Muscular Dystrophy Therapeutics: Methods and Protocols

Preface
Contents
Contributors
Part I: Basics and Introduction
	Chapter 1: Current Strategies of Muscular Dystrophy Therapeutics: An Overview
		1 Introduction
		2 Antisense Therapy
			2.1 Exon Skipping
			2.2 More Steric Blocking Approaches
			2.3 Gapmers
			2.4 Other Oligonucleotide-Based Therapies
		3 CRISPR Genome Editing
		4 Gene Replacement
		5 Cell Therapy
		6 Small Molecules
			6.1 Nonsense Suppression
			6.2 Disease Modification
		7 Conclusions
		References
	Chapter 2: Viltolarsen: From Preclinical Studies to FDA Approval
		1 Introduction
			1.1 Mutations in the DMD Gene
			1.2 Currently Approved Therapies for DMD
		2 Viltolarsen: An Overview
			2.1 Preclinical Studies
			2.2 Timeline of Drug Development
		3 Safety Profile
		4 Pharmacokinetics
		5 Efficacy of Viltolarsen
		6 Therapeutic Benefits of Viltolarsen
		7 Clinical Trials
			7.1 Phase I Clinical Trial: NCT02081625
			7.2 Phase I/II Clinical Trial: JapicCTI-163291
			7.3 Phase II Clinical Trial: NCT02740972
			7.4 Phase II Extension Study: NCT03167255
		8 Challenges
		9 Conclusion
		References
Part II: Animal Models, Sample Preparation, and Assessment
	Chapter 3: Rapid Freezing of Skeletal and Cardiac Muscles Using Isopentane Cooled with Liquid Nitrogen and Tragacanth Gum for ...
		1 Introduction
		2 Materials
			2.1 Harvesting and Processing Skeletal Muscle Tissue Samples
			2.2 Mounting Sample on Disc Corks
			2.3 Rapid Freezing Using Isopentane
			2.4 Cryosectioning the Muscle Samples
		3 Methods
			3.1 Harvesting and Processing Skeletal Muscle Tissue Samples
			3.2 Mounting Sample on Disc Corks
			3.3 Rapid Freezing Using Isopentane
			3.4 Cryosectioning the Muscle Samples
		4 Notes
		References
	Chapter 4: Cardiac and Skeletal Muscle Pathology in the D2/mdx Mouse Model and Caveats Associated with the Quantification of U...
		1 Introduction
			1.1 Considerations for Using the D2/mdx Mouse Model for Assessing Heart Pathology
				1.1.1 Phenotypic Shift in Cardiac Dysfunction
			1.2 Underlying D2/WT Pathology
			1.3 Considerations for Assessing Skeletal Muscle Pathology in D2/mdx Mice
				1.3.1 BL10/mdx Mouse vs D2/mdx Mouse Phenotypes
			1.4 Assessing Utrophin in a Lower Regenerative Context
		2 Materials
			2.1 Sample Preparation
			2.2 Histology
				2.2.1 H&E Staining
				2.2.2 Alizarin Red Staining
				2.2.3 Immunofluorescence Staining
		3 Methods
			3.1 Functional Outcomes
			3.2 Sample Collection and Processing
				3.2.1 Sample Preparation
				3.2.2 Cryosectioning
			3.3 Pathohistological Assessment
				3.3.1 H&E Staining of OCT-Embedded Muscle Sections
				3.3.2 Calcification Assessments of OCT-Embedded Muscle Sections
				3.3.3 Fibrosis Assessments in OCT-Embedded Muscle Sections
				3.3.4 Utrophin Assessments in Dystrophic Muscle
				3.3.5 Immunofluorescence
		4 Notes
		References
	Chapter 5: Physiological Assessment of Muscle, Heart, and Whole Body Function in the Canine Model of Duchenne Muscular Dystrop...
		1 Introduction
		2 Materials
			2.1 Evaluation of Bodywide Motor Function
				2.1.1 Overnight Activity Recording (See Fig. 1)
				2.1.2 Gait Analysis
			2.2 Evaluation of Skeletal Muscle Function
				2.2.1 Single Muscle Force Measurement (See Fig. 3a)
				2.2.2 Hindlimb Muscle Torque Measurement (See Fig. 4A)
			2.3 Evaluation of Heart Function
				2.3.1 Electrocardiography (ECG) (See Fig. 6)
				2.3.2 Holter Monitoring (See Fig. 7)
				2.3.3 Echocardiography (See Fig. 8)
		3 Methods
			3.1 Evaluation of Bodywide Motor Function
				3.1.1 Overnight Activity Recording
				3.1.2 Gait Analysis
			3.2 Muscle Force Assessment
				3.2.1 Single Muscle Force Measurement
				3.2.2 Hindlimb Torque Measurement
			3.3 Heart Function Assessment
				3.3.1 ECG
				3.3.2 Holter Monitoring
				3.3.3 Echocardiography
		4 Notes
		References
Part III: Antisense Oligonucleotides and Gene Replacement Therapies
	Chapter 6: Restoring Dystrophin Expression by Skipping Exons 6 and 8 in Neonatal Dystrophic Dogs
		1 Introduction
		2 Materials
			2.1 Animal Model
			2.2 Designing the Antisense Morpholinos
			2.3 Injecting the Morpholinos
			2.4 Clinical Grading (Behavior and Gait Test)
			2.5 Muscle Sampling/Necropsy
			2.6 Hematoxylin and Eosin (H&E) Staining
			2.7 RNA Extraction and RT-PCR
			2.8 Protein Extraction and Western Blot
			2.9 Immunohistochemistry
			2.10 ELISA
		3 Methods
			3.1 Designing the Antisense Morpholinos
			3.2 Injecting the Morpholino Drugs
			3.3 Behavior and Gait Test
			3.4 Muscle Sampling/Necropsy
			3.5 Hematoxylin and Eosin (H&E) Staining
			3.6 RNA Extraction and RT-PCR
			3.7 Protein Extraction and Western Blot
			3.8 Immunohistochemistry
			3.9 ELISA
		4 Notes
		References
	Chapter 7: Restoring Dystrophin Expression with Exon 44 and 53 Skipping in the DMD Gene in Immortalized Myotubes
		1 Introduction
		2 Materials
			2.1 Immortalized Skeletal Muscle Cell Culture
			2.2 PMO-Based Antisense Oligonucleotides
			2.3 RT-PCR for Evaluating Exon-Skipping Efficiency
			2.4 Western Blotting for Quantifying Dystrophin Rescue
		3 Methods
			3.1 Culture of Immortalized Healthy or Patient-Derived Skeletal Muscle Cells
			3.2 PMO Transfection
			3.3 Total RNA Extraction from the Immortalized Cell-Derived Myotubes
			3.4 RT-PCR and Evaluation of Exon-Skipping Efficiency
			3.5 Preparation of Protein Samples from Immortalized Myotubes
			3.6 SDS-PAGE and Western Blotting for Quantifying Dystrophin Rescue
		4 Notes
		References
	Chapter 8: Restoring Dystrophin Expression with Duchenne Muscular Dystrophy Exon 45 Skipping in Induced Pluripotent Stem Cell-...
		1 Introduction
		2 Materials
			2.1 iPSC Culture
			2.2 Cardiomyocytes Differentiation
			2.3 Transfection of ASOs for Exon 45 Skipping
			2.4 RT-PCR for Evaluation of Exon-Skipping Efficiency
			2.5 Western Blotting
			2.6 Immunocytochemistry
		3 Methods
			3.1 Preparation of iPSCs
			3.2 Differentiation into Cardiomyocytes and Their Enrichment
			3.3 Exon 45 Skipping for DMD iPSC-CMs Using Antisense Nucleic Acid
			3.4 Evaluation of Exon-Skipping Efficiency by RT-PCR
			3.5 Western Blot Analysis of Dystrophin Expression After Exon Skipping
			3.6 Immunofluorescence Study of Cultured Cells
		4 Notes
		References
	Chapter 9: Quantitative Evaluation of Exon Skipping in Urine-Derived Cells for Duchenne Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 Isolation and Primary Culture of UDCs
			2.2 Retroviral Construct
			2.3 Infection with MYOD1-Retroviral Vector in UDCs
			2.4 Myogenic Differentiation of MYOD1-Induced UDCs Treated with 3-Deazaneplanocin A Hydrochloride (DZNep)
			2.5 Transfection of ASO into MYOD1-Induced UDCs
			2.6 Evaluation of Exon Skipping Efficiency by RT-PCR
			2.7 Detection of Dystrophin by Western Blotting
			2.8 Detection of Dystrophin by Immunocytochemistry
		3 Methods
			3.1 Isolation and Primary Culture of UDCs (See Note 1 and Fig. 1)
			3.2 Retroviral Construct
				3.2.1 Insertion of MYOD1 into Retroviral Vector
				3.2.2 Production of Retrovirus
			3.3 Infection with MYOD1-Retroviral Vector in UDCs (See Fig. 2)
			3.4 Myogenic Differentiation of MYOD1-Induced UDCs Treated with 3-Deazaneplanocin A Hydrochloride (DZNep) (See Fig. 2)
			3.5 Transfection of ASO into MYOD1-Induced UDCs (See Fig. 2)
			3.6 Evaluation of Exon Skipping Efficiency by RT-PCR
			3.7 Detection of Dystrophin by Western Blotting
			3.8 Detection of Dystrophin by Immunocytochemistry
		4 Notes
		References
	Chapter 10: Use of Glycine to Augment Exon Skipping and Cell Therapies for Duchenne Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 HEK 293T Cell Culture
			2.2 Oligonucleotides
			2.3 Chemicals for Mechanistic Studies
			2.4 Glycine
			2.5 Animal Experiments
			2.6 Immunohistochemical Staining for Dystrophin, eMyHC, and Laminin
			2.7 Immunohistochemical Staining for FITC-Labeled PMO
			2.8 RNA Extraction and RT-PCR
			2.9 Preparation of Protein Samples
			2.10 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			2.11 Western Blotting for Dystrophin Protein
			2.12 Immunohistochemical Staining for Dystrophin-Associated Protein Complex (DAPC)
			2.13 Co-immunohistochemical Staining for PAX7 and Ki67
			2.14 Tissue Distribution
			2.15 Histology
			2.16 Isolation of Primary Myoblasts
			2.17 Myoblast Transplantation in mdx Mice
			2.18 Generation of RagB Knockout HEK293T Cells
		3 Methods
			3.1 In Vivo Experiments
				3.1.1 Local Intramuscular Injection
				3.1.2 Local Intramuscular Injection for Exon-Skipping Frequency Studies
				3.1.3 Local Intramuscular Injection for Mechanistic Studies
				3.1.4 Systemic Intravenous Injection
			3.2 Immunohistochemical Staining for Dystrophin, eMyHC, and Laminin
			3.3 Detection of FITC-Labeled PMO in Muscle Tissues
			3.4 RNA Extraction and RT-PCR
			3.5 Preparation of Protein Samples
			3.6 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
			3.7 Western Blotting for Dystrophin Protein
			3.8 Immunohistochemistry for Dystrophin-Associated Protein Complex (DAPC)
			3.9 Co-Immunohistochemical Staining for PAX7 andKi67
			3.10 Tissue Distribution
			3.11 Histology
			3.12 Isolation of Primary Myoblasts
			3.13 Myoblast Transplantation in mdx Mice
			3.14 Generation of RagB Knockout HEK293T Cells
		4 Notes
		References
	Chapter 11: Morpholino-Mediated Exons 28-29 Skipping of Dysferlin and Characterization of Multiexon-skipped Dysferlin using RT...
		1 Introduction
		2 Materials
			2.1 Cell Cultures and Morpholino Transfections
			2.2 One Step RT-PCR and cDNA Sequencing
			2.3 Protein Extraction and Dysferlin Immunoblotting Analysis
			2.4 Two-Photon Laser Wounding Assay
		3 Methods
			3.1 Cell Cultures and Morpholino Transfections
			3.2 One Step RT-PCR and cDNA Sequencing
			3.3 Protein Extraction and Dysferlin Immunoblotting Analysis
			3.4 Two-Photon Laser Wounding Assay
		4 Notes
		References
	Chapter 12: Knocking Down DUX4 in Immortalized Facioscapulohumeral Muscular Dystrophy Patient-Derived Muscle Cells
		1 Introduction
		2 Materials
			2.1 Cell Culture and Transfection of Gapmers
			2.2 RNA Extraction, cDNA Synthesis, and qPCR Analysis of Transcript Knockdown
			2.3 Immunocytochemistry and Analysis of Muscle Cell Phenotypes
		3 Methods
			3.1 Cell Culture and Transfection of Gapmers
			3.2 RNA Extraction, cDNA Synthesis and qPCR Analysis of Transcript Knockdown
			3.3 Immunocytochemistry and Analysis of Muscle Cell Phenotypes
		4 Notes
		References
	Chapter 13: Peptide-Conjugated PMOs for the Treatment of Myotonic Dystrophy
		1 Introduction
		2 Materials
			2.1 Preparation of Peptide-PMO Conjugates
			2.2 Measuring P-PMO Efficacy In Vitro: Fluorescent In Situ Hybridization (FISH) of RNA Foci in DM1 Myotubes
				2.2.1 Sterile Culture of Control and DM1 Myotubes
				2.2.2 Myotube Fixation
				2.2.3 Fluorescent In Situ Hybridization
				2.2.4 Slide Preparation
			2.3 Preparation and Intravenous Administration of P-PMO to Adult Mice
			2.4 Measuring P-PMO Toxicity in HSA-LR Mice
				2.4.1 Evaluating Toxicity Markers in HSA-LR Mice Urine
					Collection of Urine Using Metabolic Cages
					Urinary KIM-1 Quantification
				2.4.2 Evaluating Toxicity Markers in HSA-LR Mice Serum
				2.4.3 Collection and Preparation of Liver and Kidney Tissues for Histopathological Analysis
			2.5 Measuring PMO Tissue Concentration Using an Enzyme-Linked Immunosorbent Assay (ELISA)
				2.5.1 Tissue Homogenization
				2.5.2 Probes and PMO Preparation
				2.5.3 Performing the ELISA
				2.5.4 Analyzing PMO Concentration
			2.6 Measuring P-PMO Activity In Vitro And In Vivo: Analyzing Splicing Correction
				2.6.1 RT-PCR of P-PMO-Treated Human Cells or Mouse Tissue
				2.6.2 Gel Electrophoresis and Splicing Analysis
		3 Methods
			3.1 Preparation of Peptide-PMO Conjugates
				3.1.1 Synthesis of Peptides
				3.1.2 Synthesis of Peptide-PMO Conjugates on a 1.5-μmol Scale (See Note 2)
			3.2 Measuring P-PMO Efficacy In Vitro: Fluorescent In Situ Hybridization (FISH) of DM1 Myotubes RNA Foci
				3.2.1 Sterile Cell Culture of Control and DM1 Myotubes (See Note 7)
				3.2.2 Cell Fixation
				3.2.3 Fluorescent In Situ Hybridization (FISH)
				3.2.4 Slide Preparation
			3.3 Preparation and Intravenous Administration of P-PMO to Adult Mice
			3.4 Measuring P-PMO Toxicity in HSA-LR Mice
				3.4.1 Evaluating Toxicity Markers in HSA-LR Mice Urine
					Collection of Urine Using Metabolic Cages
					Urinary KIM-1 Quantification
				3.4.2 Evaluating Toxicity Markers in HSA-LR Mouse Serum
				3.4.3 Collection and Preparation of Liver and Kidney Tissues for Histopathological Analysis
			3.5 Measuring PMO Tissue Concentration Using ELISA
				3.5.1 Tissue Homogenization
				3.5.2 Preparation of Probes and PMOs
				3.5.3 Performing the ELISA (See Note 25 and Fig. 2)
				3.5.4 Analyzing PMO Concentration
			3.6 Measuring P-PMO Activity In Vitro and In Vivo: Analyzing Splicing Correction
				3.6.1 RT-PCR of P-PMO-Treated Human Cells or Mouse Tissue
				3.6.2 Gel Electrophoresis and Splicing Analysis
		4 Notes
		References
	Chapter 14: Developing Therapeutic Splice-Correcting Antisense Oligomers for Adult-Onset Pompe Disease with c.-32-13T>G Mutati...
		1 Introduction
		2 Materials
			2.1 Cell Lines, Culture Reagents, and PMO Delivery Reagents
			2.2 GAA Activity and Expression
			2.3 RNA Isolation, RT-PCR, and RT-qPCR
		3 Methods
			3.1 Cell Culture
			3.2 Nucleofection of PMOs into Fibroblasts
			3.3 Nucleofection with PMOs: Myogenic Lineages
			3.4 Cell Collection for GAA Activity and Expression
			3.5 Cell Lysis for GAA Activity Assay and Expression
			3.6 GAA Activity Assay
			3.7 GAA Expression by Western Blotting
			3.8 RNA Isolation and Analysis
		4 Notes
		References
Part IV: Gene Replacement Therapies
	Chapter 15: Molecular and Biochemical Assessment of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 Nucleic Acid and Protein Extraction
				2.1.1 DNA Extraction
				2.1.2 RNA Extraction (Fig. 1)
				2.1.3 Protein Extraction (Fig. 2)
			2.2 Complementary DNA (cDNA) Generation
			2.3 Nucleic Acid and Protein Quantification
				2.3.1 DNA Quantification
				2.3.2 RNA Quantification
				2.3.3 cDNA Quantification
				2.3.4 Protein Quantification
			2.4 Quantitative Real-Time Polymerase Chain Reaction (qPCR) (Fig. 4)
			2.5 Droplet Digital PCR (ddPCR) (Fig. 5)
			2.6 Western Blot
		3 Methods
			3.1 Nucleic Acid and Protein Extraction
				3.1.1 DNA Extraction
				3.1.2 RNA Extraction
				3.1.3 Protein Extraction
			3.2 Complementary DNA (cDNA) Generation
			3.3 Nucleic Acid and Protein Quantification
				3.3.1 DNA Quantification
				3.3.2 RNA Quantification
				3.3.3 cDNA Quantification
				3.3.4 Protein Quantification
			3.4 Quantitative Real-Time Polymerase Chain Reaction (qPCR)
			3.5 Droplet Digital PCR (ddPCR)
			3.6 Western Blot
		4 Notes
		References
	Chapter 16: Histological Assessment of Gene Therapy in the Canine DMD Model
		1 Introduction
		2 Materials
			2.1 Tissue Collection
				2.1.1 Needle Biopsy (Fig. 2)
				2.1.2 Necropsy (Fig. 4)
			2.2 Tissue Processing
				2.2.1 Snap Freezing Tissue in Optimal Cutting Temperature (OCT) Media (Fig. 5a)
				2.2.2 Snap Freezing Tissue in Liquid Nitrogen
				2.2.3 Tissue Storage in RNAlater Stabilization Solution
			2.3 Histological Staining
				2.3.1 Hematoxylin and Eosin (H&E) Staining
				2.3.2 Masson´s Trichrome (MTC) Staining (Fig. 6)
				2.3.3 Alizarin Red Staining
				2.3.4 nNOS Activity Staining
				2.3.5 Immunofluorescence Staining
				2.3.6 Immunohistochemical Staining
		3 Methods
			3.1 Tissue Collection
				3.1.1 Needle Biopsy
				3.1.2 Necropsy
			3.2 Tissue Processing
				3.2.1 Snap Freezing Tissue in Optimal Cutting Temperature (OCT) Media
				3.2.2 Snap Freezing Tissue in Liquid Nitrogen
				3.2.3 Tissue Storage in RNAlater Stabilization Solution
			3.3 Histological Staining
				3.3.1 Hematoxylin and Eosin (H&E) Staining
				3.3.2 Masson´s Trichrome (MTC) Staining
				3.3.3 Alizarin Red Staining
				3.3.4 nNOS Activity Staining
				3.3.5 Immunofluorescence Staining
				3.3.6 Immunohistochemical Staining
		4 Notes
		References
	Chapter 17: MRI Evaluation of Gene Therapy in the Canine Model of Duchenne Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 Sedation and Anesthesia Procedure (Fig. 1)
			2.2 Positioning the Dog for MRI Imaging (Fig. 2)
			2.3 MRI Imaging of the Heart and Skeletal Muscle
			2.4 Data Analysis
		3 Methods
			3.1 Sedation and Anesthesia Protocol
			3.2 Positioning the Dog for MRI Imaging
			3.3 MRI Imaging of the Heart and Skeletal Muscle
			3.4 Animal Recovery Following MRI Imaging
			3.5 Data Analysis
		4 Notes
		References
	Chapter 18: Assessment of the Gene Therapy Immune Response in the Canine Muscular Dystrophy Model
		1 Introduction
		2 Materials
			2.1 Blood Drawing (Fig. 1A)
			2.2 Blood Sample Processing (Fig. 2A)
			2.3 Multiplex Luminex Cytokine Detection Assay (Fig. 3)
			2.4 Enzyme-Linked Immunosorbent (ELISA) Assay
			2.5 Enzyme-Linked Immuno Absorbent Spot (ELISpot) Assay
		3 Methods
			3.1 Blood Drawing
			3.2 Blood Sample Processing
				3.2.1 Serum Separation
				3.2.2 Plasma Separation
				3.2.3 PBMC Isolation
			3.3 Multiplex Luminex Cytokine Detection Assay
			3.4 Enzyme-Linked Immunosorbent (ELISA) Assay
				3.4.1 ELISA Protocol to Detect Cytokine and Chemokine
				3.4.2 ELISA Protocol to Detect Anti-Cas9 Total IgG in Canine Serum
			3.5 Enzyme-Linked Immuno Absorbent Spot (ELISpot) Assay
		4 Notes
		References
	Chapter 19: Use of Mesenchymal Stem Cells to Enhance the Efficacy of Gene Therapy
		1 Introduction
		2 Materials
			2.1 AAV Preparation
				2.1.1 AAV Production
			2.2 Animal Preparation (Intravenous Injection)
			2.3 Assessment After In Vivo Gene Transduction
				2.3.1 IHC
				2.3.2 PBMC + AAV Re-stimulation
		3 Methods
			3.1 AAV Preparation, Production, and Purification
			3.2 AAV and MSCs Administration Schedule (See Fig. 1)
			3.3 AAV and MSCs Injection to Dogs
				3.3.1 AAV Preparation for Injection
				3.3.2 MSCs Preparation for Injection
				3.3.3 MSCs with AAV Solution for Injection
				3.3.4 Preparation of Dogs and Administration of Vector and/or Cell Solution
			3.4 Assessment After In Vivo Gene Transduction
				3.4.1 Immunohistochemical Staining
				3.4.2 T Cell Responses
		4 Notes
		References
	Chapter 20: Exon-Skipping for a Pathogenic COL6A1 Variant in Ullrich Congenital Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 ASO Design
			2.2 ASO Preparation
			2.3 Fibroblast Culture
			2.4 Fibroblast Transfection
			2.5 RNA Extraction and cDNA Synthesis
			2.6 Polymerase Chain Reaction (PCR) and Gel Electrophoresis
			2.7 Quantitative Reverse Transcription PCR (qRT-PCR)
			2.8 Protein Extraction and Western Blotting
			2.9 Immunofluorescence Staining for Extracellular COL6
		3 Methods
			3.1 ASO Design
			3.2 ASO Preparation
			3.3 Establishment of Primary Skin Fibroblast Culture
			3.4 Subculture of Skin Fibroblasts
			3.5 Preparation of Fibroblasts for Transfection
			3.6 Transfection of Fibroblasts with Non-PMO ASOs (2′-OMe and 2′-MOE) (See Note 8)
			3.7 Transfection of Fibroblasts with PMOs (See Note 10)
			3.8 RNA Extraction and cDNA Synthesis
			3.9 Polymerase Chain Reaction (PCR) and Gel Electrophoresis
			3.10 Real-Time Quantitative PCR
			3.11 Protein Extraction and Western Blotting
			3.12 Immunofluorescence Staining for Extracellular COL6
		4 Notes
		References
Part V: Genome Editing/CRISPR
	Chapter 21: CRISPR-Cas9 Correction of Duchenne Muscular Dystrophy in Mice by a Self-Complementary AAV Delivery System
		1 Introduction
		2 Materials
			2.1 Reagents for Cloning CRISPR sgRNAs into scAAV Plasmid
			2.2 Reagents for Injecting AAV CRISPR-Cas9 into DMD Mice
			2.3 Reagents for Skeletal and Cardiac Muscle Dystrophin Western Blot Analysis
			2.4 Reagents for Skeletal and Cardiac Muscle Dystrophin Immunostaining
		3 Methods
			3.1 Generating scAAV with Multiplexed CRISPR sgRNAs
				3.1.1 Ordering and Annealing CRISPR sgRNA Oligonucleotides
				3.1.2 Golden Gate Assembly of CRISPR sgRNAs into Donor Plasmids
				3.1.3 Golden Gate Assembly of CRISPR sgRNAs into scAAV Vector
			3.2 In Vivo CRISPR-Cas9 Gene Editing of DMD Mice Using scAAV
			3.3 Quantification of Dystrophin Restoration in DMD Mice After Systemic AAV CRISPR Gene Editing
				3.3.1 Skeletal and Cardiac Muscle Dystrophin Western Blot Analysis
				3.3.2 Skeletal and Cardiac Muscle Dystrophin Immunostaining
		4 Notes
		References
	Chapter 22: Preparation of NanoMEDIC Extracellular Vesicles to Deliver CRISPR-Cas9 Ribonucleoproteins for Genomic Exon Skipping
		1 Introduction
			1.1 CRISPR-Cas9 Genome Editing Therapy
			1.2 Duchenne Muscular Dystrophy (DMD)
			1.3 In Vivo Delivery Approaches
			1.4 Extracellular Vesicle (EV)-Based Delivery Technology
			1.5 NanoMEDIC System
		2 Materials
			2.1 Construction of CRISPR-gRNA Vectors for NanoMEDIC
			2.2 Small-Scale Transfection by Lipofection
			2.3 Large-Scale Transfection by Flow Electroporation
			2.4 Concentration by Centrifugation
			2.5 Inoculation
			2.6 Assessing Genome Editing Efficiency by Sanger Sequencing and TIDE/ICE Analysis
		3 Methods
			3.1 Designing CRISPR-gRNA for NanoMEDIC Delivery
			3.2 Construction of NanoMEDIC gRNA Vector (Fig. 2)
			3.3 Lipofection Method for NanoMEDIC Production
			3.4 Large-Scale Suspension Culture and Flow Electroporation Method
			3.5 Inoculation of NanoMEDIC
			3.6 Assessing Genome Editing Efficiency by Sanger Sequencing and TIDE/ICE Analysis (Fig. 4)
		4 Notes
			4.1 CRISPR Genome Editing Target
			4.2 Condition of Producer Cells
			4.3 Transfection (Quality of Plasmid)
			4.4 Alternative Concentration Methods
			4.5 Storage of NanoMEDIC
			4.6 In Vivo Application
			4.7 Other
		References
	Chapter 23: Restoration of Dystrophin Expression in Mdx-Derived Muscle Progenitor Cells Using CRISPR/Cas9 System and Homology-...
		1 Introduction
		2 Materials
		3 Methods
			3.1 Isolation of Skin Fibroblasts from Adult Dmd mdx Mice
			3.2 Generation of iPSCs from Dmdmdx Skin Fibroblasts
			3.3 Design and Construction of Guide Sequences to Completely Delete Exon 23 of DMD Gene [4]
			3.4 Construction of Homologous-Directed Recombination (HDR) Targeting Vector System
			3.5 Generation of Stable Cell Lines with DMD-Exon 23 Deletion
			3.6 Differentiation of Mouse iPS Cells into Myogenic Progenitor Cells (MPC) by Lenti-TRE-VP64-MyoD-T2A-dsRed Vector
			3.7 MPC Transplantation
			3.8 Immunofluorescent Tissue Staining to Detect Dystrophin Expression in Dmdmdx Mouse Model
		4 Notes
		References
Part VI: Small Molecules
	Chapter 24: Effects of Glucocorticoids in Murine Models of Duchenne and Limb-Girdle Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 Injectable Glucocorticoid Solution
			2.2 Hydroxyproline Dosing Solutions
			2.3 Grip and Treadmill Assays
			2.4 Force-Frequency Analysis in Tibialis Anterior Muscles
			2.5 Measurement of Body Weight and Glycemia
		3 Methods
			3.1 Glucocorticoid Regimens
			3.2 Quantitation of Anti-fibrotic Effects Through Hydroxyproline Dosing
			3.3 Quantitation of Regimen-Specific Effects on Grip Strength and Treadmill Performance
			3.4 Quantitation of Regimen-Specific Effects on Muscle Force
			3.5 Quantitation of Regimen-Specific Effects on Glycemia and Weight
		4 Notes
		References
	Chapter 25: High-Throughput Screening to Identify Modulators of Sarcospan
		1 Introduction
		2 Materials
			2.1 Molecular Cloning of Sarcospan Reporter Plasmids
			2.2 Generation of Stable Reporter Cell Lines
			2.3 High-Throughput Screening
			2.4 Secondary Screening
			2.5 Culture of Dystrophin-Deficient Myoblasts for Hit Validation
			2.6 Validation of Gene and Protein Expression
			2.7 Membrane Stability Assay
		3 Methods
			3.1 Molecular Cloning of SSPN Reporter Plasmids
			3.2 Generation of Stable Reporter Cell Lines
			3.3 High-Throughput Screening
			3.4 Secondary Screening
			3.5 Culture of Dystrophin-Deficient Myoblasts for Hit Validation
				3.5.1 Gene Expression Analysis
				3.5.2 Culture of Dystrophin-Deficient Human Myoblasts
			3.6 Validation of Gene and Protein Expression
				3.6.1 Gene Expression Analysis
				3.6.2 Immunoblotting
				3.6.3 ELISA to Quantify SSPN Protein Levels
			3.7 Membrane Stability Assay
		4 Notes
		References
	Chapter 26: Identifying FDA-Approved Drugs that Upregulate Utrophin A as a Therapeutic Strategy for Duchenne Muscular Dystrophy
		1 Introduction
		2 Materials
			2.1 In-Cell ELISA High-Throughput Drug Screen
			2.2 Confirmation Western Blotting
			2.3 FDA-Approved Drug Treatment in Dystrophic Mice
				2.3.1 Drug Treatment in Mice
				2.3.2 Mice Muscle Dissection and Protein Extraction
				2.3.3 Immunofluorescence
		3 Methods
			3.1 Drug Identification by an In-Cell ELISA High-Throughput FDA-Approved Drug Screen
			3.2 Confirming Drug-Induced Utrophin A Upregulation by Western Blotting
			3.3 FDA-Approved Drug Treatment in Dystrophic Mice
				3.3.1 Mdx and Wild-Type Mice Drug Treatment
				3.3.2 Mdx and Wild-Type Mice Dissections
				3.3.3 Muscle Tissue Protein Extractions
				3.3.4 Utrophin A Expression at the Muscle Fiber Sarcolemma
		4 Notes
		References
Part VII: Cell Models and Stem Cells
	Chapter 27: Monitoring Plasma Membrane Injury-Triggered Endocytosis at Single-Cell and Single-Vesicle Resolution
		1 Introduction
		2 Materials
			2.1 Cell Culture
			2.2 Live-Cell Imaging
		3 Method
			3.1 Muscle Cell Culture
			3.2 Bulk Membrane Endocytosis Studies
			3.3 Monitoring Caveolar Endosome Dynamics
			3.4 Quantification of Bulk Membrane Endocytosis
			3.5 Quantification of Single-Vesicle (Caveolae) Endocytosis
		4 Notes
		References
	Chapter 28: Evaluation of hiPSC-Derived Muscle Progenitor Cell Transplantation in a Mouse Duchenne Muscular Dystrophy Model
		1 Introduction
		2 Materials
			2.1 Dissociation and Cell Sorting
			2.2 Cryo-Injury and Transplantation
			2.3 TA Muscle Sampling and Histology
		3 Methods
			3.1 Dissociation and Cell Sorting
			3.2 Transplantation
			3.3 Euthanization and Histology
			3.4 Immunostaining and Analysis
		4 Notes
		References
	Chapter 29: Quantification of Muscle Satellite Stem Cell Divisions by High-Content Analysis
		1 Introduction
		2 Materials
			2.1 General Equipment
			2.2 Surgical Tools
			2.3 Transgenic Mouse Lines
			2.4 Plastic and Glassware for FDB Myofiber Isolation and Culture
			2.5 FDB Myofiber Isolation and Culture Reagents
			2.6 Small-Molecule Compound Reagents
			2.7 Immunofluorescence Reagents
		3 Methods
			3.1 Dissection of FDB Muscle
			3.2 Isolation of FDB Myofibers
			3.3 FDB Culture and Small-Molecule Plate Design
			3.4 Fixation and Immunostaining of FDB Myofibers
			3.5 Programming Cellomics Arrayscan for Satellite Cell Detection
			3.6 Programming MATLAB-Based Script for Satellite Cell Couplet Detection
			3.7 High-Content Screen Analysis
		4 Notes
		Funding
		References
Part VIII: Antibody
	Chapter 30: Systemic Delivery of a Monoclonal Antibody to Immunologically Block Myostatin in the A17 Mouse Model of OPMD
		1 Introduction
		2 Materials
			2.1 RK35 Antibody
			2.2 Animals
			2.3 Genotyping of A17.1 Mice
			2.4 Intraperitoneal Injection
			2.5 Postmortem Tissue Harvesting and Processing
			2.6 Immunohistochemistry Staining and Histological Analyses
			2.7 ELISA for Myostatin Expression
		3 Methods
			3.1 Genotyping of Mice (from Ear Notches)
			3.2 Intraperitoneal Injection
			3.3 Functional Tests
			3.4 Postmortem Tissue Processing
			3.5 Immunostaining for PABPN1, Laminin, Collagen VI
			3.6 Picrosirius Red Staining
			3.7 Analyses of Histological and Immunofluorescence Samples
			3.8 ELISA for Myostatin Expression
		4 Notes
		References
Index