Messenger RNA Therapeutics

Introduction: IVT Messenger RNA in the Syringe
Contents
Roadmap to the Development of mRNA Therapeutics: From Molecule Design and Delivery Strategies to Manufacturing, Quality Control, and Regulatory Considerations
	1 Introduction
	2 mRNA Design Strategies
		2.1 Reducing mRNA Immunogenicity
		2.2 Optimizing Protein Expression
		2.3 Optimizing mRNA Stability
	3 Delivery Strategies for mRNA Therapeutics
		3.1 Lipid Nanoparticles for mRNA Delivery
		3.2 Other Materials for mRNA Delivery
		3.3 mRNA Drug Product Route of Administration
	4 Manufacturing of mRNA Drug Products
		4.1 mRNA Drug Substance Manufacturing
		4.2 mRNA Drug Product Manufacturing
	5 Quality Control of mRNA Therapeutics
		5.1 Drug Substance (mRNA) CQAs and Quality Control Strategy
		5.2 Drug Product (mRNA-LNPs) CQAs and Quality Control Strategy
	6 Conclusion and Future Directions
	References
Messenger RNA for Prophylaxis
	1 Introduction
	2 mRNA Vaccines: Categories and Biological Function
	3 Advances in mRNA Constructs
		3.1 5’ Capping
		3.2 Untranslated Regions (UTRs)
		3.3 Poly(A)-Tail
		3.4 Nucleotide Modification and Codon Optimization
		3.5 Purity
		3.6 Self-Amplifying Specific Features
	4 mRNA Carrier Technologies
		4.1 Tropism and Uptake Efficiency
		4.2 The Art of Endosomal Escape
		4.3 Formulation
		4.4 Stability
	5 Conclusions
	References
Messenger RNA Therapeutics: Start of a New Era in Medicine
	1 Introduction
	2 Production of IVT mRNA
	3 Immunogenicity of IVT mRNA
	4 Strategies to Increase the Stability and Reduce the Immunogenicity of IVT mRNA
		4.1 Capping (m7GpppN or m7Gp3N)
		4.2 Tailing
		4.3 Untranslated Regions (UTRs)
		4.4 Coding Region
	5 Purification of Synthetic mRNA
	6 Synthetic mRNA Platforms and their Features
		6.1 Unmodified mRNA
		6.2 Modified mRNA
		6.3 Sequence-Optimized Unmodified mRNA
		6.4 Replicon RNA
	7 In Vivo Delivery Strategies of Exogenous mRNA
		7.1 Delivery of Naked mRNA
		7.2 Cationic Liposome-Mediated or Cationic Nanoemulsion (CNE)-based RNA Transfection
		7.3 Peptide-based Delivery
		7.4 Electroporation and Nucleoporation
		7.5 Gene Gun-Mediated Delivery of mRNA
		7.6 Use of Polymer Nanomaterials
		7.7 Virus-like Replicon Particle (VRP)-based Delivery of mRNA
		7.8 Other Lesser-Known Methods
	8 Applications of mRNA Therapeutics
		8.1 mRNA as a Therapeutic Agent for Replacement of Defective Protein within the Cell
		8.2 mRNA as Vaccines Against Cancer
		8.3 Dendritic Cell (DC) Vaccines
		8.4 mRNA Vaccines in Prevention of Diseases
		8.5 mRNA-Mediated Genome Editing
		8.6 Generation of Induced Pluripotent Stem Cells (iPSCs) using mRNA
	9 Safety of mRNA Therapeutics
		9.1 The Safety Concern Over the Use of Non-Natural Nucleotides/Nucleosides in IVT mRNA
		9.2 Safety Considerations Regarding the Encoded Protein
	10 Conclusions
	References
Hospital-Based RNA Therapeutics
	1 Introduction
	2 A Role for Hospital-Based Drug Development
	3 Manufacturing of mRNA
	4 Innovations in mRNA Therapeutics
		4.1 Reducing Immunogenicity
		4.2 Cell-Specific/Tissue-Specific Delivery and Translation
		4.3 Increasing mRNA Stability and Expression
	5 Applications of RNA Therapeutics
	6 Conclusion and Future Perspective
	References
Medical Use of mRNA-Based Directed Gene Delivery
	1 Introduction
	2 Gene Delivery with mRNA Versus DNA
		2.1 The Comparison
		2.2 Improving IVT mRNA for Clinical Use
	3 Targeted LNPs
	4 Extracellular Vesicles (EVs, Aka Exosomes)
		4.1 mRNA Loading of EVs
		4.2 Strategies to Target mRNA-Loaded EVs and Their Therapeutic Use
		4.3 Improving EVs for Clinical Use
	5 Conclusion
	References
SARS-COV-2 and Other mRNA Vaccines
	1 Introduction: The Successes of MRNA-LNP Vaccines for SARS-COV-2
	2 Pfizer-BioNTech—BNT162b2 (Comirnaty)—mRNA-LNP SARS-COV-2 Vaccine
		2.1 Clinical Phase 1 Study in Adults (19–55 years), Germany
		2.2 Clinical Phase 1 Study in Adults (18–55 and 65–85 years), U.S.
		2.3 Clinical Phase 2/3, (16 Years of Age or Older) Multi-Country
		2.4 Post-licensure Effectiveness, Safety Surveillance and Boosting
	3 Moderna—mRNA-1273 (Spikevax) mRNA-LNP SARS-COV-2 Vaccine
		3.1 Clinical Phase 1 Trial in Adults and Older Adults, U.S.
		3.2 Clinical Phase 2 Trial (18 Years of Age and Older), US
		3.3 Clinical Phase 3 Efficacy Trial in Adults (18 Years of Age or Older), U.S.
		3.4 Post-Licensure Effectiveness, Safety Surveillance and Boosting
	4 CureVac CVnCoV
		4.1 Clinical Phase 1 Trial (18–60 Years of Age), Germany and Belgium
		4.2 Clinical Phase 2b/3 Efficacy Trial (18 Years of Age and Over), Multi-Country
	5 Overall Interpretation of Safety and Efficacy Data for mRNA-LNP Vaccines Against SARS-COV-2
	6 Other mRNA SARS-COV-2 Candidate Vaccines
	7 Clinical-Stage mRNA Candidate Vaccines Against Other Non-COVID-19 Infectious Diseases
	8 Future mRNA Technology Advances
	References
Pulmonary Delivery of Messenger RNA (mRNA) Therapeutics for Respiratory Diseases
	1 Introduction
	2 Pulmonary RNA Delivery Systems
		2.1 Lipid-Based Delivery System
		2.2 Polymer-Based Delivery System
		2.3 Peptide-Based Delivery System
		2.4 Modified Naked mRNA
	3 mRNA Strategies and Respiratory Diseases
		3.1 Cystic Fibrosis
		3.2 Asthma
		3.3 Respiratory Syncytial Virus Infection
		3.4 Antiviral Effect Using Cas13
		3.5 Vaccines Against Respiratory Viral Infections
	4 Summary and Future Prospects
	References
Synthetic mRNA Gene Therapies and Hepatotropic Non-viral Vectors for the Treatment of Chronic HBV Infections
	1 Introduction
	2 Gene and Epigenome Editing Technologies to Disable HBV
		2.1 HBV Designer Nucleases and Base Editors
		2.2 HBV Epigenome Modifiers
	3 Developing mRNA as Therapeutics
		3.1 Enhancing Translation and Stability of Synthetic mRNA
		3.2 Liver-Specific Delivery of mRNA Using Non-viral Vectors
	4 The Future of HBV mRNA Therapy
	References
Preparation of Synthetic mRNAs—Overview and Considerations
	1 Introduction
		1.1 Synthetic mRNAs
		1.2 Kinds of Synthetic mRNAs
		1.3 Key Structural Attributes for a Functional Synthetic mRNA
		1.4 Chemical Modifications of Synthetic mRNA
		1.5 The mRNA Cap
	2 Platforms for mRNA Synthesis
		2.1 Restriction Enzymes for Template Linearization
		2.2 RNA Polymerase
		2.3 RNase Inhibitor
		2.4 Inorganic Pyrophosphatase
		2.5 DNase I
		2.6 Capping Enzyme
		2.7 Poly-A Polymerase
	3 Purification of the Synthetic mRNA
	4 Analyses of Synthetic mRNAs
		4.1 Capping Efficiency
		4.2 Poly-A Tail Length
	5 Perspective and Future Directions
	References
In Vitro-Transcribed mRNAs as a New Generation of Therapeutics in the Dawn of Twenty-First Century: Exploitation of Peptides as Carriers for Their Intracellular Delivery
	1 In Vitro-Transcribed mRNA as a Powerful Gene Therapy Tool
	2 Advances in the Delivery of IVT-mRNA
	3 Peptide-Based Systems for IVT-mRNA Delivery
	4 Cell-Penetrating Peptides or Protein Transduction Domains
	5 Strategies to Generate a PTD/Nucleic Acid Complex
		5.1 Non-covalent Conjugation Approach for IVT-mRNA Delivery via Peptides
		5.2 Covalent Conjugation Approach for IVT-mRNA Delivery via Peptides
	6 Future Perspectives
	References
Lipid Nanoparticle-Mediated Delivery of Therapeutic and Prophylactic mRNA: Immune Activation by Ionizable Cationic Lipids
	1 Introduction
	2 Cytosolic Delivery of mRNA
	3 Lipid Nanoparticles (LNPs) as Delivery Systems for mRNA
	4 Immune Activation by Lipids Used for mRNA Delivery
	5 Conclusions
	References
Adjuvants, the Elephant in the Room for RNA Vaccines
	1 Introduction
	2 mRNA as a Natural Adjuvant
		2.1 Endosomal RNA Recognition
		2.2 Cytoplasmic RNA Sensors
	3 Type I IFNs: The Double-Edged Sword
		3.1 Nucleoside Modifications
		3.2 Techniques for dsRNA Removal
		3.3 Role of Type I IFN Receptor
	4 mRNA Delivery Systems
	5 Conclusion
	References
Advances in mRNA Delivery and Clinical Applications
	1 Introduction
	2 Lipid Nanoparticles
	3 Polymers
	4 Lipid–Polymer Hybrid Nanoparticles
	5 Polypeptides
	6 Other Carriers
	7 Applications of Therapeutic mRNA
		7.1 Vaccine
		7.2 Protein-Replacement Therapy
		7.3 Gene Editing
	8 Discussion and Perspectives
	References
Lipid Nanoparticles to Harness the Therapeutic Potential of mRNA for Cancer Treatment
	1 Introduction
	2 Structure, Synthesis, and Purification of in Vitro Transcribed (IVT) mRNA
		2.1 Structural Organization of IVT-mRNA
		2.2 Synthesis of IVT-mRNA
		2.3 Purification of Synthetic mRNA
	3 Lipid Nanoparticles for Therapeutic mRNA Delivery
	4 mRNA-Based Cancer Immunotherapy: Antitumor Vaccines
		4.1 mRNA-Loaded LNP-Mediated Monoclonal Antibody Delivery
		4.2 mRNA-Loaded LNPs for CAR Immune Cell Engineering
	5 Future Prospective and Conclusions
	References
RNA/Polymer-Based Supramolecular Approaches for mRNA Delivery
	1 Introduction
	2 Polymeric Assemblies
		2.1 Polyplexes
		2.2 Polymeric Micelles
	3 RNA Architectonics
	4 Combination of RNA Architectonics and Polymeric Micelles
	5 Conclusion
	References
Delivery Vehicles for Self-amplifying RNA
	1 Introduction
	2 Delivery Vehicles
		2.1 Lipid Nanoparticles
		2.2 Polymeric Nanoparticles
		2.3 Cationic Nanoemulsions
		2.4 Electroporation
	3 Mechanistic Understanding of saRNA Nanoparticle Cellular Uptake and Entry
		3.1 Cellular Uptake and Release Pathways
	4 Routes of Administration
	5 Innate Immune Sensing of saRNA Nanoparticles
		5.1 saRNA Immune Sensing
		5.2 Immune Sensing of Delivery Vehicles
	6 Conclusion
	References
Nuclear Export of mRNAs with Disease Pathogenesis and Therapeutic Implications
	1 Introduction
	2 Nuclear Export of mRNA
		2.1 NXF1-Mediated mRNA Export
		2.2 CRM1-Mediated mRNA Export
		2.3 Ubiquitin–Proteasome System (UPS) Regulation of mRNA Export
	3 Nuclear Export of mRNA in Disease Pathogenesis
		3.1 Links to Cancers
		3.2 Links to Neurodegenerative Diseases
	4 Therapeutic Strategy Targeting mRNA Export Machinery/Factors
	5 Concluding Remarks
	References
Preparation of Messenger RNA-Loaded Nanomedicine Applied on Tissue Engineering and Regenerative Medicine
	1 Introduction
	2 Preparation of Therapeutic mRNA
		2.1 DNA Template
		2.2 In Vitro Transcription (IVT)
		2.3 Capping
		2.4 The 5′ and 3′ UTR, Including the PolyA Tail
		2.5 Quality Analysis
	3 Delivery Carrier Assembled as mRNA Medicine
		3.1 Lipid-Based Nanocarrier
		3.2 Polymeric Nanocarrier
		3.3 Peptide-Related Carrier
		3.4 Other Typed Carrier
		3.5 Cation-Free Administration
	4 Release Profile of mRNA Medicine
		4.1 Endosomal Escape
		4.2 Sustained Expression and Resistance to Nuclease Attack
	5 Application in Regenerative Medicine
		5.1 Brain and CSF
		5.2 Cartilage
		5.3 Bone
		5.4 Intervertebral Disc (IVD)
		5.5 Olfactory Neuron
		5.6 Liver
		5.7 Cardiac and Skeletal Muscle Cells
		5.8 Spinal Cord
		5.9 Stem Cells Engineering
	6 Conclusion
	7 Perspective
	References
Nonsequential Pre-mRNA Splicing: From Basic Understanding to Impacts on Splice-Manipulating Therapies
	1 Introduction
	2 What Influences Intron Removal Order?
	3 Genes Studied for the Order of Intron Removal
		3.1 Dystrophin
		3.2 Collagen Type I Alpha 1
		3.3 Collagen Type V Alpha 1
		3.4 Collagen Type VII Alpha 1
	4 Strategies Used to Study Intron Removal Order
		4.1 Reverse Transcription Polymerase Chain Reaction Method
		4.2 Next-Generation RNA Sequencing
	5 Pre-mRNA Splicing Order Can Impact Exon Skipping Antisense Oligonucleotide Design
	6 Conclusion
	References