Description
This book presents a comprehensive collection of detailed state-of-the-art exon skipping and splices modulation protocols. Chapters detail 14 genetic diseases, AON-mediated therapies, and CRISPR/Cas9-mediated gene editing therapies. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, Exon Skipping and Inclusion Therapies: Methods and Protocols aims to help researchers initiate the development of next-generation therapies. Part I: Basics and Introductions 1. Invention and Early History of Exon Skipping and Splice Modulation Kenji Rowel Q. Lim and Toshifumi Yokota 2. An Overview of Recent Advances and Clinical Applications of Exon Skipping and Splice Modulation for Muscular Dystrophy and Various Genetic Diseases Merryl Rodrigues and Toshifumi Yokota 3. Recent Advances and Clinical Applications of Exon Inclusion for Spinal Muscular Atrophy Hae-Won Son and Toshifumi Yokota 4. Nusinersen in the Treatment of Spinal Muscular Atrophy Kara Goodkey, Tejal Aslesh, Rika Maruyama, and Toshifumi Yokota Part II: Design of AONs and Strategy 5. Tips to Design Effective Splice-switching Antisense Oligonucleotides for Exon Skipping and Exon Inclusion Rika Maruyama and Toshifumi Yokota 6. Antisense Oligonucleotide Targeting of 3’UTR of mRNA for Expression Knockdown Golnoush Golshirazi, Lukasz Ciszewski, Ngoc Lu-Nguyen, and Linda Popplewell Part III: In Vitro Evaluation of Exon Skipping for DMD 7. Quantitative Evaluation of Exon Skipping in Immortalized Muscle Cells In Vitro Kenji Rowel Q. Lim and Toshifumi Yokota 8. Direct Reprogramming of Human DMD Fibroblasts into Myotubes for In Vitro Evaluation of Antisense-m Exon Skipping and Exons 45-55 Skipping Accompanied by Rescue of Dystrophin Expression Joshua JA Lee, Takashi Saito, William Duddy, Shin’ichi Takeda, and Toshifumi Yokota 9. In vitro Multi-exon Skipping by Antisense PMOs in Dystrophic Dog and Exon 7-Deleted DMD Patient Akinori Nakamura, Yoshitsugu Aoki, Maria Tsoumpra, Toshifumi Yokota, and Shin’ichi Takeda 10. Creation of DMD Muscle Cell Model using CRISPR-Cas9 Genome Editing to Test the Efficacy of Antisense-mediated Exon Skipping Rika Maruyama and Toshifumi Yokota 11. In vitro Evaluation of Exon Skipping in Disease Specific iPSC-derived Myocytes Mingming Zhao, Emi Shoji, and Hidetoshi Sakurai 12. Restoration of Dystrophin Protein Expression by Exon Skipping utilizing CRISPR-Cas9 in Myoblasts Derived from DMD Patient iPS Cells Masataka Ifuku, Kumiko A. Iwabuchi, Masami Tanaka, Mandy Siu Yu Lung, and Akitsu Hotta 13. Skipping of Duplicated Dystrophin Exons: in vitro Induction and Assessment Kane Greer, Sue Fletcher, and Steve D Wilton Part IV: In Vivo Evaluation of Exon Skipping for DMD 14. In Vivo Evaluation of Dystrophin Exon Skipping in mdx Mice Bo Wu, Mingxing Wang, Sapana Shah, and Qi Long Lu 15. Exon 51 Skipping Quantification by Digital Droplet PCR in del52hDMD/mdx Mice Monika Hiller, Pietro Spitali, Nicole Datson, and Annemieke Aartsma-Rus 16. Systemic Injection of Peptide-PMOs into Humanized DMD Mice and Detection by RT-PCR and ELISA Dyanna Melo, Rika Maruyama, and Toshifumi Yokota 17. In vivo Evaluation of Single- and Multi-exon Skipping in mdx52 Mice Yoshitaka Mizobe, Shouta Miyatake, Hotake Takizawa, Yuko Hara, Toshifumi Yokota, Akinori Nakamura, Shin’ichi Takeda, and Yoshitsugu Aoki 18. A Novel Zebrafish Model for Assessing In Vivo Delivery of Morpholino Oligomers Joseph Kim, Karl Clark, Carrie Barton, Robert Tanguay, and Hong Moulton Part V: Enhancing the Efficacy 19. Validation and Detection of Exon Skipping Boosters in DMD Patient Cell Models and mdx Mouse Barthelemy. Florian, Wang. Dereck, Nelson. Stanley F., Miceli, M. Carrie 20. Use of Glucose/Fructose to Enhance the Exon Skipping Efficacy Gang Han, Xianjun Gao, and HaiFang Yin 21. Systemic Intravenous Administration of Antisense Therapeutics for Combinatorial Dystrophin and Myostatin Exon Splice Modulation Ngoc Lu-Nguyen, George Dickson, and Alberto Malerba 22. The Assembly of Fluorescently Labeled Peptide-oligonucleotide Conjugates via Orthogonal Ligation Strategies John Karas, Bradley J. Turner, and Fazel Shabanpoor 23. In vivo Evaluation of Multiple Exon Skipping with Peptide-PMOs in Cardiac and Skeletal Muscles in Dystrophic Dogs Rika Maruyama, Yoshitsugu Aoki, Shin’ichi Takeda, and Toshifumi Yokota 24. Use of Tricyclo-DNA Antisense Oligonucleotides for Exon Skipping Karima Relizani and Aurelie Goyenvalle 25. Optimization of 2′,4′-BNA/LNA-based Oligonucleotides For Splicing Modulation in vitro Takenori Shimo and Satoshi Obika Part VI: Exon Inclusion for SMA 26. Pre-Mrna Splicing Modulation by Antisense Oligonucleotides Natalia N. Singh, Diou Luo, and Ravindra N. Singh 27. In vitro Evaluation of Antisense-mediated Exon Inclusion for Spinal Muscular Atrophy Aleksander Touznik, Rika Maruyama, and Toshifumi Yokota 28. Systemic Injection of Antisense oligos into SMA Mice and Evaluation Tejal Aslesh, Rika Maruyama, and Toshifumi Yokota Part VII: Exon Skipping and Splice Modulation for Various Diseases 29. Exon Skipping using Antisense Oligonucleotides for Laminin-alpha2-deficient Muscular Dystrophy Yuko Hara, Yoshitaka Mizobe, Shouta Miyatake, Hotake Takizawa, Tetsuya Nagata, Toshifumi Yokota, Shin’ichi Takeda, and Yoshitsugu Aoki 30. Exon Skipping by Ultrasound-enhanced Delivery of Morpholino with Bubble Liposomes for Myotonic Dystrophy Model Mice Yoichi Negishi, Yoko Endo-Takahashi, and Shoichi Ishiura 31. Dysferlin Exon 32 Skipping in Patient Cells Florian Barthlmy, Sbastien Courrier, Nicolas Lvy, Martin Krahn, and Marc Bartoli 32. Morpholino-mediated Exon Skipping Targeting Human ACVR1/ALK2 for Fibrodysplasia Ossificans Progressiva Rika Maruyama and Toshifumi Yokota 33. Exon Skipping of FceRI for Allergic Diseases Greer K. Arthur and Glenn Cruse 34. Antisense Oligonucleotide Design and Evaluation of Splice-modulating Properties Using Cell-based Assays Ralph Slijkerman, Hannie Kremer, and Erwin van Wijk 35. Antisense-mediated Splice Modulation to Reframe Transcripts Matthias Titeux, Sandrina Turczynski, Nathalie Pironon, and Alain Hovnanian 36. Morpholino-mediated Exon Inclusion for SMA Haiyan Zhou and Francesco Muntoni
