Design and Delivery of SiRNA Therapeutics (Methods in Molecular Biology, 2282)

This volume details protocols on rationale design of therapeutic siRNA molecules and its encapsulation with smart vehicles to overcome the barriers to an effective administration in vivo. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Design and Delivery of SiRNA Therapeutics aims to ensure successful results in the further study of this vital field. This volume details protocols on rationale design of therapeutic siRNA molecules and its encapsulation with smart vehicles to overcome the barriers to an effective administration in vivo. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Design and Delivery of SiRNA Therapeutics aims to ensure successful results in the further study of this vital field Preface Contents Contributors Chapter 1: RNA Interference: Story and Mechanisms 1 Introduction 2 The Interferon Response Hindered the Application of RNAi in Mammalian Cells 3 Molecular Mechanisms RNAi Induced by dsRNAs 4 MicroRNAs: Masters of Gene Regulation 5 piRNAs: Guardians of the Germline 6 CRISPR Interference 7 A Versatile Genome Editing Technology 8 Conclusions References Chapter 2: Selection of Chemical Modifications in the siRNA Seed Region That Repress Off-Target Effect 1 Introduction 2 Materials 2.1 Design and Preparation of siRNAs 2.2 Luciferase Reporter Vector and Oligonucleotides with CM and SM Sequences 2.3 Cell Culture 2.4 Transfection and Luciferase Reporter Assay Reagents 2.5 Calculation of Tm 2.6 Microarray Analysis 3 Methods 3.1 Designing and Preparing siRNAs with DNA or 2′-OMe Modifications in the Seed Region 3.2 Preparing Luciferase Reporters with CM and SM Sequences 3.3 RNA Silencing Activity Assay Using the Dual Luciferase Reporter System 3.4 Microarray Analysis 4 Notes References Chapter 3: Stick-Based Methods for Aptamer-Mediated siRNA Targeted Delivery 1 Introduction 2 Materials 2.1 Oligonucleotides 2.2 Construct Preparation 2.3 Non-denaturing Polyacrylamide Gel for Annealing Control 2.4 Cell Culture 2.5 Binding and Internalization Analysis by Quantitative Real-Time PCR (RT-qPCR) 2.6 Cell Lysis and Western Blotting for Functional Analysis 2.7 Gene Expression by RT-qPCR 3 Methods 3.1 Aptamer-siRNA Design 3.2 Construct Preparation (Fig. 1) 3.3 Control of the Correct Annealing by Gel Electrophoresis 3.4 Cell Culture (For Binding and Internalization or Functional Activity Assays) 3.5 Binding and Internalization Analysis by RT-qPCR 3.6 AsiC Functional Activity Analyses by RT-qPCR 3.7 AsiC Functional Activity Analyses by Immunoblotting 4 Notes References Chapter 4: Evaluation of siRNA Stability and Interaction with Serum Components Using an Agarose Gel-Based Single-Molecule FRET... 1 Introduction 2 Materials 3 Methods 3.1 Validation of Designed FRET-siRNA by RNase A Digestion 3.1.1 Evaluating Band Fluorescence Intensity by FIJI ImageJ Gel Analyzer Tool 3.2 Tracking the Integrity of FRET-siRNA in Different Percentages of Serum and Evaluating Interaction with Serum Components 3.3 Degradation Kinetics of FRET-siRNA in Serum 3.4 Evaluation of FRET-siRNA Stabilizing Effect of siRNA Carriers in Serum 4 Notes References Chapter 5: Development of siRNA Therapeutics for the Treatment of Liver Diseases 1 Introduction 2 Chemical Modifications in siRNA Drugs 3 Strategies for Delivering siRNA Drugs to the Liver 3.1 Lipid Nanoparticles 3.2 Dynamic Polyconjugates 3.3 GalNAc-siRNA Conjugates 4 Development of siRNA Drugs for Treatment of Liver Diseases 5 FDA-Approved siRNA Drugs for the Treatment of Liver Diseases 6 Concluding Remarks and Future Perspectives References Chapter 6: siRNA Design and GalNAc-Empowered Hepatic Targeted Delivery 1 Introduction 2 Materials 2.1 Software, Database, and Equipment 2.2 Reagents 2.3 Cells and Animals 3 Methods 3.1 Primary Mouse Hepatocyte Isolation 3.2 siRNA Design and Screening 3.2.1 mRNA (cDNA) Selection 3.2.2 Generation of siRNA Sequence List 3.2.3 Activity Evaluation 3.2.4 Off-Target Effect Evaluation 3.3 siRNA Chemical Modification 3.4 In Vivo Delivery of GalNAc-siRNA Conjugate 3.5 In Vivo Activity of GalNAc-siRNA Conjugate 3.6 In Vivo Toxicity of GalNAc-siRNA Conjugate 4 Notes References Chapter 7: Synthesis of GalNAc-Oligonucleotide Conjugates Using GalNAc Phosphoramidite and Triple-GalNAc CPG Solid Support 1 Introduction 2 Materials 2.1 Synthesis of Triple GalNAc TEG CPG Solid Support and GalNAc TEG Phosphoramidite 2.2 Oligonucleotide Synthesis 3 Methods 3.1 Preparation of Triple GalNAc TEG Solid Support for RNA Synthesis 3.1.1 Synthesis of 3,3′-((2-(12-(Benzyloxy)-12-oxododecanamido)-2-((2-carboxyethoxy)methyl)propane-1,3-diyl)bis(oxy))-dipropio... 3.1.2 N-(12-(Benzyloxy)-12-oxododecanamido)-tris[2,5,8,11,18-pentaoxa-14-aza-15-oxo-19-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy... 3.1.3 N-(11-Carboxy-undecanamido)-tris[2,5,8,11,18-pentaoxa-14-aza-15-oxo-19-(3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-β-d-galac... 3.1.4 N-(12-((3-O-(4,4′-Dimethoxytrityl)-2-hydroxypropyl)-amino)-12-oxododecanamido)-tris[2,5,8,11,18-pentaoxa14-aza-15-oxo-19... 3.1.5 TRIS-(GalNAc-TEG)-CPG (8) 3.2 Preparation of a Mono GalNAc TEG Phosphoramidite 3.2.1 Synthesis of 16-Amino-1-O-(4,4′-dimethoxytrityl)-4,7,10,13-tetraoxahexadecan-1,2-diol 10 3.2.2 Synthesis of 5-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)-N-(1-O-(4,4′-dimethoxytrityl)-1,2-dihydr... 3.2.3 Synthesis of 5-(3,4,6-Tri-O-acetyl-2-acetamido-2-deoxy-β-d-galactopyranosyloxy)-N-(1-O-(4,4′-dimethoxytrityl)-2-O-(N,N-d... 3.3 Synthesis of GalNAc-siRNA Conjugates 3.3.1 Synthesis of GalNAc-RNA Conjugates 3.3.2 Purification of GalNAc-RNA Conjugates 3.3.3 Analysis of GalNAc-RNA Conjugates by Polyacrylamide Gel Electrophoresis (Fig. 4b) 3.3.4 Annealing of GalNAc Modified siRNA Duplexes 4 Notes References Chapter 8: Preparation of Lipid-Conjugated siRNA Oligonucleotides for Enhanced Gene Inhibition in Mammalian Cells 1 Introduction 2 Materials 2.1 RNA Oligonucleotide Synthesis 2.2 High-Performance Liquid Chromatography (HPLC) 2.3 Desalting RNA Oligonucleotide Solutions 2.4 Ultraviolet-Visible (UV) Spectroscopy 2.5 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry 2.6 Cell Culture Models 2.7 Enzyme-Linked Immunosorbent Assay (ELISA) 3 Methods 3.1 Chemical Synthesis of Lipid-siRNA 3.1.1 Preparation of 3′- and 5′-Lipid RNA Oligonucleotide Conjugates 3.1.2 Purification of RNA Oligonucleotide Conjugates by HPLC 3.1.3 Annealing and Precipitation of siRNA Duplexes 3.2 Evaluation of the TNF-α Inhibition Properties 3.2.1 In Vitro Gene Silencing Studies Pre-binding Strategy Non-pre-binding Strategy 4 Notes References Chapter 9: Engineering of Solid Dosage Forms of siRNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles Using a Quality-by-Design A... 1 Introduction 2 Materials 3 Methods 3.1 Design of Experiment (DoE) 3.1.1 Statistical Model Optimization 3.1.2 Determining the Optimal Operating Space 3.1.3 Constructing the Plots 3.2 Freeze Drying 3.3 Spray Drying 3.3.1 Preparation of the Feedstock Dispersion for Spray Drying 3.3.2 The Spray Drying Process 3.3.3 Powder Yield 3.4 Moisture Content 3.4.1 Sample Preparation and Loading 3.5 Aerodynamic Particle Size 3.6 Surface Morphology 3.6.1 Sample Preparation and Loading 3.6.2 Scanning Electron Microscopy 3.7 Solid State Properties 3.7.1 Sample Preparation and Loading 4 Notes References Chapter 10: Preparation and Characterization of siRNA-Loaded Liposomes 1 Introduction 2 Materials 2.1 Preparation of LPH 2.2 Characterization of LPH 2.3 Preparation of LCP 2.4 Characterization of LCP 2.5 In Vivo Luciferase Silencing Effect of LCP 3 Methods 3.1 Preparation of LPH 3.2 Characterization of LPH 3.2.1 Particle Size and Zeta Potential 3.2.2 Trapping Efficiency of siRNA 3.3 Preparation of LCP 3.4 Characterization of LCP (Fig. 2) 3.4.1 Particle Size and Zeta Potential 3.4.2 Transmission Electron Microscopy 3.4.3 Trapping Efficiency of siRNA 3.5 In Vivo Luciferase Silencing Effect of LCP 4 Notes References Chapter 11: siRNA Vehicles for High Endosomal Escapability 1 Introduction 2 Materials 2.1 Preparation of Polycationic Liposome and siRNA Complex 2.2 2-(p-Toluidino)-6-napthalene sulfonic acid (TNS) Assay 2.3 Confocal Laser Scan Microscopy 3 Methods 3.1 Preparation of Polycationic Liposomes and siRNA Complexes 3.2 Measurement of pKa of Liposome 3.3 Confocal Laser Scan Microscopy 4 Notes References Chapter 12: siRNA Design and Delivery Based on Carbon Nanotubes 1 Introduction 2 Materials 2.1 Preparation of siRNA Complexes 2.2 Gel Electrophoresis for siRNA Binding Capacity 2.3 Cell Culture and Transfection 2.4 Antibody Staining and Flow Cytometry 3 Methods 3.1 Preparation of siRNA Complexes 3.2 Gel Electrophoresis for siRNA Binding Capacity 3.3 Cell Culture 3.4 Cell Transfection 3.5 Antibody Staining for Flow Cytometry Analysis 3.6 Flow Cytometry Analysis for Target Protein Expression 3.7 Data Analysis 4 Notes References Chapter 13: MRI-Visible Nanovehicle for Efficient siRNA Delivery 1 Introduction 2 Materials 2.1 Reagents 2.2 Equipment 3 Methods 3.1 Alkyl-PEI/SPIO Complexes Fabrication 3.1.1 N-Alkyl-PEI Synthesis 3.1.2 Superparamagnetic Iron Oxide Nanoparticles Synthesis 3.1.3 Alkyl-PEI/SPIO Complexes Fabrication 3.1.4 Label Alkyl-PEI/SPIO Complexes with FITC 3.2 Alkyl-PEI/SPIO-siRNA Complex Formation and Characterization (see Note 1) 3.3 Cellular Uptake of Alkyl-PEI/SPIO-siRNA Complexes 3.4 Cell Transfection and Cytotoxicity of Alkyl-PEI/SPIO-siRNA Complexes 3.5 Transfection Efficiency Evaluation 3.5.1 Luciferase Activity Assay 3.5.2 Western Blot Analysis 3.5.3 Immunofluorescent Staining (Fig. 4) 3.6 MRI Study of Transfected Cells (Fig. 5) 3.7 siRNA Delivery by Alkyl-PEI/SPIO Nanovehicle In Vivo 3.7.1 Luciferase Activity Assay 3.7.2 Tissue Western Blot Assay 4 Notes References Chapter 14: Cationic Dendrimers for siRNA Delivery: An Overview of Methods for In Vitro/In Vivo Characterization 1 Introduction 2 Chemical-Physical Characterization of the Pure NV 2.1 Dynamic Light Scattering (DLS) 2.2 Transmission Electron Microscopy (TEM) 2.3 Isothermal Titration Calorimetry (ITC) 3 siRNA/NV Complex Characterization 3.1 DLS and TEM 3.2 ITC 3.3 Agarose Gel Retardation Analysis and RNAse A Assay 4 siRNA Cellular Uptake and Release 4.1 Flow Cytometry (FC) 4.2 Confocal Microscopy 5 In Vitro Silencing Activity 5.1 Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) 5.2 Western Blotting (WB) 5.3 MTT Assay 6 In Vivo Gene Silencing Activity 6.1 In Vivo Delivery and Gene Silencing 6.2 Immunohistochemistry (IHC) 7 Toxicity 7.1 MTT and Lactate Dehydrogenase (LDH) Assay 7.2 Hemolysis Experiment 7.3 In Vivo Toxicity Evaluation 8 Conclusion References Chapter 15: ITC for Characterization of Self-Assembly Process of Cationic Dendrons for siRNA Delivery 1 Introduction 1.1 ITC Instrumentation and General Principles 1.2 ITC for Micellization Study 2 Materials 3 Methods 3.1 Sample Preparation 3.2 Checking of Micelle Formation by DLS 3.3 ITC Protocol 3.4 ITC Data Analysis 3.4.1 Raw Data Inspection and Optimization 3.4.2 CMC Determination and DeltaHmic Calculation 3.4.3 CMC Value Validation by Pyrene Assay 3.4.4 Estimation of the Micelle Aggregation Number (Nagg) 3.4.5 Calculation of the Degree of Counterion Binding (β) 3.4.6 Calculation of the Gibbs Free Energy of Micellization (DeltaGmic) and Its Entropic Contribution (TDeltaSmic) 4 Notes References Chapter 16: Cationic Dendrimers for siRNA Delivery: Computational Approaches for Characterization 1 Introduction 2 Materials 2.1 Forcefield and Software 2.2 Radius of Gyration and Asphericity 2.3 Radial Monomer Density 2.4 Solvent Accessible Surface Area and Interior Cavities 2.5 MM/PBSA 3 Methods 3.1 Box and Simulation Input Parameters Creation 3.2 Simulation Protocol 3.3 Dendrimer Parametrization 3.4 siRNA Model Optimization 3.5 Complex Creation and Simulation 3.6 Dendrimer Analysis 3.7 siRNA-Dendrimer Complex Structural Analysis 3.8 siRNA-Dendrimer Energy of Binding Analysis 4 Notes References Chapter 17: Fundamental and Practical Aspects in the Formulation of Colloidal Polyelectrolyte Complexes of Chitosan and siRNA 1 Introduction 2 General Aspects of the Polyelectrolyte Complexation 2.1 Weak and Strong Polyelectrolyte Complexes 2.2 Colloidal Polyelectrolyte Complexes 3 Chitosan 3.1 Origin of Chitosan 3.2 Biological Properties 3.3 Solution Properties 3.4 Complexation Behavior of Chitosan 4 Chitosan-Based siRNA Delivery Systems 4.1 Thermodynamics of Complexation 4.2 Physicochemical Properties of Colloidal Complexes 4.2.1 Mixing 4.2.2 Molar Mass and DA 4.2.3 Concentration and N:P 4.2.4 Colloidal and Thermodynamic Stability 4.2.5 Biological Evaluation 5 Conclusion References Chapter 18: Cell-Penetrating Peptides Delivering siRNAs: An Overview 1 Cell-Penetrating Peptides 1.1 Introducing Cell-Penetrating Peptides 1.2 Uptake Mechanisms and Endosomal Escape 2 Small Interfering RNA 2.1 A Brief Introduction to Small Interfering RNAs 2.2 Delivery Methods 3 Delivering siRNA Using CPPs: Three Strategies 3.1 CPP Covalently Conjugated to siRNA (CPP-siRNA) 3.2 Non-covalent Complexes (CPP:siRNA) and Applications 3.3 CPP-Decorated Multifunctional Nanocomplexes for siRNA Delivery 4 Conclusions References Chapter 19: Non-covalent Encapsulation of siRNA with Cell-Penetrating Peptides 1 Introduction 2 Materials 2.1 UV Spectrophotometry and Agarose Gel Electrophoresis for Interaction, Encapsulation, and Stability Assays 2.2 Circular Dichroism 2.3 Quantitative 96-Well Microplate-Based and Semi-Quantitative-Imaging Confocal Microscopy Transfection Efficiency Assay 2.4 Silencing by tdTomato-siRNA Transfection of tdTomato-Positive Engineered Cells 3 Methods 3.1 Evaluation of Candidate CPP-siRNA Interaction Using UV siRNA Peak Hypochromicity 3.2 Determination of the Optimal Encapsulation Molar Ratio for the Candidate CPP-siRNA Complex 3.3 Dextran Sulfate Sodium Competition Assay for Cell-Penetrating Peptide-siRNA Complexes 3.4 Stability Assay of Cell-Penetrating Peptide-siRNA Complexes 3.5 Circular Dichroism 3.6 Quantitative 96-Well Plate-Based Transfection Efficiency Assay 3.7 Confocal Microscopy-Based Semi-Quantitative-Imaging Transfection Efficiency Assay 3.8 Exploiting tdTomato-Transfected Fluorescent Cells for the Evaluation of siRNA-Driven Gene Silencing 4 Notes References Chapter 20: siRNA Therapeutics for Protein Misfolding Diseases of the Central Nervous System 1 Introduction 2 Materials 2.1 LSPC and PALETS Reagents, Supplies, and Equipment 2.2 LSPC Delivery Supplies 3 Methods 3.1 Preparing Liposomes 3.1.1 DOTAP LSPCs 3.1.2 DOTAP PALETS 3.1.3 DSPE PALETS 3.2 Resuspending Liposomes 3.3 Preparing LSPCs and PALETS 3.3.1 LSPCs 3.3.2 DOTAP PALETS 3.3.3 DSPE PALETS 3.4 Injecting LSPCs or PALETS 3.5 Instilling LSPCs or PALETS 3.6 Monitoring LSPC and PALET Trafficking 3.6.1 Monitoring LSPCs and PALETS in Live Mice 3.6.2 Visualizing LSPCS and PALETS in the CNS 3.6.3 Quantifying LSPC and PALETS Delivery 3.6.4 Quantifying siRNA Knockdown 4 Notes References Chapter 21: siRNA Loaded-Exosomes 1 Introduction 2 Materials 3 Methods 3.1 Dendritic Cell Primary Culture 3.2 Exosome Isolation 3.3 Exosome Characterization and Quantitation 3.4 siRNA Loading into Exosomes 3.5 Assessment of siRNA Loading into Exosomes 4 Notes References Chapter 22: Production and Characterization of Anti-CCR5 siRNA-Loaded Polycaprolactone Nanoparticles for Topical Pre-exposure ... 1 Introduction 2 Materials 2.1 Production and Characterization of Nanoparticles 2.2 Cell Culture, Silencing Efficiency, and Cytotoxicity 3 Methods 3.1 Production of Nanoparticles 3.2 Physicochemical Characterization of Nanoparticles 3.3 Biological Properties of Nanoparticles 3.3.1 Cytotoxicity 3.3.2 Silencing Efficiency 4 Notes References Chapter 23: siRNA Therapeutics in Ocular Diseases 1 Introduction 2 Ocular Structures and Modalities for siRNA Administration 2.1 Topical Administration 2.2 Intravitreal Administration 3 Design of siRNA for Ocular Therapy 4 Delivery Systems for Ocular siRNA 4.1 Lipid and Peptide-Based Delivery Systems 5 Quantitation and Biodistribution of siRNAs in the Eye 5.1 Differential Quantitation of Extra- and Intracellular Antisense Strands 6 Safety of siRNA After Ocular Administration 7 Current Status of Ocular siRNA Candidates 8 Conclusion References Chapter 24: Delivery of siRNA to the Eye: Protocol for a Feasibility Study to Assess Novel Delivery System for Topical Deliver... 1 Introduction 2 Materials 2.1 Gel Retardation Assay 2.2 Human Corneal Epithelium (HCE) Cell Line Maintenance 2.3 In Vitro Cellular Uptake of Fluorescent siRNA to Corneal Epithelial Cells 2.4 In Vivo siRNA Uptake into Mouse Cornea 3 Methods 3.1 Gel Retardation Assay 3.2 Human Corneal Epithelium (HCE) Cell Line Maintenance 3.3 In Vitro Cellular Uptake of Fluorescent siRNA to Corneal Epithelial Cells 3.4 In Vivo siRNA Uptake into Mouse Cornea 4 Notes References Index