This fully updated edition presents a collection of protocols reflecting the latest advancements in exon skipping and inclusion strategies. The book explores the design of antisense oligonucleotide therapies, in vitro and in vivo evaluation of exon skipping in Duchenne muscular dystrophy, enhancing exon skipping efficiency, as well as methods involving spinal muscular atrophy and other diseases. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

Authoritative and comprehensive, Exon Skipping and Inclusion Therapies: Methods and Protocols, Second Edition serves as a valuable resource for scientists working to refine RNA-targeted therapeutics and translate them into clinical applications.

Includes cutting-edge techniques Provides step-by-step detail essential for reproducible results Contains key implementation advice from the experts

Inhalt

Progress and Future Directions in Exon Skipping and Inclusion Therapies: The Landscape of Oligonucleotide-Based Genetic Medicine.- Evolution and Breakthroughs in Exon Skipping and Splice Modulation: From Inception to Clinical Success.- An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases.- Milasen: The Emerging Era of Patient-Customized N-of-1 Antisense Oligonucleotides as Therapeutic Agents for Genetic Diseases.- Tips to Design Effective Splice-Switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion.- In Silico Prediction and Selection of Exon-Skipping Antisense Oligonucleotide Sequences Using eSkip-Finder.- Quantitative Evaluation of Exon Skipping in Immortalized Muscle Cells In Vitro.- Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-Mediated Exon Skipping and Exons 45-55 Skipping Accompanied by Rescue of Dystrophin Expression.- In Vitro Multiexon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient.- Creation of DMD Muscle Cell Model Using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-Mediated Exon Skipping.- In Vitro Evaluation of Exon Skipping in Disease Specific iPSC-Derived Myocytes.- Skipping of Duplicated Dystrophin Exons: In Vitro Induction and Assessment.- In Vivo Evaluation of Dystrophin Exon Skipping in mdx Mice.- Guide to Selection of Muscle-Homing Peptides after In Vivo Phage Display Biopanning.- Systemic Injection of Peptide-PMOs into Humanized DMD Mice and Detection by RT-PCR and ELISA.- In Vivo Evaluation of Single- and Multi-Exon Skipping in mdx52 Mice.- A Novel Zebrafish Model for Assessing In Vivo Delivery of Morpholino Oligomers.- Electrophysiological Evaluation in mdx52 Mouse Brain after Antisense-Mediated Exon 51 or 53 Skipping.- Use of Glucose/Fructose to Enhance the Exon Skipping Efficacy.- Systemic Intravenous Administration of Antisense Therapeutics for Combinatorial Dystrophin and Myostatin Exon Splice Modulation.- The Assembly of Fluorescently Labeled Peptide-Oligonucleotide Conjugates.- In Vivo Evaluation of Multiple Exon Skipping with Peptide-PMOs in Cardiac and Skeletal Muscles in Dystrophic Dogs.- Use of Tricyclo-DNA Antisense Oligonucleotides for Exon Skipping.- Optimization of 2,4-BNA/LNA-Based Oligonucleotides for Splicing Modulation In Vitro.- Pre-mRNA Splicing Modulation by Antisense Oligonucleotides.- In Vitro Evaluation of Antisense-Mediated Exon Inclusion for Spinal Muscular Atrophy.- Systemic Injection of Antisense Oligos into Spinal Muscular Atrophy (SMA) Mice and Evaluation.- Morpholino-Mediated Exon Inclusion for Spinal Muscular Atrophy (SMA).- Exon Skipping by Ultrasound-Enhanced Delivery of Morpholino with Bubble Liposomes for Myotonic Dystrophy Model Mice.- Restoration of Dysferlin after Exon 32 Skipping in Patient Cells.- Morpholino-Mediated Exon Skipping Targeting Human ACVR1/ALK2 for Fibrodysplasia Ossificans Progressiva.- Exon Skipping of FcRI for Allergic Diseases.- Exon-Skipping Using Antisense Oligonucleotides for Laminin-Alpha2-Deficient Muscular Dystrophy.