Gapmers Methods and Protocols

This volume presents a comprehensive collection of detailed state-of-the-art protocols for gapmer-mediated RNA knockdown from leaders in the field. Chapters detail a historical and contemporary perspective of RNase H-inducing antisense oligonucleotides called gapmers and gapmer-mediated RNA knockdown for basic research and 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, Gapmers: Methods and Protocols aims to provide diverse applications of gapmers along with protocols that will assist readers in moving the frontier.

Part I: Basics and Introduction

1. Invention and Early History

Kenji Rowel Q. Lim and Toshifumi Yokota

2. Development and Clinical Applications of Antisense Oligonucleotide Gapmers

Leanna Chan and Toshifumi Yokota

3. Knocking Down Long Non-coding RNAs using Antisense Oligonucleotide Gapmers

4. Development of Antisense Oligonucleotide Gapmers for the Treatment of Huntington’s Disease

Tejal Aslesh and Toshifumi Yokota

5. Development of Antisense Oligonucleotide Gapmers for the Treatment of Dyslipidemia and Lipodystrophy

Tejal Aslesh and Toshifumi Yokota

6. Inotersen for the Treatment of Hereditary Transthyretin Amyloidosis

7. Degradation of Toxic RNA in Myotonic Dystrophy Using Gapmer Antisense Oligonucleotides

Quynh Nguyen and Toshifumi Yokota

Part II: Design of Gapmers and Strategy

8. DNA/RNA Heteroduplex Oligonucleotide for Highly Efficient Gene Silencing

Rintaro Iwata Hara, Kotaro Yoshioka, and Takanori Yokota

9. Tips for Successful lncRNA Knockdown using Gapmers

Kim A Lennox and Mark A Behlke

10. Calcium-Mediated In Vitro Transfection Technique of Oligonucleotides with Broad Chemical Modification Compatibility

Fumito Wada, Shin-ichiro Hori, Satoshi Obika, and Tsuyoshi Yamamoto

11. Evaluating the Knockdown Activity of MALAT1 ENA Gapmers in vitro

Shinzo Iwashita, Takao Shoji, and Makoto Koizumi

12. Albumin-binding Fatty Acid-modified Gapmer Antisense Oligonucleotides for Modulation of Pharmacokinetics

Yunpeng Cai, Chenguang Lou, Jesper Wengel, and Kenneth A. Howard

Part III: In Vitro/In Vivo Evaluation of Gapmers and Antisense Oligonucleotides

13. The use of Gapmers for in vivo Suppression of Hepatic mRNA Targets

14. Development of LNA Gapmer Oligonucleotide-Based Therapy for ALS/ FTD Caused by the C9orf72 Repeat Expansion

Chaitra Sathyaprakash, Raquel Manzano, Miguel A. Varela, Yasumasa Hashimoto, Matthew J. A. Wood, Kevin Talbot, and Yoshitsugu Aoki

15. Targeted Gene Silencing in Malignant Hematolymphoid Cells using GapmeR

16. Gapmer Antisense Oligonucleotides to Selectively Suppress the Mutant allele in COL6A Genes in Sominant Ullrich Congenital Muscular Dystrophy

Part IV: Detection of Gapmers

17. Detection of Locked Nucleic Acid Gapmers from Mouse Samples using ELISA

Kenji Rowel Q. Lim, Quynh Nguyen, and Toshifumi Yokota