Chapter 4: Methods for Noninvasive Monitoring of Muscle Fiber Survival with an AAV Vector Encoding the mSEAP Reporter Gene
1. Introduction
2. Materials
2.1. Generating the Cis-Plasmid for Recombinant AAV Packaging
2.2. Production of the rAAV Reporter Vector
2.3. In Vivo Vector Delivery into Muscle Tissue
2.4. mSEAP Secretion Measurement
2.5. Histological and mSEAP Histo-Enzymatic Revelation
3. Methods
3.1. Generating the Cis Plasmid for Recombinant AAV Packaging
3.2. Production of the rAAV Reporter Vector
3.3. In Vivo Vector Delivery into Muscle Tissue
3.4. mSEAP Secretion Measurement (see Note 12)
3.5. mSEAP Histo-Enzymatic Revelation
4. Notes
References
Chapter 5: Monitoring Murine Skeletal Muscle Function for Muscle Gene Therapy
1. Introduction
2. Materials
2.1. Ex Vivo Analysis of Muscle Forces of an Intact EDL Muscle
2.2. In Situ TA Muscle Force Analysis
2.3. Grip Force Measurement
2.4. Downhill Treadmill Assay
3. Methods
3.1. Ex Vivo Analysis of Muscle Forces of an Intact EDL Muscle
3.2. In Situ TA Muscle Force Analysis
3.3. Grip Force Measurement
3.4. Downhill Treadmill Assay
4. Notes
References
Chapter 6: Phenotyping Cardiac Gene Therapy in Mice
1. Introduction
2. Materials
2.1. Treadmill Performance Regimen
2.2. 12-Lead ECG Assay
2.3. Closed-Chest Left Ventricular Catheterization Hemodynamic Assay
3. Methods
3.1. Treadmill Performance Regimen
3.2. 12-Lead ECG Assay
3.3. Closed-Chest Left Ventricular Catheterization Hemodynamic Assay (See Note 10)
4. Notes
References
Chapter 7: Golden Retriever Muscular Dystrophy (GRMD): Developing and Maintaining a Colony and Physiological Functional Measurements
1. Introduction
2. Materials
2.1. Colony Development and Maintenance
2.2. Physiological Functional Measurements
3. Methods
3.1. Colony Development and Maintenance
3.2. Physiological Functional Measurements
4. Notes
References
Part II:
New Technology in Muscle Gene Therapy
Chapter 8: Directed Evolution of Adeno-Associated Virus (AAV) as Vector for Muscle Gene Therapy
1. Introduction
2. Materials
2.1. In Vitro Recombination of AAV Cap Genes by DNA Shuffling
2.2. Construction of Plasmid Library and Generation of Virus Library
2.3. Screening of Virus Library in Animal Model
2.4. Production and Characterization of Recombinant AAVs
3. Methods
3.1. DNA Shuffling
3.2. Construction of Shuffled Plasmid Library
3.3. Generation of Chimeric Virus Library
3.4. Biopanning of Virus Library in Mouse Model
3.5. Characterization of Tissue Tropism of AAV Variants
4. Notes
References
Chapter 9: Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors
1. Introduction
2. Materials
2.1. Generating Reengineered AAV2 Vectors
2.1.1. Mutagenesis
2.1.2. AAV Vector Production
2.2. Evaluating Systemic Gene Transfer to Skeletal Muscle Using AAV Vectors
2.2.1. Intravenous (Tail Vein) Administration of AAV Vectors
2.2.2. Isolated Limb Infusion of AAV Vectors
2.2.3. Live Animal Bioluminescence Imaging (See Note 5)
2.2.4. Harvesting Different Muscle Groups
2.2.5. Tissue Lysate Luciferase Expression Assay
3. Methods
3.1. Generating Reengineered AAV2 Vectors
3.1.1. Mutagenesis (See Note 7)
3.1.2. AAV Vector Production
3.2. Evaluating Systemic Gene Transfer to Skeletal Muscle Using AAV Vectors
3.2.1. Intravenous (Tail Vein) Administration of AAV Vectors
3.2.2. Isolated Limb Infusion of AAV Vectors
3.2.3. Live Animal Bioluminescence Imaging (See Note 5)
3.2.4. Harvesting Different Muscle Groups
3.2.5. Luciferase Expression Assay with Tissue Lysates
4. Notes
References
Chapter 10: Bioinformatic and Functional Optimization of Antisense Phosphorodiamidate Morpholino Oligomers (PMOs) for Therapeutic Modulation of RNA Splicing in Muscle
2.1.2. Engineering Modified U7 snRNA Specific to Dystrophin Exons
2.1.3. Generating AAV Vectors Encoding the Modified U7 snRNA
2.1.4. Generating the Lentiviral Vectors Encoding the Modified U7 snRNA
2.2. In Vitro Evaluation of Engineered U7 snRNA Construct in Human Myoblasts
2.2.1. Lentiviral Transduction of Human Myoblasts
2.2.2. Skipping Analysis by RT-PCR
2.2.3. Quantifying Dystrophin Expression by Western Blot
2.3. In Vivo Evaluation of AAV-U7 snRNA Vectors
2.3.1. Intramuscular Injection
2.3.2. Skipping Analysis by RT-PCR
3. Methods
3.1. Generating the Modified U7 snRNA Vectors
3.1.1. Engineering U7smOpt from wt U7 snRNA Gene
3.1.2. Engineering Modified U7 snRNA Specific to Dystrophin Exons
3.1.3. Generating AAV Vectors Encoding the Modified U7 snRNA
3.1.4. Generating the Lentiviral Vectors Encoding the Modified U7 snRNA
3.2. In Vitro Evaluation of Engineered U7 snRNA Construct in Human Myoblasts
3.2.1. Lentiviral Transduction of Human Myoblasts
3.2.2. Skipping Analysis by RT-PCR
3.2.3. Quantifying Dystrophin Expression by Western Blot
3.3. In Vivo Evaluation of AAV-U7 snRNA Vectors
3.3.1. Intramuscular Injection in the TA Muscle
3.3.2. Skipping Analysis by RT-PCR
4. Notes
References
Chapter 12: Application of MicroRNA in Cardiac and Skeletal Muscle Disease Gene Therapy
1. Introduction
2. Materials
2.1. Detecting the Expression of Muscle miRNAs by Northern Blotting Analysis
2.2. Studying the Regulation of Muscle miRNAs on Their Targets by Luciferase Reporter Assays
2.3. Overexpression and Knockdown of Muscle miRNAs In Vitro
3. Methods
3.1. Detecting the Expression of Muscle miRNAs by Northern Blotting Analysis
3.2. Studying the Regulation of Muscle miRNAs on Their Targets by Luciferase Reporter Assays
3.3. Overexpression or Knockdown of Muscle miRNAs In Vitro
4. Notes
References
Chapter 13: Molecular Imaging of RNA Interference Therapy Targeting PHD2 for Treatment of Myocardial Ischemia
1. Introduction
2. Material
2.1. Generating the shPHD2 Construct
2.2. Testing the shPHD2 ConstructIn Vitro
2.3. Evaluation of the shPHD2 Construct In Vivo
3. Methods
3.1. Generating the shPHD2 Construct
3.2. Testing the shPHD2 Construct In Vitro
3.3. Evaluationof the shPHD2 ConstructIn Vitro
3.4. Testing the shPHD2 Efficacy In Vivo
4. Notes
References
Chapter 14: Lentiviral Vector Delivery of shRNA into Cultured Primary Myogenic Cells: A Tool for Therapeutic Target Validation
1. Introduction
2. Materials
2.1. Generation of shRNA-Expressing Lentiviral Vectors
2.1.1. Selection and Cloning of shRNA Cassettes
2.1.2. Production of Self-Inactivated Recombinant Lentiviral Vectors
2.2. Isolation of Primary Satellite Cells
2.3. Evaluation of Silencing Efficiency by Quantitative RT-PCR Analysis
3. Methods
3.1. Generation of shRNA-Expressing Lentiviral Vectors
3.2. Isolation of Primary Satellite Cells
3.3. Evaluation of Silencing Efficiency by Quantitative RT-PCR Analysis
4. Notes
References
Part III:
Methods for Muscle Gene Transfer in Large Animal Models
Chapter 15: Fetal Muscle Gene Therapy/Gene Delivery in Large Animals
1. Introduction
2. Materials
2.1. Generation of Time-Mated Sheep
2.2. Confirmation of Pregnancyand Gestational Age by Ultrasound
2.3. Sheep Anesthesia
2.4. Ultrasound-Guided Procedures
2.5. Vector Dose
3. Methods
3.1. Generation of Time-Mated Sheep
3.2. Confirmation of Pregnancy and Gestational Age by Ultrasound
3.3. Sheep Anesthesia (see Note 6)
3.4. Ultrasound-Guided Procedures
3.5. Vector Dose
4. Notes
References
Chapter 16: Electroporation of Plasmid DNA to Swine Muscle
1. Introduction
2. Materials
2.1. Plasmid Preparation
2.2. Intramuscular Injection and Electroporation Procedure
3. Methods
3.1. Plasmid Preparation
3.2. Intramuscular Injection and Electroporation Procedure
4. Notes
References
Chapter 17: Local Gene Delivery and Methods to Control Immune Responses in Muscles of Normal and Dystrophic Dogs
1. Introduction
2. Materials
2.1. Immunosuppression Regimen
2.2. In Vivo Evaluation of Effectiveness of Immunosuppression
2.3. Evaluation of Cellular Immunity and Gene Expression
2.3.1. Quantifying CD3+ Cells by Flow Cytometry
2.3.2. Examine Muscle Histology by Hematoxylin and Eosin–Phloxine Staining (H&E)
2.3.3. Detection of T cells and Expression of Canine Factor IV and Dystrophin by Immunofluorescence
3. Methods
3.1. Immunosuppressive Regimens
3.2. In Vivo Evaluation of Effectiveness of Immunosuppression
3.3. Evaluation of Cellular Immunity and Gene Expression
3.3.1 Quantifying CD3+ Cells by Flow Cytometry
3.3.2. Examine Muscle Histology by Hematoxylin and Eosin–Phloxine Staining (H&E)
3.3.3. Detection of T cells and Expression of Canine Factor IV and Microdystrophin by Immunofluorescence
4. Notes
References
Chapter 18: Gene Transfer to Muscle from the Isolated Regional Circulation
1. Introduction
2. Materials
3. Methods
4. Notes
References
Chapter 19: AAV-Mediated Gene Therapy to the Isolated Limb in Rhesus Macaques
1. Introduction
2. Materials
2.1. Generation of Microdystrophin.FLAG Vector
2.2. Biotesting of Microdystrophin.FLAG Vector
2.3. IFLP to Target the Gastrocnemius of the Macaque
3. Methods
3.1. Generation
of Microdystrophin.FLAG Vector
3.2. Biotesting of Microdystrophin.FLAG Vector in Rhesus Macaques
3.3. IFLP to Target the Gastrocnemius
of the Macaque
4. Notes
References
Chapter 20: Antisense Oligo-Mediated Multiple Exon Skipping in a Dog Model of Duchenne Muscular Dystrophy
1. Introduction
2. Materials
2.1. Design of Antisense Oligos
2.2. Transfection of Antisense 2’OMePS into Dog Myoblasts
2.3. Intramuscular Injections of Antisense Oligos in Dogs
2.4. Systemic Injections of Antisense Morpholinos
2.5. RNA Extraction
2.6. RT-PCR
2.7. cDNA Sequencing
2.8. Muscle Sampling from Necropsy of Dogs
2.9. Immunostaining for Dog Muscles
2.10. Western Blotting from Dog Muscles
2.11. Clinical Grading of Dogs
3. Methods
3.1. Design of Antisense Oligos
3.2. Transfection of Antisense 2’OMePS into Dog Myoblasts
3.3. Intramuscular Injections of Antisense Oligos in Dogs
3.4. Systemic Injections of Antisense Morpholinos
3.5. RNA Extraction from Myotubes
3.6. RT-PCR
3.7. cDNA Sequencing
3.8. Muscle Sampling from Necropsy of Dogs
3.9. Immunostaining for Dog Muscles
3.10. Western Blotting from Dog Muscles
3.11. Clinical Grading of Dogs
4. Notes
References
Chapter 21: Whole Body Skeletal Muscle Transduction in Neonatal Dogs with AAV-9
1. Introduction
2. Materials
2.1. Delivering an AAV-9 AP Vector Through the Jugular Vein in Newborn Dogs
2.2. Evaluating AP Expression
3. Methods
3.1. Delivering an AAV-9 AP Vector Through the Jugular Vein in Newborn Dogs
3.2. Evaluating AP Expression
4. Notes
References
Chapter 22: A Translatable, Closed Recirculation System for AAV6 Vector-Mediated Myocardial Gene Delivery in the Large Animal
1. Introduction
2. Materials
2.1. Vector
2.2. Preoperative Care and Preparation
2.3. Cardiopulmonary Bypass with Isolated Cardiac Circuit
2.4. Intramyocardial Injection
2.5. Postoperative Care
2.6. Euthanasia, Necropsy, and Tissue Harvest
2.7. PCR: Tissue Isolation and Polymerase Chain Reaction Sequence
2.8. Western Blotting
3. Methods
3.1. Vector Design and Production
3.2. Preoperative Care and Preparation
3.3. Cardiopulmonary Bypass with Isolated Cardiac Circuit
3.4. Intramyocardial Injection
3.5. Postoperative Care
3.6. Euthanasia, Necropsy, and Tissue Harvest
3.7. PCR: Tissue Isolation and Polymerase Chain Reaction Sequence
3.8. Western Blotting
4. Notes
References
Chapter 23: Method of Gene Delivery in Large Animal Modelsof Cardiovascular Diseases
1. Introduction
1.1. Antegrade Injection
1.2. Retrograde Injection
1.3. Direct Injection
1.4. Pericardial Delivery
2. Materials
2.1. Antegrade Injection
2.2. Retrograde Injection
2.3. Surgical Direct Injection
2.4. Percutaneous Direct Injection
2.5. Surgical Pericardial Delivery
2.6. Percutaneous Pericardial Delivery
3. Methods
3.1. Antegrade Injection
3.2. Retrograde Injection
3.3. Surgical Direct Injection
3.4. Percutaneous Direct Injection
3.5. Surgical Pericardial Delivery (Surgically and Percutaneously)
3.6. Percutaneous Pericardial Delivery
4. Notes
References
Chapter 24: Percutaneous Transendocardial Delivery of Self-Complementary Adeno-Associated Virus 6 in the Canine
1. Introduction
2. Materials
2.1. Cardiac Catheterization and Vector Delivery
2.2. Assessment of Cardiac Gene Transfer Efficiency
3. Methods
3.1. Cardiac Catheterization and Vector Delivery
3.2. Assessment of Cardiac Gene Transfer Efficiency
4. Notes
References
Index
Nội dung
[...]... failure Gene therapy for muscle diseases such as DMD requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles This can be achieved by the use of regulatory cassettes composed of enhancers and promoters that contain combinations of muscle- specific and ubiquitous Dongsheng Duan (ed.), Muscle Gene Therapy: ... Microdystrophin Gene for Duchenne Muscular Dystrophy Gene Therapy Takis Athanasopoulos, Helen Foster, Keith Foster, and George Dickson Abstract Duchenne muscular dystrophy (DMD) is a severe muscle wasting X-linked genetic disease caused by dystrophin gene mutations Gene replacement therapy aims to transfer a functional full-length dystrophin cDNA or a quasi micro/mini -gene into the muscle A number... Skeletal and Cardiac Muscles Charis L Himeda, Xiaolan Chen, and Stephen D Hauschka Abstract Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with musclespecific... partially characterized The muscle creatine kinase (MCK) gene has served as a useful model of muscle- specific gene transcription since its protein product is specifically and abundantly expressed in striated muscle, and its regulatory regions have been extensively characterized MCK is also expressed at different levels in different anatomical skeletal muscles, and in skeletal vs cardiac muscle (3, 4) This allows... 2 1 S D (1996) Analysis of muscle creatine kinase gene regulatory elements in skeletal and cardiac muscles of transgenic mice Mol Cell Biol 16, 1649–1658 Shield, M A., Haugen, H S., Clegg, C H., and Hauschka, S D (1996) E-box sites and a proximal regulatory region of the muscle creatine kinase gene differentially regulate expression in diverse skeletal muscles and cardiac muscle of transgenic mice Mol... constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expression in cardiac muscle, and in... expression of a reporter gene (13) (see Note 4) 8 Himeda, Chen, and Hauschka 2 Multimerize enhancers Multimerizing a single enhancer such as that from the MCK gene can provide significant increases in cassette activity (see Note 5) Tissue- or cell-type-specific enhancers from other striated muscle genes (such as the a-myosin heavy chain gene and the cardiac troponin T [cTnT] gene) can also be added... important for expression in particular muscle types In order to boost expression, new elements can be introduced Introduce additional positive control elements by site-directed mutagenesis (nucleotide mutation or insertion) These elements include general muscle elements (A/T-rich/MEF2, CArG/SRF, MCAT/TEF-1, MEF3/Six, NFAT), skeletal muscle elements (E-box), slow/ fast skeletal muscle elements (SURE/FIRE regions... activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types Key words: Skeletal muscle, Cardiac muscle, Regulatory cassette, Muscular dystrophy, Gene therapy, Transcriptional regulation, Muscle creatine kinase 1 ntroduction I Duchenne muscular dystrophy... analyses of the muscle creatine kinase enhancer Trex control element in skeletal and cardiac muscle indicate differences in gene expression between muscle types Transgenic Res 12, 337–349 7 Bischoff, R (1989) Analysis of muscle regen1 eration using single myofibers in culture Med Sci Sports Exerc 21, S164–S172 8 Bischoff, R (1990) Interaction between satel1 lite cells and skeletal muscle fibers Development . incurable. Compared to retinal gene therapy, muscle gene therapy faces a number of unique challenges. Muscle is one of the most abundant tissues in the body. An effective therapy will require systemic. in mind, we compiled this collection of muscle gene therapy methods and protocols with the intention of bridging the translational gap in muscle gene therapy. The book is divided into three. survival, and physiology assays of skeletal muscle and heart function. Technology breakthroughs are the driving force in muscle gene therapy. Early muscle gene transfer studies were largely performed