Cardiac Gene Therapy: Methods and Protocols

Preface
Contents
Contributors
Part I: Overview
Chapter 1: Updates on Cardiac Gene Therapy Research and Methods: Overview of Cardiac Gene Therapy
1 Introduction
1.1 Cardiac Gene Therapy Approaches
1.2 Vectors and Promotors
1.3 Cardiac Gene Delivery Methods
1.4 Towards Clinical Translation
2 Conclusion
References
Part II: Gene Suppression, Editing, and Reprogramming
Chapter 2: Tough Decoy-Mediated Cardiac Gene Suppression
1 Introduction
2 Materials
2.1 Construction of Recombinant Adenovirus Expression Vector
2.2 Production of Recombinant Adenovirus
2.3 Luciferase Reporter Assay
2.4 Quantitative Real-Time PCR
2.5 Western Blotting
3 Methods
3.1 Obtain Shuttle Vector Carrying miRNA Tough Decoy
3.2 Linearization of a Shuttle Vector
3.3 Recombination in Bacteria
3.4 Purification of Recombinant Adenoviral Genome
3.5 Generation of Infectious Adenovirus
3.6 Preparation and Amplification of Primary Viral Stock
3.7 Purification of Virus
3.8 Luciferase Reporter Assay
3.9 Quantitative Real-Time PCR Analysis
3.10 Western Blotting
4 Notes
References
Chapter 3: Direct Reprogramming of Adult Human Cardiac Fibroblasts into Induced Cardiomyocytes Using miRcombo
1 Introduction
2 Materials
2.1 Reagents for Cell Culture
2.2 Reagents for microRNA Transfection
2.3 Reagents for RNA Purification and cDNA Synthesis
2.4 Reagents for Droplet Digital PCR
2.5 Reagents for Flow Cytometry
2.6 Reagents for Calcium Transient Imaging
3 Methods
3.1 Culture of Adult Human Cardiac Fibroblasts
3.2 AHCF Transfection with microRNAs
3.3 RNA Extraction
3.4 Evaluation of Transfection Efficiency: Day 2
3.5 Evaluation of Cell Reprogramming: Day 7
3.6 Evaluation of Cell Reprogramming: Day 15
3.7 Evaluation of Cell Reprogramming: Day 30
4 Notes
References
Chapter 4: CRISPR/Cas9 Gene Editing of RYR2 in Human iPSC-Derived Cardiomyocytes to Probe Ca2+ Signaling Aberrancies of CPVT A...
1 Introduction
2 Materials
2.1 Construction of Plasmid for RNA-Guided Cas9 Exonuclease and CRISPR/Cas9 Gene Edit
2.2 Cardiac Differentiation of hiPSCs and Dissociation of hiPSC-CMs
3 Methods
3.1 Design and Constructions of Materials for CRISPR/Cas9 Gene Editing
3.2 Introducing the Plasmid DNA and Repair Template into hiPSCs by Electroporation
3.3 Screening for Correctly Gene-Edited Cells
3.4 Cardiac Differentiation of the Mutant hiPSCs
3.5 Cardiomyocyte Dissociation for Single-Cell Experiments
3.6 Cardiac Differentiation of the Mutant hiPSCs in Maturation Media
4 Notes
References
Chapter 5: Enhancing Cardiomyocyte Transcription Using In Vivo CRISPR/Cas9 Systems
1 Introduction
2 Materials
2.1 Cloning
2.2 Validation
2.3 Downstream Application
3 Methods
3.1 Design gRNAs for Transcriptional Activation
3.2 Validate gRNAs for Transcriptional Activation In Vitro
3.3 Apply CRISPRa In Vivo
4 Notes
References
Chapter 6: AAV-Mediated Somatic Gene Editing for Cardiac and Skeletal Muscle in a Large Animal Model
1 Introduction
2 Materials
2.1 Vector Production
2.2 Vector Delivery in Pigs
3 Methods
3.1 Vector Design
3.2 Transfection of HEK 293 T Cells for rAAV Production
3.3 Iodixanol Purification
3.4 Gravity Flow Size Exclusion Purification
3.5 Virus Concentration
3.6 Anesthesia
3.7 Antegrade Coronary Injection
3.8 Selective Pressure Regulation of Retroinfusion
4 Notes
References
Part III: Cardiac Gene Therapy Vectors and Promotors
Chapter 7: Optimization of Synthesis of Modified mRNA
1 Introduction
2 Materials
2.1 Equipment
2.2 Solutions and Supplies
2.3 Synthesis of modRNA
2.3.1 Tailed DNA Template Preparation
2.3.2 PCR Product Purification Using QIAquick PCR Purification Kit
2.3.3 In Vitro Transcription (IVT) Reaction
2.3.4 RNA Phosphatase Treatment
2.3.5 modRNA Purification Using MEGAclear
2.3.6 modRNA Concentration for In Vivo Use Using Amicon Ultra-4 Centrifugal Filters
2.3.7 Quality Control of modRNA Using TapeStation
3 Methods
3.1 Tailed DNA Template Preparation
3.2 PCR Product Purification Using QIAquick PCR Purification Kit
3.3 In Vitro Transcription (IVT) Reaction
3.4 RNA Phosphatase Treatment
3.5 Purify modRNA Using MEGAclear
3.6 modRNA Concentration for In Vivo Use Using Amicon Ultra-4 Centrifugal Filters
3.7 Quality Control of modRNA Using TapeStation
4 Notes
References
Chapter 8: Design and Production of Heart Chamber-Specific AAV9 Vectors
1 Introduction
1.1 AAV9: Research Tool and Gene Therapy
1.2 AAV9: Structure
1.3 AAV9 vs. Transgenesis
1.4 AAV9: Tissue-Specific Enhancements
1.5 Atria vs. Ventricle
1.6 Objective: Generation of Chamber-Specific AAV9 Using Nppa and Myl2 Minimal Promoters
1.7 Identification of the Nppa and Myl2 Minimal Promoters
2 Materials
2.1 Cloning Reagents
2.2 AAV9 Production Reagents
2.3 AAV9 Purification Reagents
2.4 AAV9 Preparation for In Vivo Injection: Reagents and Delivery Equipment
3 Methods
3.1 Construction of Cardiac Chamber-Specific AAV Plasmids
3.2 Production of Cardiac Chamber-Specific AAV9
3.3 Purification of Cardiac Chamber-Specific AAV9
3.4 Preparation of Cardiac Chamber-Specific AAV9 for Usable and Safe Injection In Vivo
4 Notes
References
Chapter 9: Generation of Atrial-Specific Construct Using Sarcolipin Promoter-Associated CRM4 Enhancer
1 Introduction
2 Materials
2.1 Generation of SLN Promoter/CRM-Associated AAV Construct (See Note 1)
2.1.1 pTR.SLN.Luc./pTR.SLN.EGFP
2.1.2 pTR.CRM4.SLN.Luc
2.1.3 pTR.CRM4.SLN.EGFP
2.2 AAV Production
2.3 In Vitro Gene Transfer
2.4 In Vivo Gene Transfer
2.5 Luciferase Assay
2.6 Fluorescence Imaging
2.7 Western Blot Analysis
3 Methods
3.1 Generation of pTR.SLN.Luc Plasmid
3.2 Generation of pTR.SLN.EGFP Plasmid
3.3 Generation of pTR.CRM4.SLN.Luc Plasmid
3.4 Generation of pTR.CRM4.SLN.EGFP Plasmid
3.5 Generation of AAV Viral Vector
3.6 Preparation of PEI Stock Solution
3.7 Validation of In Vitro Gene Transfer (See Fig. 3a; See Notes 6 and 7)
3.8 In Vivo Sample Preparation for Luciferase Assay
3.9 BCA Assay for Protein Quantification
3.10 Luciferase Assay (See Fig. 3b)
3.11 Biodistribution by Western Blotting (See Fig. 4)
3.12 Fluorescent Imaging (See Fig. 5)
4 Notes
References
Part IV: Cardiac Gene Delivery Methods
Chapter 10: Cardiac Targeted Adeno-Associated Virus Injection in Rats
1 Introduction
2 Materials
2.1 Anesthesia
2.2 AAV Administration
3 Methods
3.1 Anesthesia, Intubation, and Ventilation of Rats (See Notes 1-3)
3.2 Local Intramyocardial Injection into the Ventricular Wall of the Heart
3.3 Intracoronary Injection (Fig. 1)
3.4 Postoperative Recovery
4 Notes
References
Chapter 11: Cardiac Gene Delivery in Large Animal Models: Antegrade Techniques
1 Introduction
1.1 Slow Intracoronary Perfusion
1.2 Intracoronary Perfusion + Coronary Artery Occlusion
1.3 Intracoronary Perfusion + Coronary Artery Occlusion + Coronary Sinus Occlusion
2 Materials
2.1 Slow Intracoronary Perfusion
2.2 Intracoronary Perfusion + Coronary Artery Occlusion
2.3 Intracoronary Perfusion + Coronary Artery Occlusion + Coronary Sinus Occlusion
3 Methods
3.1 Slow Intracoronary Perfusion
3.2 Intracoronary Perfusion + Coronary Artery Occlusion
3.3 Intracoronary Perfusion + Coronary Artery Occlusion + Coronary Sinus Occlusion
4 Notes
References
Chapter 12: Locked Nucleic Acid AntimiR Therapy for the Heart
1 Introduction
2 Materials
2.1 Arterial and Venous Peripheral Access
2.2 Angiography Equipment (Fig. 2)
2.3 Catheterization of the Coronary Artery (LAD or Left Circumflex Artery (RCx))
2.4 Infusion of LNA
2.5 Emergency Medication/Tools
2.6 AntimiR
3 Methods
3.1 Venous and Arterial Assess
3.2 Blocking of the Anterograde Flow in Coronary Arteries (LAD and RCx)
3.2.1 For the LAD Approach
3.2.2 For the RCx Approach
3.3 Anterograde Application (LAD and RCx)
4 Notes
References
Chapter 13: Cardiac Gene Delivery in Large Animal Models: Selective Retrograde Venous Injection
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 14: Endocardial Gene Delivery Using NOGA Catheter System
1 Introduction
2 Materials
2.1 Preparing the NOGA/MyoStar System
2.2 Preparing the Patient or the Experimental Animal
2.3 Electroanatomical Mapping
2.4 Gene Transfers
2.5 Removing the Femoral Sheath
3 Methods
3.1 Preparing the NOGA/MyoStar System
3.2 Preparing the Patient
3.3 Electroanatomical Mapping of the Left Ventricle
3.4 Excluding Points
3.5 Determining Injection Area
3.6 Gene Transfers
3.7 Removing Femoral Sheath
4 Notes
References
Chapter 15: Surgical Methods for Cardiac Gene Delivery in Large Animals
1 Introduction
2 Materials
2.1 Anesthesia and Pre- and Perioperative Medications
2.2 Direct Intramyocardial Gene Delivery
2.3 Surgical Gene Delivery with Heart-Lung Machine
3 Methods
3.1 Preoperative Care and Preparation
3.2 Direct Intramyocardial Gene Delivery (See Fig. 1)
3.3 Gene Delivery Using Heart-Lung Machine Including Method of Molecular Cardiac Surgery with Recirculating Delivery (See Fig....
3.4 Postoperative
4 Notes
References
Chapter 16: Atrial Gene Painting in Large Animal Model of Atrial Fibrillation
1 Introduction
2 Materials
2.1 Preparation
2.2 Surgery
3 Methods
3.1 Poloxamer Saline Solution Preparation
3.2 Animal Surgery
4 Notes
References
Chapter 17: Stent-Based Gene Delivery for Coronary Disease
1 Introduction
2 Materials
2.1 Priming Ad Vectors for Reversible Surface Immobilization on Thiolated Surfaces
2.2 Priming AAV Vectors for Reversible Surface Immobilization on Thiolated Surfaces
2.3 Formulation of Thiolated Metal Surfaces for Immobilization of Thiol-Reactive Viral Vectors
2.4 Assessment of Ad and AAV Vector Immobilization on the Metal Surfaces by Fluorescence Microscopy
2.5 In Vitro Reporter Gene Transduction Experiments with Stent- or Mesh Disk-Immobilized Ad and AAV Vectors
2.6 In Vitro Gene Transduction with Mesh-Immobilized Ad-SOD3: Immunocytochemistry
2.7 In Vitro Gene Transduction with Mesh-Immobilized Ad-SOD3: ROS Production
2.8 Deployment of Gene-Eluting Stent in the Carotid Artery (a Rat Model)
2.9 Optical Imaging of Vascular Gene Expression After Deployment of Gene-Eluting Stents
3 Methods
3.1 Priming Ad Vectors for Reversible Surface Immobilization on Thiolated Surfaces
3.2 Priming AAV Vectors for Reversible Surface Immobilization on Thiolated Surfaces
3.3 Formulation of Thiolated Metal Surfaces for Immobilization of Thiol-Reactive Viral Vectors
3.4 Assessment of Ad and AAV Vector Immobilization on the Metal Surfaces by Fluorescence Microscopy (See Fig. 2)
3.5 In Vitro Reporter Gene Transduction Experiments with Stent- or Mesh Disk-Immobilized Ad and AAV Vectors (See Figs. 3, 4, a...
3.6 In Vitro Gene Transduction with Mesh-Immobilized Ad-SOD3: Immunocytochemistry (See Fig. 6)
3.7 In Vitro Gene Transduction with Mesh-Immobilized Ad-SOD3: ROS Production (See Fig. 7)
3.8 Deployment of Gene-Eluting Stent in the Carotid Artery (a Rat Model)
3.9 Optical Imaging of Vascular Gene Expression After Deployment of Gene-Eluting Stents (See Fig. 8)
4 Notes
References
Chapter 18: Selective Anti-AAV Antibody Depletion by Hemapheresis and Immunoadsorption
1 Introduction
2 Materials
2.1 AAV-Immunoadsorbent Matrix Preparation (AAV Beads)
2.2 In Vitro Immunoadsorption
2.3 Catheter Implantation
2.4 In Vivo Hemapheresis and Immunoadsorption
2.5 Neutralization Luciferase Assay
2.6 ELISA
2.7 In Vivo Imaging
3 Methods
3.1 AAV-Immunoadsorbent Matrix Preparation (AAV Beads)
3.2 In Vitro Immunoadsorption
3.3 Elution of Anti-AAV Immunoglobulins from the Immunoadsorbent
3.4 Catheter Implantation
3.5 In Vivo Hemapheresis and Immunoadsorption
3.6 Neutralization and Luciferase Assay
3.7 ELISA
3.8 In Vivo Imaging
4 Notes
References
Chapter 19: Ex Vivo Delivery of Viral Vectors by Organ Perfusion for Cardiac Transplantation Gene Therapy
1 Introduction
2 Materials
2.1 Donor Blood Washing with Cell Saver Device
2.2 Priming the Ex Vivo Perfusion Device/Preparation of the Perfusate (See Note 1)
2.3 Ex Vivo Perfusion of the Cardiac Graft (See Note 1)
2.4 Administration of the Viral Vector to the Circuit
2.5 Removal of Cardiac Graft from Ex Vivo Perfusion Device
3 Methods
3.1 Donor Blood Washing of Neutralizing Components (See Note 2)
3.2 Preparing the Perfusate and Priming the Ex Vivo Perfusion Device
3.3 Ex Vivo Normothermic Perfusion of Cardiac Graft
3.4 Administration of the Viral Vector to the Circuit (See Note 7)
3.5 Removal of Cardiac Graft from Ex Vivo Perfusion Device
4 Notes
References
Part V: Pulmonary Hypertension
Chapter 20: Intra-Airway Gene Delivery for Pulmonary Hypertension in Rodent Models
1 Introduction
2 Materials
2.1 Rat Models of Pulmonary Artery Hypertension
2.1.1 Animals
2.1.2 Anesthesia
2.1.3 Surgery, PH Induction, Gene Delivery
2.2 Mouse Models of Pulmonary Artery Hypertension
2.2.1 Animals
2.2.2 PH Induction and Gene Delivery
2.3 Functional PH Characterization
2.4 Histological PAH Characterization
3 Methods
3.1 Animal Preparation and PH Model Creation in Rats
3.1.1 Monocrotaline-Induced PAH Model
3.1.2 Sugen-Chronic Hypoxia-Induced PAH Model
3.1.3 Pneumonectomy-Induced PAH Model
3.2 Animal Preparation and PH Model Creation in Mice
3.2.1 Sugen-Chronic Hypoxia-Induced PAH Model
3.3 Airway Gene Delivery
3.4 PAH Assessment and Gene Transduction Efficiency
4 Notes
References
Chapter 21: Endobronchial Gene Delivery for Pulmonary Hypertension in a Large Animal Model
1 Introduction
2 Materials
2.1 Animal Preparation
2.2 Bronchoscopy and Gene Delivery
3 Methods
3.1 Experiment Preparation
3.2 Animal Preparation
3.3 Bronchoscopy and Gene Delivery
4 Notes
References
Part VI: Patient Screening and Measuring the Efficacy of Cardiac Gene Therapy
Chapter 22: Cell-Based Determination of Neutralizing Antibodies Against Adeno-Associated Virus in Cardiac Gene Therapy
1 Introduction
2 Materials
2.1 Cell Culture and Maintenance
2.2 Neutralizing Antibody Assay
2.3 Luciferase Assay
3 Methods
3.1 Cell Culture and Cell Maintenance
3.2 ``Cell Plate´´ Preparation
3.3 Neutralizing Antibody Assay
3.4 Luciferase Assay
4 Notes
References
Chapter 23: Left Ventricular Pressure Volume Assessment Using Carotid Artery Access in the Rat
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 24: Assessing the Effect of Cardiac Gene Therapy Using Catheter-Based Pressure-Volume Measurement in Large Animals
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 25: ELISpot Assay for Gene Therapy in Large Animal Studies
1 Introduction
2 Materials
2.1 PBMC Isolation
2.2 ELISpot Assay
3 Methods
3.1 PBMC Isolation
3.2 ELISpot Assay
4 Notes
References
Chapter 26: Assessing Recombinant AAV Shedding After Cardiac Gene Therapy
1 Introduction
2 Materials
2.1 Storage Medium Preparation
2.2 Culture Medium Preparation
2.3 Specimen Sampling
2.3.1 Blood Sampling
2.3.2 Feces Sampling
2.3.3 Urine Sampling
2.3.4 Saliva Sampling
2.3.5 Nasal Mucus Sampling
2.3.6 qPCR Analysis
2.4 Infectious Replication Assay
3 Methods
3.1 Preparation of Storage Medium
3.2 Preparation of Culture Medium
3.3 Specimen Sampling
3.3.1 Blood Sampling (See Note 1)
3.3.2 Feces Sampling
3.3.3 Urine Sampling
3.3.4 Saliva Sampling
3.3.5 Nasal Mucus Sampling (Only Under Sedation)
3.4 qPCR Analysis to Determine Vector Concentration in Specimen Samples
3.4.1 Preparation of the rAAV DNA for the Standard Curve
3.4.2 Preparation of the Positive and Negative Control Samples
3.4.3 qPCR Protocol
3.5 Infectious Replication Assay
3.5.1 Day 1 (Morning): Seed Culture Plate with HeLa RC32 Cells
3.5.2 Day 2 (Morning): Prepare Specimen Samples and Adenovirus Dilutions to Add to Cells
3.5.3 Day 4 (Morning): Collect Cell Pellets
3.5.4 Total DNA Extraction from Infectious Replication Assay Cell Pellet
3.5.5 qPCR Analysis to Determine the Vector Concentration in Samples
4 Notes
References
Index