Main 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.

Contents:

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 Cel ls

Florian Barthelemy, Sebastien Courrier, Nicolas Levy, 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 Fc RI 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