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
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
Table of ContentsPart 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, Mª Francisca Coutinho, and Sandra Alves
6. Conjugation of Nucleic Acids and Drugs to Gold Nanoparticles
Paula Milán-Rois, Ciro Rodriguez-Diaz, Milagros Castellanos, and Álvaro 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-Castañeda, 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 Suárez-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
Arístides López-Márquez, Ainhoa Martínez-Pizarro, Belén Pérez, 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, Rubén Artero, and Estefanía Cerro-Herreros
14. Evaluation of Exon Skipping and Dystrophin Restoration in In Vitro Models of Duchenne Muscular Dystrophy
Andrea López-Martínez, Patricia Soblechero-Martín, 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 Vázquez-Domínguez 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
Lucía Echevarría 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 Sørensen, and Bård Smedsrød
Part VI: Intellectual Property
28. Patent Considerations When Embarking on New Antisense Drug Programs
Laurence D.S. Gainey
