Main description:

This open access volume gathers a variety of models, delivery systems, and approaches that can be used to assess RNA technology for exploiting antisense as a therapeutic intervention. Beginning with a section on the design of antisense technology and their delivery, the book continues by covering model systems developed to evaluate efficacy, both in vivo and in vitro, as well as methods to evaluate preclinically the toxicity associated with these new potential drugs, and intellectual property considerations. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Antisense RNA Design, Delivery, and Analysis provides basic knowledge and a large collection of methods to facilitate the work of newcomers to this vibrant and expanding field.
This book was conceived thanks to the network DARTER (Delivery of Antisense RNA Therapeutics). DARTER is funded by the EU Cooperation of Science and Technology (COST), which aims to enhance interaction and collaborations between researchers in Europe and other countries.

Contents:

Part I: Overview

1. Introduction and History of the Chemistry of Nucleic Acids Therapeutics

Michael J. Gait and Sudhir Agrawal

2. Antisense RNA Therapeutics: A Brief Overview

Virginia Arechavala-Gomeza and Alejandro Garanto

Part II: Design and Formulation of Antisense Technology

3. Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion

Haiyan Zhou

4. Design and Delivery of SINEUP: A New Modular Tool to Increase Protein Translation

Michele Arnoldi, Giulia Zarantonello, Stefano Espinoza, Stefano Gustincich, Francesca Di Leva, and Marta Biagioli

5. How to Design U1 snRNA Molecules for Splicing Rescue

Liliana Matos, Juliana I. Santos, M Francisca Coutinho, and Sandra Alves

6. Conjugation of Nucleic Acids and Drugs to Gold Nanoparticles

Paula Milan-Rois, Ciro Rodriguez-Diaz, Milagros Castellanos, and Alvaro Somoza

7. Determination of Optimum Ratio of Cationic Polymers and Small Interfering RNA with Agarose Gel Retardation Assay

Omer Aydin, Dilek Kanarya, Ummugulsum Yilmaz, and Cansu Umran Tunc

8. Generation of Protein-Phosphorodiamidate Morpholino Oligomer Conjugates for Efficient Cellular Delivery via Anthrax Protective Antigen

Valentina Palacio-Castaneda, Roland Brock, and Wouter P.R. Verdurmen

Part III: In Vitro Model Systems

9. Development and Use of Cellular Systems to Assess and Correct Splicing Defects

Nuria Suarez-Herrera, Tomasz Z. Tomkiewicz, Alejandro Garanto, and Rob W.J. Collin

10. Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells

Aristides Lopez-Marquez, Ainhoa Martinez-Pizarro, Belen Perez, Eva Richard, and Lourdes R. Desviat

11. In Vitro Models for the Evaluation of Antisense Oligonucleotides in Skin

Jeroen Bremer and Peter C. van den Akker

12. In Vitro Delivery of PMOs in Myoblasts by Electroporation

Remko Goossens and Annemieke Aartsma-Rus

13. Rapid Determination of MBNL1 Protein Levels by Quantitative Dot Blot for Evaluation of Antisense Oligonucleotides in Myotonic Dystrophy Myoblasts

Nerea Moreno-Cervera, Irene Gonzalez-Martinez, Ruben Artero, and Estefania Cerro-Herreros

14. Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy

Andrea Lopez-Martinez, Patricia Soblechero-Martin, and Virginia Arechavala-Gomeza

15. Generation of Human iPSC-Derived Myotubes to Investigate RNA-Based Therapies In Vitro

Pablo Herrero-Hernandez, Atze J. Bergsma, and W.W.M. Pim Pijnappel

16. Eye on a Dish Models to Evaluate Splicing Modulation

Kwan-Leong Hau, Amelia Lane, Rosellina Guarascio, and Michael E. Cheetham

17. Establishment of In Vitro Brain Models for AON Delivery

Elena Daoutsali and Ronald A.M. Buijsen

Part IV: In Vivo Model Systems

18. Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides

Irene Vazquez-Dominguez and Alejandro Garanto

19. Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modulation Therapies

Renske Schellens, Erik de Vrieze, Ralph Slijkerman, Hannie Kremer, and Erwin van Wijk

20. Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment

Ngoc Lu-Nguyen, Alberto Malerba, and Linda Popplewell

21. In Vivo Models for the Evaluation of Antisense Oligonucleotides in Skin

Jeroen Bremer and Peter C. van den Akker

22. Delivery of Antisense Oligonucleotides to the Mouse Retina

Alejandro Garanto

23. Delivery of Antisense Oligonucleotides to the Mouse Brain by Intracerebroventricular Injections

Tom Metz, Elsa C. Kuijper, and Willeke M.C. van Roon-Mom

24. Intrathecal Delivery of Therapeutic Oligonucleotides for Potent Modulation of Gene Expression in the Central Nervous System

Zachary Kennedy, James W. Gilbert, and Bruno M.D.C. Godinho

Part V: Safety and Toxicology

25. Preclinical Safety Assessment of Therapeutic Oligonucleotides

Patrik Andersson

26. Preclinical Evaluation of the Renal Toxicity of Oligonucleotide Therapeutics in Mice

Lucia Echevarria and Aurelie Goyenvalle

27. Protocol for Isolation and Culture of Mouse Hepatocytes (HCs), Kupffer Cells (KCs), and Liver Sinusoidal Endothelial Cells (LSECs) in Analyses of Hepatic Drug Distribution

Kjetil Elvevold, Ingelin Kyrrestad, Karen Kristine Sorensen, and Bard Smedsrod

Part VI: Intellectual Property

28. Patent Considerations When Embarking on New Antisense Drug Programs

Laurence D.S. Gainey