Description
Book SynopsisTable of ContentsList of Contributors xix
Preface xxiii
Section I Introduction 1
1 Introduction to AAV-based in vivo Gene Therapy 3
Oscar Segurado
1.1 Introduction 3
1.1.1 History of Gene Therapy 3
1.1.2 AAV-based in vivo Gene Therapy: A Revolution in Medicine 4
1.1.3 The AAV Vector Structure 11
1.1.4 Cell Entry and Transduction Pathway 12
1.2 Advantages and Disadvantages for AAV in vivo 13
1.2.1 Effectiveness and Advantages of AAV Vectors for in vivo Gene Therapy 13
1.2.2 Challenges of AAV Vectors for in vivo Gene Therapy 14
1.3 Technology Platforms of AAV-based in vivo Gene Therapy 14
1.3.1 cDNA Replacement 15
1.3.2 Genome Editing 15
1.3.2.1 Zfn 16
1.3.2.2 TALENs 16
1.3.2.3 CRISPR/Cas 9 16
1.3.3 Base Editing and Prime Editing 17
1.3.4 RNAi Gene Silencing 17
1.3.5 Gene Addition 18
1.4 AAV Serotypes and Tissue Affinity 18
1.4.1 The Liver as a Biofactory 19
1.4.2 The CNS as a Biofactory 19
1.4.3 The Muscle as a Biofactory 19
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 19
1.5.1 Gene Therapy Clinical Trials: Spotlight on Hemophilia A 20
1.6 Predictions for Scientific and Medical Progress 22
1.6.1 Predictions for Challenges in the Field 22
1.6.2 Addressing Durability 23
1.6.3 Addressing Immunogenicity 24
1.6.4 Addressing Malignancy 24
1.7 Predictions for Market Adoption 24
1.7.1 Patients and Patient Advocacy Groups 25
1.7.2 Physicians, Clinical Guidelines, Regulatory Agencies 25
1.7.3 Payers 26
1.8 Final Thoughts 26
1.8.1 Can We Afford in vivo Gene Therapies? 26
1.8.2 Can in vivo Gene Editing Replace Gene Therapy? 27
References 28
2 Recent Development in in vivo Clinical Gene Therapy Platforms 35
John Murphy and Jane Owens
2.1 Introduction 35
2.1.1 rAAV-cDNA Replacement Therapies 35
2.1.1.1 Introduction: Approved rAAV-cDNA Replacement Therapies 36
2.1.1.2 Glybera (alipogene tiparvovec), Marketed by uniQure 36
2.1.1.3 Luxturna (voretigene neparvovec-rzyl), Marketed by Spark Therapeutics 38
2.1.1.4 Zolgensma (onasemnogene abeparvovec), Marketed by Novartis 40
2.1.2 Introduction: rAAV-cDNA (gene) Therapy Candidates in Clinical Development 46
2.1.2.1 AAV-Gene Replacement Clinical Trials for the Eye 47
2.1.2.2 Clinical Trials for Heart Disease 47
2.1.2.3 Clinical Trials for Hematologic and Metabolic Disease (Targeting the Liver) 48
2.1.2.4 Clinical Trials for Skeletal Muscle 48
2.1.3 Introduction: rAAV-as a Vehicle for in vivo Gene Editing 48
2.1.3.1 Non-nuclease Mediated Methods 48
2.1.3.2 Nuclease-mediated Homology Directed Repair 52
2.1.4 Nuclease-mediated Gene Disruption following AAV Delivery 54
2.1.5 Challenges and Opportunities with AAV as a Delivery Vehicle for Nuclease-Mediated Gene Editing 56
References 56
Section II Translational Biomarkers for Gene Therapy 61
3 Biomarker and Bioanalytical Readouts for the Development of AAV Gene Therapy 63
Yanmei Lu and Wibke Lembke
3.1 Introduction 63
3.1.1 AAV-Mediated in vivo Gene Therapy 63
3.1.2 Biomarker Category and Utility 65
3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD) Biomarkers 66
3.2.1 Viral Biodistribution and Shedding 66
3.2.2 Transgene mRNA Expression 68
3.2.3 Transgene and Target Protein Activity and Concentration 68
3.2.4 Substrate and Other Distal PD Biomarkers 70
3.3 Safety and Monitoring Biomarkers and Readouts 71
3.3.1 Assessment of genotoxicity 72
3.3.1.1 AAV Integration/Insertional Mutagenesis Risk 72
3.3.1.2 AAV Germline Transmission Risk 73
3.3.1.3 Off-Target Gene Editing 73
3.3.2 Biomarkers for Immune-Mediated Toxicity 74
3.3.2.1 Hepatotoxicity 74
3.3.2.2 Thrombotic Microangiopathy 76
3.3.2.3 Muscle Toxicity 77
3.3.2.4 Immunogenicity Assessment for rAAV Gene Therapy 77
3.3.3 Safety Biomarkers for Nonimmune Organ-Specific Toxicity 78
3.3.3.1 Dorsal Root Ganglia Toxicity 78
3.3.3.2 Other Target Organ Toxicity Biomarkers 79
3.4 Predictive and Diagnostic Biomarkers for Study Enrollment and Patient Stratification 80
3.4.1 Preexisting Anti-Capsid Antibody 80
3.4.1.1 Companion Diagnostic 81
3.4.2 Preexisting Anti-Transgene Protein Antibody 81
3.5 Summary 82
References 82
4 Nonclinical and Clinical Study Considerations for Biodistribution, Shedding, and Pharmacokinetics/Pharmacodynamics 87
Manuela Braun and Kefeng Sun
4.1 Biodistribution and Viral Shedding 87
4.1.1 Introduction to Biodistribution and Viral Shedding 87
4.1.1.1 Definition and Terminology for Biodistribution and Shedding 88
4.1.1.2 Global Regulatory Guidance on Conducting Biodistribution and Shedding Studies 88
4.1.2 Nonclinical Biodistribution and Shedding Studies for AAV Vectors 89
4.1.2.1 Design, Execution, and Reporting 90
4.1.2.2 Examples 95
4.1.3 Clinical Biodistribution and Shedding Studies for AAV Vectors 96
4.1.3.1 General Considerations in Viral Shedding Studies in the Clinical Setting 97
4.1.3.2 Biodistribution Characterization in Human: Necessity and Concerns 98
4.1.3.3 Examples 98
4.1.4 Gaps and Challenges on Biodistribution and Shedding Characterization 99
4.2 Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling and Clinical Dose Selection of Gene Therapy 100
4.2.1 Overview on PK/PD and Dose Selection Strategies for Gene Therapy 100
4.2.1.1 AAV Dosing Regimen – Safety Relationship and Safety-based Clinical Dose Projection 101
4.2.1.2 AAV Dose – Pharmacodynamics/Efficacy Relationship and Projection of Pharmacologically-Active Dose (PAD) 102
4.2.2 Dose Scaling Approaches: Allometric and Activity-Based Methods 102
4.2.3 Mechanistic Approaches to Modeling Gene Therapy 105
4.2.3.1 Modeling and Simulation of AAV Biodistribution 106
4.2.3.2 Modeling Transgene Product PK and PD of the Transgene Product 106
4.2.4 Clinical Pharmacology Considerations for Gene Therapy 106
4.2.4.1 Variability in Transgene Product Levels and/or Treatment Response 106
4.2.4.2 Durability of Transgene Expression and/or Treatment Response 107
4.2.5 Gaps and Challenges on PK/PD and Clinical Dose Selection 108
4.2.5.1 Interspecies difference in AAV Transduction and Immunogenicity 108
4.2.5.2 Availability of Clinical Samples and Bioanalytical Assays 109
4.2.5.3 Availability of Long-Term Follow-Up Data 109
4.3 Summary 109
References 110
5 Immunogenicity of AAV Gene Therapy Products 117
Vibha Jawa and Bonnie Wu
5.1 Innate and Adaptive Immunity Induced by AAV-Based Gene Therapies 117
5.1.1 Innate Immune Response 117
5.1.2 Adaptive Immune Response 119
5.2 Preclinical Immunogenicity Risk Assessment 119
5.2.1 Product-related Risk Factors 120
5.2.2 Process and Manufacturing-Related Risk Factors 120
5.2.3 Patient-Related Risk Factors 121
5.2.4 Nonclinical Assessment of Immunogenicity 121
5.2.5 Animal Models for Assessing Innate Immunity 122
5.2.6 Animal Models for Assessing Adaptive Immunity 122
5.2.7 Impact of Immunogenicity on Animal Selection and Interpretation of Study Results 123
5.3 Clinical Manifestation Associated with Immunogenicity 123
5.3.1 Pre-existing Immunity Against AAV Vector May Compromise Therapeutic Efficacy and Patient Safety 124
5.3.2 Treatment Induced Anti-AAV Capsid Antibodies may Prevent Re-dosing 124
5.3.3 Antibody Specific to Transgene Protein could lead to Toxicity or Unwanted Immunity 125
5.3.4 Risk of Immunogenicity Associated with Different Administration Routes 125
5.3.4.1 Gene Delivery to the Eye or Central Nervous System 126
5.3.4.2 Gene Delivery to Liver 126
5.3.4.3 Gene Delivery to Muscle 126
5.3.5 Product- and Process-related Impurity Related Immunogenicity 127
5.4 Clinical Mitigation Strategy 127
References 129
Section III Bioanalysis for Gene Therapy 135
6 Bioanalytical Methods to Detect Preexisting and Post-administration Humoral Immune Responses Against AAV Capsid Proteins 137
Christian Vettermann and Boris Gorovits
6.1 Introduction 137
6.2 Considerations for AAV Total Antibody Assays 138
6.2.1 Nature of AAV TAb Assay Analyte 138
6.2.2 Primary Analytical Methodologies applied for AAV TAb Detection 139
6.2.3 Tab Assay Critical Reagent Considerations 140
6.2.3.1 Positive and Negative Control Selection 140
6.2.3.2 Capture and Detection Reagents 141
6.2.3.3 Sample Testing Strategy 142
6.2.4 Key Assay Qualification/Validation Parameters 142
6.2.4.1 Assay Sensitivity 142
6.2.4.2 Serotype Specificity 142
6.2.4.3 Precision 143
6.2.4.4 Matrix Interference and Selectivity 143
6.2.4.5 Assay Cut-Point 143
6.2.5 TAb Assay Data Interpretation 144
6.3 Considerations for Cell-based Transduction Inhibition Assays 145
6.3.1 Principle and Methodology of Cell-based AAV TI Assays 145
6.3.2 AAV TI Assay Development: Designing for Clinical Relevance 146
6.3.3 Key Assay Validation Parameters 147
6.3.3.1 Screening and Titer Cut-Points 147
6.3.3.2 Limit of Detection 148
6.3.3.3 Precision 150
6.3.3.4 Specificity 150
6.3.3.5 Confirmatory Steps to Ensure Specific Detection of Neutralizing AAV Antibodies 150
6.3.3.6 Selectivity/Matrix Interference 151
6.3.3.7 Stability 151
6.3.4 Sample Testing Strategy and Monitoring Assay Performance 152
6.3.5 Data Interpretation: Preexisting TI Titer and Clinical Efficacy 152
6.3.6 Value and Challenges of Standardizing TAb and TI Assays 156
References 157
7 Bioanalytical Methods to Study Biodistribution and Shedding of AAV-Based Gene Therapy Vectors 163
Christian Vettermann and Russell Soon
7.1 Introduction 163
7.2 Choice of Platform: qPCR vs. Digital PCR 164
7.3 Aspects of Method Development 168
7.4 Back-Calculation Formulas and Extraction Efficiency Assessments 172
7.5 Sensitivity Requirements 177
7.6 Specificity Requirements 179
7.7 Standard Curve Performance, Colinearity, Precision, and Accuracy 180
7.8 Selectivity Assessment and Matrix Interference 181
7.9 Sample Stability Considerations 182
7.10 Data Reporting Formats, Acceptance Criteria, and Trending 184
7.11 Immunocapture qPCR: An Ultra-Sensitive Method to Detect Intact AAV Capsids 187
References 189
8 Transgene mRNA Expression Analysis 193
Venkata Vepachedu and Hsing-Yin Liu
8.1 Purpose of Measuring Transgene mRNA 193
8.1.1 Transgene Encodes Therapeutic Protein Entity 194
8.1.2 Transgene Encodes Other Entities 196
8.2 Technologies to Quantify Transgene Expression in Tissues 196
8.2.1 RT-qPCR or RT-dPCR 196
8.2.1.1 RNA Extraction (Separate vs. DNA/RNA Co-extraction), Quality Testing, and Quantification 197
8.2.1.2 Co-extraction of DNA and RNA from same Sample 199
8.2.1.3 Quantification and Quality Testing of total RNA in Purified Extracts 200
8.2.1.4 Quantification Using DNA vs. RNA Standards 201
8.2.1.5 Assay Qualification/Validation and Report 201
8.2.1.6 Reporting 205
8.2.2 In Situ Hybridization (ISH) 206
8.2.2.1 Values of ISH for Discovery Studies 207
8.2.2.2 Semi-quantitative, Tissue Fixation, Probe to Reference Classic Procedure 208
8.3 Summary 211
References 211
9 Quantification of Transgene Protein Expression and Biochemical Function 215
Robert Dodge and Liching Cao
9.1 Introduction 215
9.2 Transgene Protein Concentration Determination 216
9.2.1 Human Transgene in Preclinical Species 216
9.2.2 Human Transgene Assessment for Intracellular Proteins 216
9.2.3 Human Transgene Protein Assessment for Non-secreted Proteins 218
9.2.4 Human Transgene Protein Assessment for Secreted Proteins 220
9.2.5 Human Transgene Protein Assessment for Expressed Therapeutics 221
9.2.6 Transgene Protein Assay Format Considerations 221
9.2.6.1 Immunoassays 222
9.2.6.2 Mass Spectrometry Assays 222
9.2.6.3 Semiquantitative Assay Formats 223
9.3 Transgene Protein Activity Determination 224
9.3.1 Method Development Considerations 224
9.3.1.1 Enzyme Kinetics, the Initial Rate of Reaction, and Substrate Concentration 224
9.3.1.2 Reference Standard 226
9.3.1.3 Sample Processing 228
9.3.1.4 Buffers and Incubation Temperature 230
9.3.1.5 Assay Dynamic Range, Minimum Required Dilution, Matrix Interference, and Parallelism 230
9.3.1.6 Specificity and Selectivity 231
9.3.1.7 Quality Controls (QCs) 232
9.3.2 Method Validation 234
9.4 Summary 234
References 235
10 Substrate and Distal Pharmacodynamic Biomarker Measurements for Gene Therapy 239
Liching Cao, Kai Wang, John Lin, and Venkata Vepachedu
10.1 Introduction 239
10.2 Technologies to Quantify Substrate and Distal PD Biomarker 241
10.2.1 Liquid Chromatography/Tandem Mass Spectrometry (lc-ms/ms) 241
10.2.1.1 Method Development Challenges and Resolutions 241
10.2.1.2 Method Validation by LC-MS/MS 245
10.2.2 Histology 246
10.2.3 Functional Activity and Immunoassays 248
10.2.3.1 Method Validation of Immunoassay 249
10.2.4 mRNA Detection of Downstream Target Expression as a PD Biomarker 253
10.2.4.1 RT-qPCR for Relative Gene Expression Analysis 254
10.2.4.2 RNA-seq 259
10.2.4.3 Nanostring Technology 260
10.2.4.4 Regulatory Considerations for RNA Quantitation in GLP Studies 261
10.2.5 Single-cell Analysis 263
10.3 Summary 265
References 266
11 Detection of Cellular Immunity to Viral Capsids and Transgene Proteins 271
Maurus de la Rosa and Magdalena Tary-Lehmann
11.1 Introduction 271
11.1.1 Humoral and Cellular Immune Responses to Gene Therapy 271
11.1.2 Selected Clinical Observations Showing the Lack of Understanding About T-Cell-Mediated Immune Responses and the Need for Sensitive T-Cell Analytics 272
11.2 Methods for the Detection of Cellular Immune Responses 274
11.2.1 Methods to Detect T-Cell Responses in Clinical Trials 274
11.2.1.1 Enzyme-Linked Immunosorbent Spot Assay 274
11.2.1.2 Intracellular Cytokine Staining 276
11.2.1.3 Tetramer Staining 276
11.2.1.4 Proliferation Assays 276
11.2.1.5 Cytokine Bead Array 276
11.2.1.6 Gene Expression Profiling 277
11.2.1.7 Multiplexed Epitope Mapping 277
11.2.1.8 Conclusion 277
11.2.2 Technical Challenges of Detecting Cellular Immune Responses 277
11.3 Validation of Cellular Assays Using PBMC (Example ELISPOT) 278
11.3.1 Validation Strategies 278
11.3.1.1 Precision 279
11.3.1.2 Specificity 279
11.3.1.3 Limit of Detection and Range 280
11.3.1.4 Common Exceptions for ELISPOT Validation: Accuracy, Linearity, and Reproducibility 281
11.3.2 Parameters Affecting ELISPOT Assay Performance 282
11.3.2.1 PBMC Sample Handling: Temperature, Resting, and Serum 282
11.3.2.2 Antigen Concentration and Number of Replicates 285
References 286
12 Detection of Humoral Response to Transgene Protein and Gene Editing Reagents 291
George Buchlis and Boris Gorovits
12.1 Pre- and Post-dose Humoral Immunity to Transgene-expressed Proteins 291
12.1.1 Risk-based Analysis of Response Probability and Impact 291
12.1.1.1 Route of Administration 291
12.1.1.2 Biodistribution of Vector, Vector Serotype, Dose, and Expression Level 293
12.1.1.3 Patient Immune Status: Age, Prior Exposure, No Endogenous Production, Immunosuppression, and Autoimmunity 293
12.1.1.4 Response Induction vs. Response Boosting 294
12.2 Relevance of Analytical Protocols Applied in Determining Immune Response to Protein Therapeutics to the Detection of Anti-Transgene Protein Responses 294
12.3 Analysis of Immune Response by Binding and Functional Antibody Assay Protocols 295
12.4 Comparative Analysis of the Immune Response Evaluation for Transgene Proteins that are Expressed Extracellularly vs. Intracellularly 297
12.5 Humoral Immune Response to Gene Editing Reagents 298
12.5.1 Diversity of Gene Editing Systems 298
12.5.2 Immunological Potential of CRISPR-Cas System 299
12.5.3 Detection of Anti-Cas9 Protein Immunity in Animal and Human Matrix 301
12.5.4 Strategies Proposed to Mitigate Anti-Cas9 Immunity 304
References 304
13 rAAV Integration: Detection and Risk Assessment 317
Jing Yuan, Irene Gil-Farina, Raffaele Fronza, and Laurence O. Whiteley
13.1 Introduction 317
13.1.1 Biology of AAV Vectors as it Relates to Mechanisms of AAV Integration 318
13.1.2 Literature Review of AAV Studies in Relation to Neoplasia Development 318
13.2 Review of Regulatory Guidance and Discussion Points that Are Raised on AAV Carcinogenesis 324
13.2.1 Factors to Consider in the Design of Nonclinical Studies Evaluating AAV Integration 325
13.2.2 Methods for rAAV Integration Analysis 326
13.2.3 AAV Data Analysis Methods 328
13.2.3.1 AAV Primary Analysis 331
13.2.3.2 Impurity Analysis 332
13.2.3.3 AAV Genome Rearrangements 332
13.2.3.4 Integration Site Analysis 332
13.2.3.5 Clonality Analysis 333
13.2.3.6 Genotoxic Integrations 334
13.3 Assessing the Biologic Relevance of AAV Integration Profile 335
13.4 Conclusion and Future Direction 337
References 338
14 Detection and Quantification of Genome Editing Events in Preclinical and Clinical Studies 347
Marina Falaleeva, Shengdar Tsai, Kathleen Meyer, and Yanmei Lu
14.1 Introduction 347
14.1.1 Genome Editing Modalities and Molecular Outcomes 348
14.1.2 Clinical Trials Using Genome Editing Technologies 350
14.2 Regulatory Guidance on Engineered Nuclease On- and Off-target Assessment 352
14.3 Strategies and Methodologies to Evaluate On-target and Off-target Activities 353
14.3.1 Strategies to Evaluate Off-target Sites in Preclinical and Clinical Studies 353
14.3.2 Techniques to Identify Genome Wide Off-target Sites 355
14.3.3 Targeted Approaches to Measure Short Insertions and Deletions 356
14.3.3.1 Droplet Digital™ PCR 365
14.3.3.2 Endonuclease Mismatch Cleavage Assays 366
14.3.3.3 Sanger Sequencing Combined with Sequence Trace Decomposition 368
14.3.3.4 Indel Detection by Amplicon Analysis (IDAA) 369
14.3.4 Technologies to Measure Large Genomic Rearrangements 369
14.3.5 Discussion 374
14.4 Concluding Remarks 376
References 376
Section IV Companion Diagnostic Development for Gene Therapy 383
15 Introduction to Companion Diagnostics for Gene Therapy 385
Paul Bartel and Jennifer Granger
15.1 Introduction to Companion Diagnostics 385
15.2 Role in Gene Therapy 386
15.3 Overall Strategy 387
15.4 Development Process 387
15.5 Considerations for Commercialization 390
15.6 Conclusion 391
References 391
16 Validation for Gene Therapy Companion Diagnostics 393
Karen L. Richards and Kennon Daniels
16.1 Introduction 393
16.1.1 Overview of FDA Oversight for the Use of Assays in Gene Therapy Clinical Trials and the Path to Commercialization with Corresponding Level of Validation 393
16.1.2 Summary of Validation Requirements for Gene Therapy Companion Diagnostics (GTx CDx) 395
16.1.3 Role of CDx in Therapeutic Development and Unique Challenges to Validating GTx CDx 395
16.1.4 Key Considerations for Developing GTx cdx 396
16.2 Development of CTAs for Use in GTx Clinical Trials 397
16.2.1 Stratification vs. Selection 397
16.2.2 Regulatory Risk Determination: Significant or Nonsignificant? 398
16.2.3 CTA Design Considerations 400
16.2.4 CTA Validation Requirements 401
16.3 Best Practices for Sample Banking and Consent of Subjects 401
16.3.1 Validation Strategies for CDxs for Commercial Use 401
16.4 Design Considerations 402
16.4.1 Single-site vs. Distributable Kit 402
16.4.2 Validation Requirements 402
16.5 Bridging Studies 404
16.6 Commensurate Regulatory Review and Approval of GTx cdx 406
16.7 Concluding Sections 406
16.7.1 Summary of Validation Considerations for CTAs/CDx in GTx Clinical Trials 406
16.7.2 Summary of Validation Considerations for CTAs/CDx to Enable GTx Marketing 407
References 407
17 Regulatory Considerations for Gene Therapy Companion Diagnostics 409
Mica Elizalde and Paul Bartel
17.1 Introduction 409
17.2 US Fda 409
17.2.1 Clinical Trials for Investigational Device Exemption 410
17.2.2 US FDA Marketing Authorization Pathways 413
17.2.2.1 510(k) process 413
17.2.2.2 PMA Process 414
17.2.2.3 HDE Process 414
17.2.2.4 Differences Between 510(k) and PMA 415
17.2.3 US FDA Pre-submission Feedback 416
17.3 European Union 416
17.3.1 European Union Clinical Trials 416
17.3.2 European Union Marketing Authorization Pathways 418
17.4 Other Regulated Markets 420
17.4.1 Global Regulatory Strategy 421
17.5 Development Strategy with the Therapeutic 422
17.5.1 Considerations for Rare Disease Indications 423
17.6 Partner Relationship 424
17.6.1 Importance of the Partner Relationship 424
17.7 Commercial and Post-Approval Considerations 425
17.7.1 Future Proofing the Companion Diagnostic 425
17.7.2 Modifications of the Companion Diagnostic 426
17.8 Final Word 426
References 426
Section V Regulatory Perspectives on Gene Therapy 429
18 Current Regulatory Landscape for Gene Therapy Product Development and the Role of Biomarkers 431
Laura I. Salazar-Fontana PhD and Mike Havert PhD
18.1 Introduction 431
18.2 What is Gene Therapy? 432
18.3 Biomarkers Defined 433
18.4 Early Gene Therapy Biomarkers 434
18.5 Current Expectations for Gene Therapy Biomarkers 437
18.6 Safety Biomarkers for Gene Therapy Products 438
18.6.1 Immune Toxicities to in vivo gene therapy 438
18.6.2 Immune Toxicities to Ex Vivo GT 441
18.6.3 Long-Term Risks 442
18.7 Concluding Remarks 442
References 443
Index 449
