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 2ʹ,4ʹ-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 Barthélémy, Sébastien Courrier, Nicolas Lévy, 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 FcεRIβ 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
