA Vaccines
		1 Introduction
			1.1 The Goals of Safety and Toxicity Testing in Drug Development
			1.2 Regulatory Background
				1.2.1 Some Terms and Definitions
				1.2.2 Regulatory Framework
		2 Some Basic Requirements of Safety/Toxicity Testing
			2.1 Study Protocol (GLP Compliant)
			2.2 Study Report (GLP Compliant)
			2.3 Archiving
		3 Methods
			3.1 What Potential Main/First Line Safety Concerns with Vaccines or Adjuvants Can Be Expected in General?
			3.2 What Potential Main Safety Concerns with Nucleic Acid/RNA-Based Vaccines Are Expected?
			3.3 Prerequisites
			3.4 Selection of the Adequate Animal Model
			3.5 Testing Strategy for the Safety Assessment
				3.5.1 Repeated Dose Toxicity
				3.5.2 Safety Pharmacology
				3.5.3 Immunotoxicity
				3.5.4 Environmental Risk Assessment (ERA)
		References
Index