Copyright â Springer - 2015
Chapter 1: Adeno-Associated Vectors for Gene Delivery to the Nervous System
1.2 Viral Capsid Design Considerations for AAV Production
1.2.1 Choice of AAV Serotypes
1.3.1 Packaging Large Genes
1.3.2 Self-ưComplementary AAV
1.3.3 Long-Term Expression
1.4 Production, Purification, and Titration of AAV
3.1 Growing and Amplification of Insert and Capsid Plasmids
3.2 DNA Preparations from Bacteria Cultures
3.3 Larger Preparations for Viral Production
3.4 Determining Plasmid Concentration by Spectrophotometry
3.5 Transfection Using Calcium Phosphate Precipitation
3.6 Preparation of Cell Lysate for Ultracentrifugation in Iodixanol Gradient
3.7 Polyethyenimine (PEI) Transfection
3.8 AAV Purification by Iodixanol Density Gradient
3.9 Concentration and Desalting of AAV Preparations
3.10 Analysis of Vector Purity and Identity by SDS-PAGE and Western Blotting
3.11 Determination of rAAV Titer by Dot-Blot Hybridization Assay
3.11.3 Preparation of Dot Blots
3.11.4 Membrane Hybridization Using ECL Nucleic Acid Direct Labeling and Detection System
3.11.5 Calculation of Titers
3.12 Titration of AAV by Real-Time PCR
3.12.1 Sample Preparation for Real-Time PCR
3.12.2 Taqman PCR Reaction
Chapter 2: Lentiviral Vectors for Gene Delivery to the Nervous System
1.2 Ex Vivo and In Vivo Gene Delivery
1.3 Ideal Gene Delivery Vehicle
1.4 Lentiviral Vectors in Gene Therapy
1.7 Cell Type-ưSpecific Targeting
1.8 Lentiviral Vectors and Gene Therapy
3.1 Preparation and Quantification of Plasmids Required for Viral Production
3.1.1 Plasmid DNA Extraction/Purification
3.1.2 Determination of DNA Quality and Concentration by Spectrophotometry
3.1.3 Supercoiled DNA gel Quantification by Densitometry
3.1.4 Restriction Enzyme Analysis of DNA Samples
3.2 Lentivirus Production
3.2.1 Propagation, Passaging, and Preservation of Packaging Cell Line HEK 293T
Thawing the HEK 293T Cells
3.2.2 Transfecting HEK 293T Cells
3.2.3 Concentrating Viral Supernatants
3.2.4 Small-Scale Production of Lentiviral Vectors (Single Dish Transfection)
3.2.5 Labeling Lentiviral Particles with Lipophilic Dyes
3.3 Titration of Lentiviral Vectors
3.3.1 FACS Based Titration
3.3.2 Titration by p24 ELISA
3.3.3 Titration by qRT-PCR
Assay to Determine Relative Vector Particle Numbers Based on Virion RNA (One-Step qRT-PCR)
Quantitative PCR Assay to Determine the Number of Vector Copies Associated with Genomic DNA Extracted from Transduced Cells
3.3.4 Comparison of Titration Methods
3.4 General Considerations of In Vitro Gene Transduction
3.4.1 Transduction Procedure
3.5 In Vivo Application of Lentiviral Vectors
3.5.1 Intrastriatal Delivery of Lentiviral Vectors
3.5.2 Intramuscular Delivery of Lentiviral Vectors
Chapter 3: Gene Therapy for Parkinson’s Disease: AAV5-Mediated Delivery of Glial Cell Line-Derived Neurotrophic Factor (GDNF)
1.1 GDNF as a Dopaminergic Neurotrophic Factor
2.4 Analysis of Transgene Expression
Chapter 4: Gene Delivery and Gene Therapy for Alzheimer’s Disease
1.1 Background: Adeno-ưAssociated Virus
1.2 Strategies to Increase Efficiency and Specificity of Gene Transfer: AAV Vectorology
1.3 Advancements in rAAV Production
1.4 Gene Delivery to the Central Nervous System
1.5 Animal Models of Alzheimer’s Disease
1.6 Assessing the Outcomes of AD Gene Therapy: Behavioral Testing and Neurophysiology
1.7 Current AAV Therapies for Alzheimer’s Disease in Animal Studies and Clinical Trials
3.1 Generation of rAAV Vectors
3.1.1 Subcloning of Genome of Interest into pAAV2-MCS-WPRE
3.2 Generation and Purification of Recombinant AAV
3.2.1 Transfection into AAV-293 Cells to Generate Hybrid rAAV
3.2.2 Crude AAV Particle Extraction
3.2.3 Iodixanol Gradient Purification (See Note 3)
3.2.4 HiTrap Q HP Column Chromatography and Concentration (See Note 5)
Calculation of Physical Titer (Viral Particles/ml)
Real-Time qPCR for Quantification of rAAV Genome Copy Number
3.3 Delivery of Virus to Mouse Brain In Vivo
3.3.1 Stereotaxic Coordinates
3.3.2 Anesthesia and Brain Microinjections
Aseptic Procedures and Anesthesia
3.3.3 Intracranial Injection
3.4 Bromo-deoxyuridine (BrdU) Administration to Measure Neurogenesis
3.5 Behavioral Testing Using Radial Arm Water Maze
3.5.1 Maze Construction and Setup
3.5.2 Testing Animals in the Maze
3.6 Analysis of Brain Tissues
3.6.1 Histological Analysis of Gene Expression, Neurogenesis, and Amyloid Pathology with Immunofluorescence
3.6.2 Analysis of Neurogenesis
3.6.3 Analysis of Amyloid Pathology
Chapter 5: Gene Therapy for Huntington’s Disease
1.1.1 Nerve Growth Factor
1.1.2 Brain-Derived Neurotrophic Factor
1.1.3 Ciliary Neurotrophic Factor
1.1.4 Glial Cell Line-Derived Neurotrophic Factor
1.3 RNA Interference Gene Silencing
1.3.2 Short Interfering RNAs
1.3.4 Allele-Specific Targeting
1.5 Adeno-ưAssociated Viral Vectors: Tools for HD Gene Therapy Research
2.1 Cell Culture and Transfection
2.2 Purification of rAAV Vectors
2.2.2 Heparin Column Purification
2.2.3 Iodixanol Purification
2.3 Titration of rAAV Vectors
3.1 rAAV Vector Packaging
3.1.1 HEK293 Cell Culture
3.1.2 Transfection of HEK293 Cells
3.1.3 Purification of rAAV Vectors: Heparin Column Purification for Chimeric AAV1:2 Vectors
3.1.4 Iodixanol Purification Method for All AAV Serotypes
3.2 SDS-PAGE to Assess Purity of Viral Vector Stocks
3.3 Titration of rAAV Vectors
3.3.1 rAAV Vector Genomic DNA Preparation
3.3.2 Quantitative Real-Time PCR
3.4 Stereotaxic Delivery of Vectors into the Rodent Brain
3.5.1 Spontaneous Locomotor Activity in an Open Field
3.5.3 Rotarod Performance
Chapter 6: Gene Therapy Approaches to Promoting Axonal Regeneration After Spinal Cord Injury
1.1 Delivery of Neurotrophic Factors
1.2 Manipulating Transcription Factors
1.3 Blocking Inhibitory Molecules
1.4 Manipulating Cell Adhesion Molecules
1.5 Targeting PTEN/mTOR Pathway
1.6 Combinatorial Treatments
3.1 Production of Lentiviral Vectors
3.2 Gene Delivery to Red Nucleus in a Rubrospinal Tract Transection Model
3.2.1 Assembling UltraMicroPump and Hamilton Syringe
3.2.2 Surgical Procedure for Injection of Viral Vectors
3.2.3 Transection of Rubrospinal Tract
3.2.5 Immunohistocheưmistry and Quantification of RST Axons
3.3 Gene Delivery to the Spinal Cord in a Dorsal Hemisection Model
3.3.1 Surgical Procedure for Dorsal Hemisection
3.3.2 Injection of the Viral Vector into Spinal Cord
3.3.3 Injection of Retrograde Tracers to the Sciatic Nerve
3.3.4 Immunohistocheưmistry and Quantification of Regenerating Axons
3.4 Transplantation of Genetically Modified Schwann Cells in a Corticospinal Tract Injury Model
3.4.1 Culture and Transduction of Schwann Cells
3.4.2 Dorsal Hemisection, Transplantation of Schwann Cells, and Injection of Viral Vectors
3.4.3 Behavioral Assessment for Motor Function
3.4.4 Injection of Anterograde Tracer
3.4.5 Immunohistocheưmistry
Chapter 7: Gene Delivery to Neurons of the Dorsal Root Ganglia Using Adeno-Associated Viral Vectors
2.1 Gene Delivery to DRG Neurons
2.2 Direct Injection of DRG with AAV
2.3 Method A. Direct Injection of AAV into L4/L5 DRG
2.3.1 Assembly of the Stereotactic Frame
2.3.3 Loading the Viral Vector
2.3.4 Injecting the Viral Vector
2.4 Intrathecal Delivery of Viral Vector
2.5 Method B. Intrathecal AAV Delivery Targeting the Lumbar DRG Using a Catheter
2.5.2 Insertion of the Catheter
2.5.3 Risks of Intrathecal Injections
2.6 Comparison of the Two Methods
Chapter 8: Targeted Gene Therapy for Ischemic Stroke
2.1 Hypoxia-ưInducible Gene Expression AAV Vector Construction
2.2 Permanent Distal Middle Cerebral Artery Occlusion (pMCAO) Model and Viral Vector Injection
2.2.1 Assorted Surgical Instruments
2.4 Tissue Stains and Morphometry
2.4.1 β-gal Staining (Frozen Cryostat Sections)
2.4.2 Cresyl Violet Staining
3.1 Hypoxia-ưInducible Gene Expression AAV Vector
3.3 Intravenous Injection of AAV Vectors (See Note 4)
3.3.1 Tail Vein Injection (Fig. 3a, See Note 5)
3.3.2 Jugular Vein Injection
3.4 Behavioral Tests (See Note 6)
3.4.2 Adhesive Removal Test
3.5 Tissue Stains and Morphometry
3.5.1 Assay Transgene Expression (X-gal Staining, Fig. 3b)
3.5.2 Assay Infarct/Atrophic Volume (Cresyl Violet Staining)
Chapter 9: Adeno-Associated Viral Gene Therapy for Retinal Disorders
2 Strategies for Vector Design
2.4 Self-ưComplementary Vectors
3 Gene Therapy of Large Genes Using AAV
4 Virus Production: Methodology
4.1 Cloning and Transformation
4.1.1 Transformation Protocol
4.3 Transfecting Cells for Virus Production
4.4 Harvesting and Lysing Cells
4.5 Virus Purification by Iodixanol Gradient
4.7 Virus Titer Using qPCR
4.8 Site-Directed Mutagenesis to Form Mutant Capsid Variants
5 Testing AAV Vectors In Vitro
5.2 Ex Vivo Retinal Culture
6 Testing AAV Vectors In Vivo
6.1 Intraocular Injections
6.2 Confocal Scanning Laser Ophthalmoscopy
6.3 Tissue Collection and Processing
Chapter 10: Gene Therapy for Epilepsies
2.1 AAV Vector Production
2.2 Genomic Titering of AAV Vectors
2.3 Rodent Stereotaxic Neurosurgery
3.1 AAV Vector Production
3.2 Genomic Titering of rAAV
3.3 Rodent Stereotaxic Neurosurgery
3.4.1 Kainic Acid Model: Intrahippocampal Administration
3.4.2 Kainic Acid Model: Intracerebroventricular Administration
3.4.3 Kainic Acid Model: Systemic Administration
4.1 AAV Vector Production
4.2 Genomic Titering for AAV Vector
4.3 Rodent Stereotaxic Neurosurgery
Chapter 11: AAV Gene Therapy Strategies for Lysosomal Storage Disorders with Central Nervous System Involvement
2 Infusion of AAV Vectors into the Mouse Brain
2.1 Stereotaxic Injection into Adult Mouse Brain
2.1.1 Materials and Reagents
2.2 Injection of AAV Vectors into the Cerebral Lateral Ventricles of Neonatal Mice
2.2.1 Material and Reagents
3 Infusion of AAV Vector into the Brain of Large Animal Models of LSDs
3.1 Materials and Reagents
3.2 Stereotaxic Injection of AAV Vectors into the Brain of Feline Models of Gangliosidoses
3.3 Stereotaxic Injection of AAV Vectors into the Brain of Tay–Sachs Disease Sheep
4 Infusion of AAV Vectors into the Brains of Nonhuman Primates
4.1 Stereotaxic Injection of AAV Vectors into the Brain of a Nonhuman Primate
Chapter 12: Gene Therapy in Spinal Muscular Atrophy (SMA) Models Using Intracerebroventricular Injection into Neonatal Mice
1.1 Molecular Genetics of SMA
1.1.2 SMN Protein and Function
1.1.4 Therapeutic Interventions in SMA
1.1.5 SMA Gene Replacement Therapy
Time Point of Application
1.2 Summary of Our Research in SMA Gene Therapy
1.2.1 Development of ssAAV2 Vectors
1.2.2 Development of scAAV9 Vectors
1.3 ICV Injection into the Mouse Brain at Early Postnatal Days
3.1 ICV Injection into the Mouse Brain at Early Postnatal Days
3.1.1 Preparation of the Needles for ICV Injection
3.1.2 Preparation of the Injection Stock Solution
3.1.3 Loading the Injection Solution into the Glass Micropipette
3.1.5 Verifying a Successful ICV Injection
Chapter 13: Gene Therapy for Chronic Pain: How to Manipulate and Unravel Pain Control Circuits from the Brain?
2.2 Stereotaxic Injection and Immunodetection of Transduced Neurons
2.3 Effects of HSV-1-ưMediated Gene Delivery
3.1 Vector Construction and Production
3.1.1 Transfection for Generation of Recombinant Virus
3.1.2 Titration of the Recombinant Virus
3.1.3 Limiting Dilution for Virus Purification
3.1.4 Virus Stock Preparation with Sucrose Gradient Purification
3.2 Stereotaxic Injection of HSV-1 Vectors and Immunodetection of Transduced Neurons
3.2.1 Stereotaxic Injections
3.2.2 Immunodetection of Transduced Neurons
Immunodetection of β-Galactosidase
Double Immunodetection of β-Galactosidase and Tyrosine Hydroxylase
3.3 Effects of HSV-1-ưMediated Gene Delivery
3.3.1 Nociceptive Behavioral Effects
Neuropathic Pain Induction and Behavioral Assessment
Reversal of Behavioral Effects
3.3.2 Neurochemical Effects
Chapter 14: Gene Therapy Approaches Using Reproducible and Fully Penetrant Lentivirus-Mediated Endogenous Glioma Models
3.1 Lentiviral Vector Production
3.2 Generation of Glioma Models: Intracranial Lentiviral Injection in Rats
3.3 Testing of a Conditional Cytotoxic Gene Therapy, i.e., Herpes Simplex Virus Type I Thymidine Kinase (HSV1-TK), Encoded Within a First-ưGeneration Adenovirus Vector (Ad-TK) in Combination with Systemic Delivery of Ganciclovir (GCV)
4.1 Lentiviral Vector Production
4.2 Glioma Models: Intracranial Lentiviral Injection in Rats
4.3 Testing of a Conditional Cytotoxic Gene Therapy, i.e., Herpes Simplex Virus Type I Thymidine Kinase (HSV1-TK) Encoded Within a First-ưGeneration Adenovirus Vector (Ad-TK) in Combination with Systemic Delivery of Ganciclovir (GCV) in the LV-Induced GB
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