Antisense RNA Design, Delivery, and Analysis

Preface
Acknowledgments
Contents
Contributors
Part I: Overview
	Chapter 1: Introduction and History of the Chemistry of Nucleic Acids Therapeutics
		1 Introduction to Synthetic Antisense Oligonucleotides and siRNA
		2 Oligonucleotide Synthesis
		3 Synthetic Oligonucleotide Analogues in Antisense and siRNA
			3.1 Backbone Analogues
				3.1.1 Phosphorothioates
				3.1.2 Charge-Neutral Analogues
			3.2 Sugar Analogues
				3.2.1 2′-O-Alkyl Sugars
				3.2.2 Locked/Bridged Nucleic Acids
			3.3 Heterocyclic Base Analogues
		4 RNase H Active Gapmer Chemistry for Use as Drugs
		5 siRNA Chemistry for Use as Drugs
		6 Immune Responses to Nucleic Acids
		7 Conjugates and Delivery
		8 Further Developments in Therapeutics
		9 Summary
		References
	Chapter 2: Antisense RNA Therapeutics: A Brief Overview
		1 Introduction
		2 Mechanisms of Action
			2.1 Splicing Modulation
				2.1.1 Exon Exclusion (Shortened Proteins)
				2.1.2 Exon Inclusion
				2.1.3 Splicing Redirection
				2.1.4 Deep-Intronic Variants
			2.2 Transcript Degradation
				2.2.1 RNase H1-Activating Antisense Oligonucleotides (Gapmers)
				2.2.2 Disrupting Reading Frame
		3 Therapeutic Potential
			3.1 Examples of Clinical Trials for Muscle Diseases
			3.2 Examples of Clinical Trials for Eye Diseases
		4 Future of AON Trials and Personalized Medicine: n = 1 Trials?
		5 Hurdles
		6 Conclusions
		References
Part II: Design and Formulation of Antisense Technology
	Chapter 3: Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion
		1 Introduction
		2 Materials
			2.1 AON Design
			2.2 Synthesis and Preparation of Bifunctional Oligonucleotides
			2.3 Culture of Skin Fibroblasts from SMA Patient
			2.4 Fibroblast Transfection
			2.5 RNA Extraction
			2.6 cDNA Synthesis
			2.7 Polymerase Chain Reaction (PCR)
			2.8 Quantitative Real-Time PCR
			2.9 Western Blotting
		3 Methods
			3.1 Design of Bifunctional Oligonucleotides
			3.2 Transfection of SMA Fibroblasts
			3.3 Splicing Assay of Bifunctional AONs on SMN2 Exon 7 Inclusion at RNA Level
			3.4 Bifunctional AONs on Restoring SMN Protein Measured by Western Blotting
		4 Notes
		References
	Chapter 4: Design and Delivery of SINEUP: A New Modular Tool to Increase Protein Translation
		1 Introduction
		2 Materials
			2.1 Design and Cloning of SINEUP
			2.2 SINEUP Delivery into Cellular Model
			2.3 RNA and Protein Analysis
		3 Methods
			3.1 Binding Domain Design and Cloning
			3.2 Effector Domain Design and Cloning
			3.3 SINEUP Delivery into Cellular Model
			3.4 SINEUP Efficacy Assessment: Transcription and Translation Evaluation
		4 Notes
		References
	Chapter 5: How to Design U1 snRNA Molecules for Splicing Rescue
		1 Introduction
		2 Materials
			2.1 Generating Modified U1 snRNA Vectors Adapted to the 5′ss of Interest
			2.2 In Vitro Therapeutic Evaluation of Modified U1 snRNA Vectors in Human Fibroblasts
				2.2.1 Transfection of Modified U1 snRNA Vectors in Human Fibroblasts
				2.2.2 Analysis of Splicing Rescue by RT-PCR
		3 Methods
			3.1 Generating the Modified U1 snRNA Vectors
				3.1.1 Engineering Modified U1 snRNA Vectors Adapted to the 5′ss of Interest
			3.2 In Vitro Therapeutic Evaluation of Modified U1 snRNA Vectors in Human Fibroblasts
				3.2.1 Modified U1 snRNA Vectors Transfection in Human Fibroblasts
				3.2.2 Analysis of Splicing Rescue by RT-PCR
				3.2.3 Assessment of the Effect of U1 snRNA-Induced Splicing Rescue at Protein Level
		4 Notes
		References
	Chapter 6: Conjugation of Nucleic Acids and Drugs to Gold Nanoparticles
		1 Introduction
		2 Materials
			2.1 AuNP Synthesis
				2.1.1 Materials
				2.1.2 Reagents
				2.1.3 Equipment
			2.2 Dithiolane-Based Linkers Synthesis
				2.2.1 Materials
				2.2.2 Reagents
				2.2.3 Equipment
			2.3 Functionalization of AuNPs
				2.3.1 Materials
				2.3.2 Reagents
				2.3.3 Equipment
		3 Methods
			3.1 AuNP Synthesis
			3.2 Dithiolane-Based Linkers Synthesis
				3.2.1 Compound 1 [2,5-Dioxopyrrolidin-1-yl(R)-5-(1,2-Dithiolan-3-yl) Pentanoate]
				3.2.2 PEG (2)
				3.2.3 Drug-Modified Linker (3)
				3.2.4 Compound 4: N-(1,3-Dihydroxybutan-2-yl)-5-(1,2-Dithiolan-3-yl)Pentanamide
				3.2.5 Compound 5: N-(1-(bis(4-Methoxyphenyl)(Phenyl)Methoxy)-3-Hydroxybutan-2-yl)-5-(1,2-Dithiolan-3-yl)Pentanamide
				3.2.6 Compound 6: 4-((3-(5-(1,2-Dithiolan-3-yl)Pentanamido)-4-(bis(4-Methoxyphenyl)(Phenyl)Methoxy)Butan-2-yl)oxy)-4-Oxobutano...
				3.2.7 Compound 7: 4-((3-(5-(1,2-Dithiolan-3-yl)Pentanamido)-4-(bis(4-Methoxyphenyl)(Phenyl)Methoxy)Butan-2-yl)oxy)-4-Oxobutana...
			3.3 AuNP Functionalization with Thiol-Modified Oligonucleotides
			3.4 Oligonucleotide Quantification
				3.4.1 Standard Curve Preparation
				3.4.2 Oligonucleotide Quantification in AuNPs
			3.5 AuNP Functionalization with Dithiolane-Modified Oligonucleotides or Drugs
				3.5.1 AuNP Functionalization with Dithiolane-Modified Oligonucleotides
				3.5.2 AuNP Functionalization with Dithiolane-Modified Drugs
		4 Notes
		References
	Chapter 7: Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay
		1 Introduction
		2 Materials
			2.1 Agarose  Gel
			2.2 Polymer and siRNA
		3 Methods
			3.1 Calculation of Nitrogen to Phosphate (N/P) Ratio for Complexation for siRNA/Cationic Polymer Carriers
				3.1.1 Calculation of the Amine Groups of Polymer
				3.1.2 Calculation of Phosphate Groups of siRNAs
				3.1.3 Calculation of N/P Ratios
			3.2 Preparation of 1% Agarose Gel Electrophoresis
			3.3 Loading siRNA/Cationic Polymer Carrier Complex into 1% Agarose  Gel
		4 Notes
		References
	Chapter 8: Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax...
		1 Introduction
		2 Materials
			2.1 Protein Expression
			2.2 Protein Purification
			2.3 Protein-PMO Conjugation
		3 Methods
			3.1 Expression of PA and LF-cys
			3.2 Purification of PA and LF-cys
			3.3 Purification of PA by Size-Exclusion Chromatography (SEC)
			3.4 Functionalizing PMO with a Maleimide Moiety
			3.5 Coupling of Anthrax Lethal Factor to Maleimide-Functionalized PMO
		4 Notes
		References
Part III: In Vitro Model Systems
	Chapter 9: Development and Use of Cellular Systems to Assess and Correct Splicing Defects
		1 Introduction
		2 Materials
			2.1 Design of Midigene and Maxigene Splice Vector
			2.2 Site-Directed Mutagenesis
			2.3 Culture Conditions and Cell Lines
			2.4 Midigene and AON Transfection
			2.5 RT-PCR
		3 Methods
			3.1 Design of Midigene Splice Vectors
				3.1.1 Gateway Cloning
				3.1.2 Side-Directed Mutagenesis
			3.2 Design of Maxigene Splice Vectors
				3.2.1 In Silico Design of Maxigene Strategy
				3.2.2 Cloning of Maxigene Vectors
				3.2.3 Site-Directed Mutagenesis
			3.3 In Vitro Evaluation of Splice Vectors in Cell Lines
				3.3.1 Transfection in HEK293T
				3.3.2 Transfection in WERI-Rb-1
				3.3.3 Validation of Splicing Events by Reverse Transcriptase PCR (RT-PCR)
			3.4 Correcting Splicing Defects in Artificial Systems
				3.4.1 Midigene Vector and AON Co-transfection
				3.4.2 Quantification of Splicing Redirection with Image J
				3.4.3 How to Know If an AON Is Effective?
		4 Notes
		References
	Chapter 10: Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Ribonucleoprotein (RNP) Transfection
			2.3 Fluorescence Activated Cell Sorting
			2.4 Genomic DNA Isolation
			2.5 Polymerase Chain Reaction (PCR)
			2.6 Restriction Fragment Length Polymorphism Assay (RFLP)
			2.7 RNA Isolation and Reverse Transcription
			2.8 Web Resources
		3 Methods
			3.1 HepG2 Cell Culture
			3.2 Design of Guide RNAs and Donor Template
			3.3 Preparation of RNA Duplex
			3.4 Preparation of the Ribonucleoprotein Complex (RNP)
			3.5 Reverse Transfection of RNP and DNA Donor Template
			3.6 Fluorescent-Activated Cell Sorting (FACS)
			3.7 Generation of the Single-Cell Colonies
			3.8 Genomic DNA Extraction and RFLP Analysis
			3.9 Sequencing Analysis of Candidate Clones and Off-Targets Analysis
			3.10 RNA Isolation
			3.11 RT-PCR and Sequencing Analysis to Confirm the Splicing Defect
		4 Notes
		References
	Chapter 11: In Vitro Models for the Evaluation of Antisense Oligonucleotides in Skin
		1 Introduction
		2 Materials
			2.1 Cell Culture
		3 Methods
			3.1 Isolation and Culture of Epidermal Keratinocytes and Dermal Fibroblasts
			3.2 Transfection of Primary Fibroblasts and Keratinocytes
				3.2.1 Fibroblasts
				3.2.2 Keratinocytes
		4 Notes
		References
	Chapter 12: In Vitro Delivery of PMOs in Myoblasts by Electroporation
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Electroporation
			2.3 RNA Isolation
			2.4 cDNA Synthesis
			2.5 RT-PCR Analysis of Skipping Efficiency
			2.6 RT-qPCR of Skipping Efficiency
		3 Methods
			3.1 Nucleofection and Maintenance of Cells
			3.2 RNA Isolation
			3.3 cDNA Synthesis
			3.4 RT-PCR Analysis
			3.5 RT-qPCR Analysis
		4 Notes
		References
	Chapter 13: Rapid Determination of MBNL1 Protein Levels by Quantitative Dot Blot for the Evaluation of Antisense Oligonucleoti...
		1 Introduction
		2 Materials
			2.1 Cell Culture and Transfection
			2.2 QDB Assay
		3 Methods
			3.1 Cell Transfection
			3.2 Sample Collection and Quantification
			3.3 Sample Preparation
			3.4 Sample Application and Transference
			3.5 Primary Antibody Incubation
			3.6 Secondary Antibody Incubation
			3.7 Quantification
			3.8 Data Analysis
		4 Notes
		References
	Chapter 14: Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy
		1 Introduction
			1.1 Evaluating Exon Skipping at RNA Level
			1.2 Evaluating Dystrophin Restoration in Cell Culture
		2 Materials
			2.1 Cell Culture and Transfection
			2.2 RT and Nested PCR Analysis
			2.3 Myoblots
		3 Methods
			3.1 Cell Culture and Transfection
			3.2 Nested PCR and Gel Image Analysis Method
				3.2.1 Reverse Transcription
				3.2.2 Nested  PCR
				3.2.3 Band Semi-Quantification
			3.3 Myoblot Method
				3.3.1 Myoblot Procedure
				3.3.2 Myoblot Analysis
			3.4 Concluding Remarks
		4 Notes
		References
	Chapter 15: Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro
		1 Introduction
		2 Materials
			2.1 Skeletal Muscle Progenitor Cell Culture
			2.2 Cell Culture Media
			2.3 Antisense Oligonucleotide Design and Delivery
			2.4 Immunofluorescence
			2.5 RNA Isolation, cDNA Synthesis, and Quantitative RT-PCR (RT-qPCR)
		3 Methods
			3.1 Expansion, Cryopreservation, and Differentiation of  MPCs
				3.1.1 Expansion
				3.1.2 Freeze-Thaw
				3.1.3 Differentiation into Multinucleated Myotubes
			3.2 Delivery and Efficacy of Antisense Oligonucleotides in Patient-Derived Myotubes
				3.2.1 Transfection
				3.2.2 Immunofluorescence
				3.2.3 RT-qPCR
		4 Notes
		References
	Chapter 16: Eye on a Dish Models to Evaluate Splicing Modulation
		1 Introduction
		2 Materials
			2.1 General Materials
			2.2 iPSC Culture
			2.3 EB Suspension Protocol
			2.4 EB Adherent Protocol
			2.5 Non-EB Adherent Protocol
			2.6 RNA Extraction
		3 Methods
			3.1 EB Suspension Protocol
			3.2 EB Adherent Protocol
			3.3 Non-EB Adherent Protocol
			3.4 AON Treatment of Organoids
			3.5 RNA Extraction from Organoids
			3.6 Read-out
		4 Notes
		References
	Chapter 17: Establishment of In Vitro Brain Models for AON Delivery
		1 Introduction
		2 Materials
			2.1 Neuroectodermal Differentiation
			2.2 Neurospheres
			2.3 Organoids
			2.4 Fixation and Embedding
			2.5 Cryosectioning and Immunofluorescent Staining
		3 Methods
			3.1 Neuroectodermal Differentiation
			3.2 Neurosphere Embedding
			3.3 Organoids
			3.4 Fixation and Embedding
			3.5 Cryosectioning and Immunofluorescent Staining
		4 Notes
		References
Part IV: In Vivo Model Systems
	Chapter 18: Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides
		1 Introduction
		2 Human and Mouse: Species Are Similar But Not Equal
		3 Checkpoints Before Generating a Humanized Mouse Model
			3.1 Literature Research
			3.2 Comparison of the Gene Sequence
			3.3 Assessment of Gene Expression in Mouse
			3.4 Humanization Feasibility
			3.5 In Vitro Validation
				3.5.1 In Vitro Splicing Assays Considerations
		4 Current Examples of Humanized Mouse Models
			4.1 Insertion of the Entire Human  Gene
			4.2 Replacing Part of the Mouse Gene by the Human  Gene
		5 Conclusions
		References
	Chapter 19: Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modu...
		1 Introduction
		2 Materials
			2.1 In Silico Splice Site Analysis
			2.2 Cloning of a Minigene Splice Vector
			2.3 Mutagenesis  PCR
			2.4 Cell Culture
			2.5 RNA Isolation
			2.6 cDNA Synthesis
			2.7 Polymerase Chain Reaction
			2.8 Agarose Gel Electrophoresis
			2.9 Donor Template Vector Design and Cloning
			2.10 Animals
			2.11 CRISPR/Cas9 Injections
			2.12 Genotyping Materials
			2.13 Quantitative RT-PCR
		3 Methods
			3.1 Splice Site Optimization
			3.2 Generation of an Engineered Zebrafish-Specific Minigene Splice Vector
			3.3 Validation of the Optimized Splice Site Using an Engineered Zebrafish-Specific Minigene Splice Assay
			3.4 Guide RNA Design for CRISPR/Cas9 Injection
			3.5 Generation of Donor Template for CRISPR/Cas9-Induced Homology Directed DNA Repair
			3.6 Generation of the Humanized Zebrafish  Line
			3.7 Genotyping After Fin Clipping
			3.8 Visualization of Human (Pseudo)Exon Inclusion in Zebrafish
			3.9 Quantification of Human (Pseudo)Exon Inclusion Using Quantitative RT-PCR (RT-qPCR)
		4 Notes
		References
	Chapter 20: Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment
		1 Introduction
		2 Materials
			2.1 PMO and PMO Conjugate
			2.2 Animals
			2.3 Intravenous Tail Vein Injection
			2.4 Postmortem Tissue Processing
			2.5 RNA Extraction and RT-PCR
			2.6 Protein Extraction and Western  Blot
			2.7 Immunohistochemistry Staining and Histological Analyses
		3 Methods
			3.1 Intravenous Tail-Vein Injection (See Note 3)
			3.2 Functional Tests
			3.3 Postmortem Tissue Processing
			3.4 RNA Extraction and RT-PCR Quantifying Exon Skipping Efficiency
			3.5 Protein Extraction and Western Blot Quantifying Dystrophin Expression
			3.6 Immunostaining for Dystrophin, Laminin, or Collagen  VI
			3.7 Picro Sirius Red Staining for Detecting Collagen I and  III
			3.8 Histological Analyses
		4 Notes
		References
	Chapter 21: In Vivo Models for the Evaluation of Antisense Oligonucleotides in Skin
		1 Introduction
		2 Materials
		3 Methods
			3.1 Anesthesia and Handling
			3.2 Surgical Procedure
			3.3 Removal of the Grafting Chambers
		4 Notes
		References
	Chapter 22: Delivery of Antisense Oligonucleotides to the Mouse Retina
		1 Introduction
		2 Materials
			2.1 For Injections
			2.2 For Harvesting  Eyes
		3 Methods
			3.1 Before Starting
			3.2 Preparation for the Procedure
			3.3 The Procedure
			3.4 After the Procedure
			3.5 Follow-Up
			3.6 Harvesting the Tissue and Read-Outs
		4 Notes
		References
	Chapter 23: Delivery of Antisense Oligonucleotides to the Mouse Brain by Intracerebroventricular Injections
		1 Introduction
		2 Materials
			2.1 Mice
			2.2 Disposables
			2.3 Fixed Equipment
			2.4 Solutions
		3 Methods
			3.1 Preparation
			3.2 Surgical Procedure
			3.3 Consecutive Injection
		4 Notes
		References
Part V: Safety and Toxicology
	Chapter 24: Intrathecal Delivery of Therapeutic Oligonucleotides for Potent Modulation of Gene Expression in the Central Nervo...
		1 Introduction
		2 Materials
			2.1 Preparation of Test Oligonucleotides
			2.2 Direct Intrathecal Injections
		3 Methods
			3.1 Preparation of Test Oligonucleotides
			3.2 Direct Intrathecal Injection
			3.3 Suggestions for Tissue Collection and Processing
		4 Notes
		References
	Chapter 25: Preclinical Safety Assessment of Therapeutic Oligonucleotides
		1 Introduction: Oligo Classes, Chemistries, and Designs
		2 Delivery
		3 Safety Assessment of Therapeutic Oligos
			3.1 Discovery Phase: Selecting the Oligo Candidate with the Best Balance Between Potency and Safety
				3.1.1 Sequence and Hybridization Dependent Effects: Assessing On- and Off-Target Safety
				3.1.2 Sequence and Hybridization Independent Effects: Coagulation Time and Complement Activation
				3.1.3 Sequence Dependent, But Hybridization Independent: Inflammation, Liver, and Kidney Toxicities
			3.2 Development Phase: In-Depth Characterization and Documentation of the Oligo Candidate
				3.2.1 Preclinical Safety Assessment During the Development Phase
				3.2.2 Regulatory Perspective
		References
	Chapter 26: Preclinical Evaluation of the Renal Toxicity of Oligonucleotide Therapeutics in Mice
		1 Introduction
		2 Materials
			2.1 Evaluation of Creatinine Levels in Urine
			2.2 Evaluation of Total Protein Level in Urine
			2.3 Evaluation of Albumin Level in Urine
			2.4 Evaluation of Acute Kidney Injury Biomarkers (AKI) Level in Urine
		3 Methods
			3.1 Evaluation of Creatinine Levels in Urine
			3.2 Evaluation of Total Protein Level in Urine
			3.3 Evaluation of Albumin Levels in Urine
			3.4 Evaluation of Acute Kidney Injury Biomarkers (AKI) Level in Urine
				3.4.1 Evaluation of β2-Microglobulin, Renin, Kim-1, IP-10, and VEGF Levels in Mouse Urines
				3.4.2 Evaluation of Cystatin C, Epidermal Growth Factor (EGF), Lipocalin-2-NGAL, Clusterin, and Osteopontin (OPN) Level in Uri...
		4 Notes
		References
	Chapter 27: Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endotheli...
		1 Introduction
		2 Materials
			2.1 Perfusion System (Fig. 1)
			2.2 Materials for Perfusion
			2.3 Buffers, Density Medium, Liberase, Cell Culture Media, Coating of Cell Culture Dishes with Fibronectin, Magnetic-Activated...
		3 Methods
			3.1 Preparing the Perfusion System
			3.2 Running the Liver Perfusion to Generate Single Cell Suspension of Liver Cells
			3.3 Cell Isolation
				3.3.1 MACS Isolation of KCs and LSECs
				3.3.2 Culturing LSECs (Continue Here After Step 5 in Subheading 3.3.1)
				3.3.3 Culturing KCs (Continue Here After Step 5 in Subheading 3.3.1)
				3.3.4 Isolation and Culturing of HCs (Continue Here After Step 4 in Subheading 3.3)
		4 Notes
		References
Part VI: Intellectual Property
	Chapter 28: Patent Considerations When Embarking on New Antisense Drug Programs
		1 Introduction
		2 What Is a Patent?
		3 Why Patent Inventions?
			3.1 Exclusive Market Position
			3.2 Opportunity to License or Sell the Invention
			3.3 Collateral for Raising Funding
			3.4 Incentive to Invest in Research and Development
		4 Types of Inventions to Consider
		5 Patenting Requirements
			5.1 Novelty
			5.2 Inventive Step (or Non-Obviousness)
			5.3 Industrial Utility
			5.4 Sufficiency/Written Description
		6 Confidentiality
		7 Patent Timeline
		8 Data Requirements
		9 Searching
		10 Freedom to Operate (FTO)
		11 Summary
		12 Glossary
		13 Notes
		References
Index