Retinitis Pigmentosa

Foreword
Preface
Contents
Contributors
Chapter 1: Introduction and Discovery of Retinitis Pigmentosa
1 Introduction to Retinitis Pigmentosa
2 Discovery of Night Blindness
3 Discovery of RP
4 Allied Diseases
5 Visual Field Defect in Retinitis Pigmentosa
6 Electroretinogram (ERG) in Retinitis Pigmentosa
7 Definition of Retinitis Pigmentosa
8 The Natural Course of Retinitis Pigmentosa
9 Future Prospective in Retinitis Pigmentosa
References
Chapter 2: Etiology of Retinitis Pigmentosa
1 Introduction
2 Non-syndromic RP
3 Autosomal Recessive Mutations of RP
4 Autosomal Dominant Forms of RP
5 X-Linked Forms of RP
6 Other Inheritance Patterns of RP
6.1 Syndromic Forms of RP
6.2 Usher Syndrome
6.3 Bardet-Biedl Syndrome
6.4 Alport Syndrome
6.5 Kearns-Sayre Syndrome/Ragged Red Fiber Myopathy
6.6 Abetalipoproteinemia
6.7 McLeod Syndrome
6.8 Refsum Disease
6.9 Laurence-Moon Syndrome
6.10 Cockayne Syndrome
6.11 Alstrom Syndrome
6.12 Leber Congenital Amaurosis
7 Secondary RP
8 Conclusion
References
Chapter 3: Clinical Evaluation of Patients with Retinitis Pigmentosa
1 Introduction
1.1 History
2 Materials
3 Methods
3.1 Visual Acuity
3.2 Refraction
3.3 Visual Fields to Confrontation
3.4 Intraocular Pressure Measurement
3.5 Aqueous Humor
3.6 Lens
3.7 Vitreous
3.8 Fundus Examination
3.9 Summary
References
Chapter 4: Molecular Genetic Testing Approaches for Retinitis Pigmentosa
1 Introduction
1.1 Next-Generation Sequencing
1.2 Panel Testing
1.3 Whole Exome Sequencing
1.4 Whole Genome Sequencing
1.5 Testing Strategies
2 Materials
3 Methods
3.1 Library Preparation
3.1.1 DNA Fragmentation
3.1.2 End Repair
3.1.3 AMPure Bead Cleanup
3.1.4 Adenylate 3′ Ends
3.1.5 Ligate Adapters
3.1.6 Enrich DNA Fragments
3.1.7 Validate Library
3.1.8 SureSelect Enrichment
3.1.9 Magnetic Bead Preparation
3.1.10 Select Hybrid Capture with SureSelect
3.1.11 Amplify the Captured Library to Add Index Tags
3.1.12 Sample Purification Using Agencourt AMPure XP Beads (see Note 1)
3.1.13 Validation of Enriched Library
3.1.14 Library Quantification by qPCR
3.2 DNA Sequencing
3.2.1 Denature Template DNA
3.2.2 Dilute Denatured DNA
3.2.3 Denature and Dilute PhiX Control
3.2.4 Mixing of Sample Library and PhiX Control
3.3 Data Analysis
4 Conclusion
5 Notes
References
Chapter 5: Pedigree Analysis of Families and Patients Affected by Retinitis Pigmentosa
1 Introduction
2 Materials
3 Methods
3.1 Protocol
3.2 Examples of Pedigrees Containing IRDs Can Be Seen in Figs. 1, 2, and 3
3.3 Conclusion
References
Chapter 6: Whole Genome Sequencing for Detection of Structural Variants in Patients with Retinitis Pigmentosa
1 Introduction
2 Materials
3 Methods
3.1 Library Preparation and Sequencing
3.1.1 Fragment DNA
3.1.2 Repair DNA Damage and Ends
3.1.3 Ligate SMRTbell Adapters and Purify Templates
3.1.4 Anneal Sequencing Primer and Bind Polymerase
3.2 Read Mapping/Calling Structural Variants
3.3 Visualization of Results
4 Notes
References
Chapter 7: Fundus Photography Methodologies to Assess RP Patients
1 Introduction
2 Materials
3 Methods
3.1 Color Fundus Photography
3.2 Red-Free Fundus Photography
3.3 Fundus Autofluorescence
References
Chapter 8: The Use of Optical Coherence Tomography in Evaluation of Retinitis Pigmentosa
1 Introduction
2 Materials
3 Methods
3.1 Clinical Diagnosis
3.2 OCT and Outer Retina
3.3 OCT and Inner Retina
3.4 Summary
References
Chapter 9: Optical Coherence Tomography Angiography (OCTA) Findings in Retinitis Pigmentosa
1 Introduction
2 Comparison to Traditional Intravenous Fluorescein Angiography and Indocyanine Green Angiography
3 Changes in Microvasculature of Retina, Choroid, and Optic Nerve Head in RP
3.1 RP in the Superficial and Deep Capillary Plexus of the Retina
3.2 Choriocapillaris
3.3 Optic Nerve Head Perfusion in RP
4 Neovascularization
5 RP with Macular Edema
6 Correlation with Function and Progression of Disease
7 Future Directions
References
Chapter 10: Electroretinogram (ERG) to Evaluate the Retina in Cases of Retinitis Pigmentosa (RP)
1 Introduction
1.1 Overview of the Visual System
1.2 Historical Perspective
1.3 The Components of ERG
1.3.1 The Origin of a-Wave
1.3.2 The Origin of b-Wave
1.3.3 The Origin of c-Wave
1.3.4 The Origin of d-Wave
2 Materials
2.1 Types of Recording Electrodes
2.1.1 Recording Electrodes
2.1.2 Reference Electrodes
2.1.3 Common Electrode
3 Methods
3.1 The Clinical ERG Procedures
3.2 The ISCEV Standard for Clinical ERG
3.3 The Standard Procedure for Performing ERGs on Patients
3.4 ERG in Infants
3.5 Interpretation of ERG Responses
3.6 The Typical ERG Findings in Patients with RP
3.7 The Specialized Protocols for Differential Diagnosis
3.8 Prolonged Dark Adaption
3.9 S-Cone ERG
3.10 Predict Visual Prognosis in Patients with Retinitis Pigmentosa
4 Notes
References
Chapter 11: Visual Function Tests: Visual Fields
1 Introduction
2 Materials
3 Methods
3.1 Taking a VF Test
3.1.1 Testing Stimuli
3.1.2 Testing Algorithms to Estimate Threshold Intensities
3.2 Interpreting the VF Test Printout
3.3 Challenges and Limitations with VF Testing
References
Chapter 12: Visual Function Tests
1 Introduction
1.1 Visual Acuity
1.2 Visual Field (VF)
2 Materials
3 Methods
3.1 Goldmann Visual Field
3.2 Humphrey Visual Field
3.3 Full-Field Stimulus (FST) Testing
3.4 Visual Function Questionnaire (VFQ-25)
3.5 Summary
References
Chapter 13: Visual Acuity as a Measurement of Visual Function
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 14: Measure of Visual Function
1 Introduction
1.1 Full-Field Stimulus Testing (FST)
1.2 Near-Infrared Autofluorescence (NIR-AF) (HRA2 Heidelberg)
1.3 Quantitative Fundus Autofluorescence (qAF) Heidelberg HRA2, qAF Module
1.4 Quantitative Near-Infrared Autofluorescence (qNIR-AF) Heidelberg HRA2
1.5 Adaptive Optics (AO) Imaging
2 Materials
3 Methods
References
Chapter 15: Ophthalmic Fluorescein Angiography
1 Introduction
1.1 History of FA
2 Materials
2.1 Sodium Fluorescein Dye
2.1.1 Preparation
2.1.2 Working Solution of Fluorescein 102 Preparation
2.2 Other Required Materials
3 Methods
3.1 Performing FA
3.2 Interpretation of FA
3.2.1 Hyperfluorescence
3.2.2 Hypofluorescence
3.3 Safety of FA
4 Notes
References
Chapter 16: Protocol for Indocyanine Green Angiography
1 Introduction
1.1 History of ICG Angiography
2 Materials
3 Methods
3.1 Performing ICG Angiography
3.2 Interpretation of ICG Angiograms
3.2.1 Increased ICG Fluorescence
3.2.2 Decreased ICG Fluorescence
3.3 Safety of ICG
4 Notes
References
Chapter 17: Use of the Medmont Dark-Adapted Chromatic Perimeter for Assessing Rod Function in Retinitis Pigmentosa
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 18: Multi-luminance Mobility Testing Endpoint
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 19: Preclinical Models of Retinitis Pigmentosa
1 Introduction
2 Models of RP by Inheritance, Origin, and Organism
2.1 Autosomal Dominant RP
2.2 Naturally Occurring Models of adRP
2.2.1 Canine
2.2.2 Feline
2.2.3 Murine
2.3 Transgenic Models of adRP
2.3.1 Murine
2.3.2 Piscine
2.3.3 Porcine
2.4 Autosomal Recessive Retinitis Pigmentosa
2.5 Naturally Occurring Models of arRP
2.5.1 Avian
2.5.2 Canine
2.5.3 Feline
2.5.4 Murine
2.6 Transgenic Models of arRP
2.6.1 Murine
2.6.2 Piscine
2.6.3 Other
2.7 X-Linked Retinitis Pigmentosa
2.8 Naturally Occurring Models of XLRP
2.8.1 Avian
2.8.2 Canine
2.8.3 Murine
2.9 Transgenic Models of XLRP
2.9.1 Murine
2.9.2 Piscine
2.10 Syndromic RP
2.11 Alström Syndrome
2.12 Bardet-Biedl Syndrome
2.13 Bietti Syndrome
2.14 Cockayne Syndrome
2.15 Gyrate Atrophy of the Choroid and Retina (GACR)
2.16 Joubert Syndrome
2.17 Neuropathy, Ataxia, and RP (NARP) Syndrome
2.18 Refsum Disease
2.19 Senior-Løken Syndrome
2.20 Usher Syndrome
3 Future Directions
References
Chapter 20: Electroretinogram (ERG) to Evaluate the Retina Using Mouse Models
1 Introduction
2 Materials
2.1 Equipment
2.2 Other Materials
2.3 Mice
3 Methods
3.1 One Day Before Examination
3.2 Preparation of ERG Recordings
3.3 ERG Recording Protocols
3.3.1 Full-Field ERG
3.3.2 Serial Intensity ERGs
3.4 ERG Response Analysis
4 Notes
References
Chapter 21: Autofluorescence Imaging to Evaluate Retinal Disease Progression in Rodent
1 Introduction
2 Methods
2.1 Anesthesia of Mice
2.2 FAF Imaging
2.3 Recovery
3 Notes
References
Chapter 22: Large Animal Models of Retinitis Pigmentosa in Therapy Development and Preclinical Testing
1 Introduction
2 Materials
2.1 Animals
2.2 Anesthesia
2.3 Intravitreal Injection
2.4 Subretinal Injection
2.5 Vision Testing
2.6 Electroretinography
2.7 Spectral Domain-Optical Coherence Tomography
2.8 Fundus Imaging or Ophthalmic Examination
3 Methods
3.1 Animals
3.2 Anesthesia
3.3 Intravitreal Injection
3.4 Subretinal Injection
3.5 Vision Testing
3.6 Electroretinography
3.7 Spectral Domain-Optical Coherence Tomography (SD-OCT) and Confocal Scanning Laser Ophthalmoscopy (cSLO) (e.g., Spectralis,...
3.8 Fundus Imaging and/or Ophthalmic Examination
4 Notes
References
Chapter 23: CRISPR Manipulations in Stem Cell Lines
1 Introduction
2 Materials
3 Methods
3.1 iPSC Preparation for Nucleofection
3.2 Pre-nucleofection
3.3 Plate and Buffer Preparation
3.4 Cell Preparation
3.5 Cell Counting
3.6 Nucleofection
3.7 Post-nucleofection
3.8 Design of Targeting Components and the Use of the CRISPR Design Tool
3.9 Design of the ssODN Template
4 Notes
References
Chapter 24: Metabolite Extraction from RPE Cells and Retinas Related to Retinitis Pigmentosa
1 Introduction
2 Materials
2.1 Basic Supplies
2.2 Materials for RPE Cell Culture
2.2.1 Supplies for Cell Culture
2.2.2 Preparation of RPE Cells
2.3 Material for Retina Dissection
3 Methods
3.1 Metabolite Extraction from Cultured RPE Cells for Liquid or Gas Chromatography-Mass Spectrometry
3.2 Metabolite Extraction from the Isotope-Labeled Culture Medium of RPE Cells for Liquid or Gas Chromatography-Mass Spectrome...
3.3 Metabolite Extraction from the Retina of Mice for Liquid or Gas Chromatography-Mass Spectrometry
4 Notes
References
Chapter 25: CRISPR-Mediated Genome Engineering in Cell Lines
1 Introduction
2 Materials
3 Methods
3.1 CRISPR gRNA Design for Gene Targeting
3.2 Cell Culture (iPSC as Sample Here)
3.2.1 Gene Targeting by Nucleofection
3.3 Screening CRISPR/Cas9 Clones for Deletions and Clone Selection
3.3.1 Colony Picking
3.3.2 Sanger Sequencing
3.3.3 PCR Program
3.3.4 TIDE Method for Quantitative of Genome Editing
4 Notes
References
Chapter 26: CRISPR Off-Target Analysis Platforms
1 Introduction
2 How Off-Targeting Effects of CRISPR-Cas9 Occur and Why They Matter
3 Detecting Off-Target Mutations
3.1 HTGTS
3.2 GUIDE-Seq
3.3 BLESS
3.4 Digenome-Seq
4 Reducing Off-Target Effects
5 Future Directions and Conclusions
References
Chapter 27: Generation of Human iPSC-Derived Retinal Organoids for Assessment of AAV-Mediated Gene Delivery
1 Introduction
2 Materials
2.1 Cell Culture and Reagents
2.2 Equipment
3 Methods
3.1 Human Pluripotent Stem Cell Culture (See Note 2)
3.1.1 Matrigel Coating
3.1.2 Cell Thawing
3.1.3 Cell Passaging
3.1.4 Cryopreservation
3.2 Retinal Organoid Differentiation and Long-Term Culture
3.2.1 Day 0: Embryoid Body Production
3.2.2 Alternative Embryoid Body Production Method: AMASS (Agarose Microwell Array Seeding and Scraping)
3.2.3 Day 7: Transfer Embryoid Bodies to Matrigel-Coated Plates
3.2.4 Day 28: Retinal Organoid Detachment and Sorting
3.2.5 Day 42: Retinal Organoid Maturation and Maintenance (See Notes 15 and 16)
3.3 AAV Transduction of Retinal Organoids and Accompanying Retinal Pigment Epithelium
3.4 Harvesting Retinal Organoids for Immunohistochemistry
4 Notes
References
Chapter 28: Culture of Human Retinal Explants for Ex Vivo Assessment of AAV Gene Delivery
1 Introduction
2 Materials
2.1 Cell Culture and Reagents
2.2 Equipment and Supplies
3 Methods
3.1 Isolation of Human Retina from Eye Cup
3.2 Culture of Human Retinal Biopsy Punches
3.3 AAV Transduction of Human Retinal Explants
3.4 Harvesting Human Retinal Explants for Immunohistochemistry
4 Notes
References
Chapter 29: Prime Editing for the Installation and Correction of Mutations Causing Inherited Retinal Disease: A Brief Methodol...
1 Introduction
2 Materials
2.1 pegRNA and nsgRNA Assembly
2.2 Additional Plasmid Constructs
2.3 Cell Culture and Reagents
2.4 Equipment and Plasticware
2.5 DNA Extraction, PCR Reagents, and Sample Preparation
3 Methods
3.1 Prime Editing Guide RNA (pegRNA) and Nicking Single Guide RNA (nsgRNA) Design
3.2 pegRNA and nsgRNA Construction
3.2.1 Digest pU6-pegRNA-GG-Vector and pU6-Spacer-Acceptor Plasmids
3.2.2 Anneal Oligonucleotides
3.2.3 pegRNA and nsgRNA Assembly
3.2.4 Transformation of Assembled Plasmids
3.3 In Vitro Validation of Prime Editing Strategy
3.3.1 Seeding Cells the Day Before Transfection
3.3.2 Cell Transfection of N2A Cells
3.3.3 DNA Isolation
3.4 Analysis of Prime Editing Efficiency
3.4.1 Sanger Sequencing
3.4.2 Next-Generation Sequencing: Amplicon-EZ
4 Notes
References
Chapter 30: Large-Scale Single-Nucleus RNA Sequencing Compatible with Complex Archived Samples
1 Introduction
2 Materials
2.1 Cryosection
2.2 RNA Quality Test
2.3 Nuclei Extraction
2.4 Library Construction Reagents
3 Methods
3.1 Sample Processing and Quality Control
3.2 RNA Quality Check
3.3 Nuclei Extraction and Purification
3.4 Single Nucleus Library Preparation, Sequencing, and Exploratory Analysis
4 Notes
References
Chapter 31: Vector Shedding and Immunogenicity Sampling in AAV-Based Gene Therapy for Retinitis Pigmentosa Patients
1 Introduction
2 Materials
2.1 Lacrimal Sampling Kit
2.2 Saliva Sampling Kit
2.3 Urine Sampling Kit
2.4 Blood Collection Kit
2.5 Blood Processing Kit
2.6 Serum Processing Kit
2.7 Other Necessary Items/Equipment
3 Methods
3.1 Lacrimal Sampling
3.2 Saliva Sampling
3.3 Urine Sampling
3.4 Blood Sampling
3.5 Anticoagulated Blood Sample Processing
3.6 Serum Sampling from Whole Blood
4 Notes
References
Chapter 32: Current Management Options for Patients with Retinitis Pigmentosa
1 Introduction
2 Nutritional Supplementation
3 Managing Associated Ocular Pathology
3.1 Cystoid Macular Edema
3.2 Posterior Subcapsular Cataracts
3.3 Refractive Errors
4 Counseling
5 Emerging Treatment Modalities
5.1 Gene Therapy
5.2 Retinal Prosthesis
5.3 Stem Cell Transplantation
6 Conclusion
References
Chapter 33: Challenges of Treatment Methodologies and the Future of Gene Therapy and Stem Cell Therapy to Treat Retinitis Pigm...
1 Introduction
2 Materials
3 Methods
3.1 Gene Therapy
3.1.1 Gene Therapy for Loss-of-Function RP
3.1.2 Gene Therapy for Gain-of-Function RP
3.2 Gene Editing
3.2.1 Challenges to Using Gene Editing for RP Treatment
3.3 Stem Cell Therapy
3.4 Therapy for Advanced Photoreceptor Degeneration
4 Conclusion
References
Chapter 34: Ocular Injection Techniques for Retinitis Pigmentosa: Intravitreal, Subretinal, and Suprachoroidal
1 Introduction
2 Methods
2.1 Intravitreal Injection
2.1.1 Background
2.1.2 Applications in Gene Therapy
2.1.3 Techniques of Intravitreal Injection
2.1.4 Post-Injection Monitoring and Follow-Up
2.1.5 Complications
2.1.6 Special Considerations
2.2 Subretinal Injection
2.2.1 Background
2.2.2 Applications in Gene Therapy
2.2.3 Technique (See Fig. 2)
2.2.4 Post-Injection Monitoring and Follow-Up [30]
2.3 Suprachoroidal Injection
2.3.1 Background
2.3.2 Applications in Gene Therapy
2.3.3 Technique
2.4 Conclusion
References
Chapter 35: Surgical Approach with Pars Plana Vitrectomy for Subretinal Gene Therapy
1 Introduction
2 Materials
3 Methods
3.1 Preoperative Surgical Protocols
3.2 Anesthesia
3.3 First Incision
3.4 Vitrectomy
3.5 Subretinal Injection
3.6 Conclusion of Surgery
4 Notes
Reference
Index