Contents

• Preface
• Acknowledgments
• Contributors
• I. Overview
  • 1. Introduction and History of the Chemistry of Nucleic Acids Therapeutics
    Michael J. Gait and Sudhir Agrawal.
    Published online: February 25, 2022.
    • 1. Introduction to Synthetic Antisense Oligonucleotides and siRNA
    • 2. Oligonucleotide Synthesis
    • 3. Synthetic Oligonucleotide Analogues in Antisense and siRNA
    • 4. RNase H Active Gapmer Chemistry for Use as Drugs
    • 5. siRNA Chemistry for Use as Drugs
    • 6. Immune Responses to Nucleic Acids
    • 7. Conjugates and Delivery
    • 8. Further Developments in Therapeutics
    • 9. Summary
    • Acknowledgments
    • References
  • 2. Antisense RNA Therapeutics: A Brief Overview
    Virginia Arechavala-Gomeza and Alejandro Garanto.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Mechanisms of Action
    • 3. Therapeutic Potential
    • 4. Future of AON Trials and Personalized Medicine: n = 1 Trials?
    • 5. Hurdles
    • 6. Conclusions
    • Acknowledgments
    • References
• II. Design and Formulation of Antisense Technology
  • 3. Design of Bifunctional Antisense Oligonucleotides for Exon Inclusion
    Haiyan Zhou.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 5. How to Design U1 snRNA Molecules for Splicing Rescue
    Liliana Matos, Juliana I. Santos, Mª. Francisca Coutinho, and Sandra Alves.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 6. Conjugation of Nucleic Acids and Drugs to Gold Nanoparticles
    Paula Milán-Rois, Ciro Rodriguez-Diaz, Milagros Castellanos, and Álvaro Somoza.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 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 Ümran Tunç.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
• 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 11. In Vitro Models for the Evaluation of Antisense Oligonucleotides in Skin
    Jeroen Bremer and Peter C. van den Akker.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 12. In Vitro Delivery of PMOs in Myoblasts by Electroporation
    Remko Goossens and Annemieke Aartsma-Rus.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 13. Rapid Determination of MBNL1 Protein Levels by Quantitative Dot Blot for the Evaluation of Antisense Oligonucleotides in Myotonic Dystrophy Myoblasts
    Nerea Moreno, Irene González-Martínez, Rubén Artero, and Estefanía Cerro-Herreros.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 16. Eye on a Dish Models to Evaluate Splicing Modulation
    Kwan-Leong Hau, Amelia Lane, Rosellina Guarascio, and Michael E. Cheetham.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 17. Establishment of In Vitro Brain Models for AON Delivery
    Elena Daoutsali and Ronald A. M. Buijsen.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
• IV. In Vivo Model Systems
  • 18. Considerations for Generating Humanized Mouse Models to Test Efficacy of Antisense Oligonucleotides
    Irene Vázquez-Domínguez and Alejandro Garanto.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Human and Mouse : Species Are Similar But Not Equal
    • 3. Checkpoints Before Generating a Humanized Mouse Model
    • 4. Current Examples of Humanized Mouse Models
    • 5. Conclusions
    • Acknowledgments
    • References
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 20. Use of Small Animal Models for Duchenne and Parameters to Assess Efficiency upon Antisense Treatment
    Ngoc Lu-Nguyen, Alberto Malerba, and Linda Popplewell.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 21. In Vivo Models for the Evaluation of Antisense Oligonucleotides in Skin
    Jeroen Bremer and Peter C. van den Akker.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • References
  • 22. Delivery of Antisense Oligonucleotides to the Mouse Retina
    Alejandro Garanto.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 23. Delivery of Antisense Oligonucleotides to the Mouse Brain by Intracerebroventricular Injections
    Tom Metz, Elsa C. Kuijper, and Willeke M. C. van Roon-Mom.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
• V. Safety and Toxicology
  • 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.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 25. Preclinical Safety Assessment of Therapeutic Oligonucleotides
    Patrik Andersson.
    Published online: February 25, 2022.
    • 1. Introduction: Oligo Classes, Chemistries, and Designs
    • 2. Delivery
    • 3. Safety Assessment of Therapeutic Oligos
    • References
  • 26. Preclinical Evaluation of the Renal Toxicity of Oligonucleotide Therapeutics in Mice
    Lucía Echevarría and Aurelie Goyenvalle.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
  • 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, and Bård Smedsrød.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. Materials
    • 3. Methods
    • 4. Notes
    • Acknowledgments
    • References
• VI. Intellectual Property
  • 28. Patent Considerations When Embarking on New Antisense Drug Programs
    Laurence D. S. Gainey.
    Published online: February 25, 2022.
    • 1. Introduction
    • 2. What Is a Patent?
    • 3. Why Patent Inventions?
    • 4. Types of Inventions to Consider
    • 5. Patenting Requirements
    • 6. Confidentiality
    • 7. Patent Timeline
    • 8. Data Requirements
    • 9. Searching
    • 10. Freedom to Operate (FTO)
    • 11. Summary
    • 12. Glossary
    • 13. Notes
    • References