Main description:

This detailed volume describes a spectrum of methods and protocols that can be used for the bench-to-bedside development and evaluation of retinal gene therapy. Methods for the successful delivery of these gene therapy vector systems to the retina are examined, as well as assays to test the efficacy in vitro in cell cultures, for gene augmentation and gene editing in vivo on rodents, pigs, and monkey retinas, and on human retinal explants as well as in human clinical studies. 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 protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and practical, Retinal Gene Therapy: Methods and Protocols provides a wide range of readers from students to research experts with vital information on ocular gene therapy vector technology, in vitro and in vivo biological assays, and clinical protocols, to promote further studies for the benefit of children and adults with inherited retinal disease.

Contents:

Part I: Retinal Gene Therapy Vector Production and Biological Assays In Vitro

1. Small Scale Production of Recombinant Adeno-Associated Viral Vectors for Gene Delivery to the Nervous System

Joost Verhaagen, Barbara Hobo, Erich M.E. Ehlert, Ruben Eggers, Joanna A. Korecka, Stefan A. Hoyng, Callan L. Attwell, Alan R. Harvey, and Matthew R.J. Mason

2. Small and Micro-Scale Recombinant Adeno-Associated Virus Production and Purification for Ocular Gene Therapy Applications

Christopher A. Reid and Daniel M. Lipinski

3. Design and Development of AAV-Based Gene Supplementation Therapies for Achromatopsia and Retinitis Pigmentosa

Christian Schoen, Elvir Becirovic, Martin Biel, and Stylianos Michalakis

4. Development of Multigenic Lentiviral Vectors for Cell-Specific Expression of Antiangiogenic miRNAs and Protein Factors

Anne Louise Askou and Thomas J. Corydon

5. Design and In Vitro Use of Antisense Oligonucleotides to Correct Pre-mRNA Splicing Defects in Inherited Retinal Dystrophies

Alejandro Garanto and Rob W.J. Collin

6. Three-Dimensional Co-Culture Bioassay for Screening of Retinal Gene Delivery Systems

Ding Wen Chen, Kathleen Pauloff, and Marianna Foldvari

7. Retinal Gene Therapy for Choroideremia: In Vitro Testing for Gene Augmentation Using an Adeno-Associated Viral (AAV) Vector

Maria I. Patricio and Robert E. MacLaren

Part II: Assays for Gene Augmentation and Editing In Vivo on Rodent and Macaque Retina

8. In Vivo Electroporation of Developing Mouse Retina

Jimmy de Melo and Seth Blackshaw

9. Methods for In Vivo CRISPR/Cas Editing of the Adult Murine Retina

Sandy S. Hung, Fan Li, Jiang-Hui Wang, Anna E. King, Bang V. Bui, Guei-Sheung Liu, and Alex W. Hewitt

10. AAV Gene Augmentation Therapy for CRB1-Associated Retinitis Pigmentosa

C. Henrique Alves and Jan Wijnholds

11. Dual AAV Vectors for Stargardt Disease

Ivana Trapani

12. Optogenetic Retinal Gene Therapy with the Light Gated GPCR Vertebrate Rhodopsin

Benjamin M. Gaub, Michael H. Berry, Meike Visel, Amy Holt, Ehud Y. Isacoff, and John G. Flannery

13. CRISPR Repair Reveals Causative Mutation in a Preclinical Model of Retinitis Pigmentosa: A Brief Methodology

Wen-Hsuan Wu, Yi-Ting Tsai, Sally Justus, Galaxy Y Cho, Jesse D Sengillo, Yu Xu, Thiago Cabral, Chyuan-Sheng Lin, Alexander G. Bassuk, Vinit B. Mahajan, and Stephen H. Tsang

14. In-Depth Functional Analysis of Rodents by Full-Field Electroretinography

Vithiyanjali Sothilingam, Regine Muhlfriedel, Naoyuki Tanimoto, and Mathias W. Seeliger

15. Advanced Ocular Injection Techniques for Therapy Approaches

Regine Muhlfriedel, Marina Garcia Garrido, Christine Wallrapp, and Mathias W. Seeliger

16. Neutralizing Antibodies Against Adeno-Associated Virus (AAV): Measurement and Influence on Retinal Gene Delivery

Melissa Desrosiers and Deniz Dalkara

17. Screening for Neutralizing Antibodies against Natural and Engineered AAV Capsids in Non-Human Primate Retinas

Timothy P. Day, Leah C. Byrne, John G. Flannery, and David V. Schaffer

18. Subretinal and Intravitreal Retinal Injections in Monkeys

Daniyar Dauletbekov, K. Ulrich Bartz-Schmidt, and M. Dominik Fischer

Part III: Clinical Protocols and Retinal Gene Therapy Vector Testing on Human Retina

19. Production of iPS-derived Human Retinal Organoids for Use in Transgene Expression Assays

Peter M. Quinn, Thilo M. Buck, Charlotte Ohonin, Harald Mikkers, and Jan Wijnholds

20. AAV Serotype Testing on Cultured Human Donor Retinal Explants

Thilo M. Buck, Lucie P. Pellissier, Rogier M. Vos, Elon H.C. van Dijk, Camiel J.F. Boon, and Jan Wijnholds

21. Human Retinal Explant Culture for Ex-Vivo Validation of AAV Gene Therapy

Harry O. Orlans, Thomas L. Edwards, Samantha R. De Silva, Maria I. Patricio, and Robert E. MacLaren

22. Visual Acuity Testing Before and After Intravitreal Injection of rAAV2- ND4 in Patients

Bin Li and Chenmian Wu

23. Recording and Analysis of the Human Clinical Electroretinogram

Mathieu Gauvin, Allison L. Dorfman, and Pierre Lachapelle

24. Recording and Analysis of Goldmann Kinetic Visual Fields

Mays Talib, Gislin Dagnelie, and Camiel J.F. Boon

25. Measuring Central Retinal Sensitivity Using Microperimetry

Mays Talib, Jasleen K. Jolly, and Camiel J.F. Boon

26. Inspection of the Human Retina by Optical Coherence Tomography

Thomas Theelen and Michel M. Teussink

27. Vector Shedding and Immunogenicity Sampling for Retinal Gene Therapy

Alun R. Barnard, Anna N. Rudenko, and Robert E. MacLaren