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List 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
1.2 Advantages and Disadvantages for AAV in vivo 13
1.3 Technology Platforms of AAV-based in vivo Gene Therapy 14
1.4 AAV Serotypes and Tissue Affinity 18
1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 19
1.6 Predictions for Scientific and Medical Progress 22
1.7 Predictions for Market Adoption 24
1.8 Final Thoughts 26 2 Recent Development in in vivo Clinical Gene Therapy Platforms 35 John Murphy and Jane Owens
2.1 Introduction 35 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
3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD) Biomarkers 66
3.3 Safety and Monitoring Biomarkers and Readouts 71
3.4 Predictive and Diagnostic Biomarkers for Study Enrollment and Patient Stratification 80
3.5 Summary 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
4.2 Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling and Clinical Dose Selection of Gene Therapy 100
4.3 Summary 109 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
5.2 Preclinical Immunogenicity Risk Assessment 119
5.3 Clinical Manifestation Associated with Immunogenicity 123
5.4 Clinical Mitigation Strategy 127 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
6.3 Considerations for Cell-based Transduction Inhibition Assays 145 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 8 Transgene mRNA Expression Analysis 193 Venkata Vepachedu and Hsing-Yin Liu
8.1 Purpose of Measuring Transgene mRNA 193
8.2 Technologies to Quantify Transgene Expression in Tissues 196
8.3 Summary 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
9.3 Transgene Protein Activity Determination 224
9.4 Summary 234 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
10.3 Summary 265 11 Detection of Cellular Immunity to Viral Capsids and Transgene Proteins 271 Maurus de la Rosa and Magdalena Tary-Lehmann
11.1 Introduction 271
11.2 Methods for the Detection of Cellular Immune Responses 274
11.3 Validation of Cellular Assays Using PBMC (Example ELISPOT) 278 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
12.2 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 13 rAAV Integration: Detection and Risk Assessment 317 Jing Yuan, Irene Gil-Farina, Raffaele Fronza, and Laurence O. Whiteley
13.1 Introduction 317
13.2 Review of Regulatory Guidance and Discussion Points that Are Raised on AAV Carcinogenesis 324
13.3 Assessing the Biologic Relevance of AAV Integration Profile 335
13.4 Conclusion and Future Direction 337 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
14.2 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
14.4 Concluding Remarks 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 16 Validation for Gene Therapy Companion Diagnostics 393 Karen L. Richards and Kennon Daniels
16.1 Introduction 393
16.2 Development of CTAs for Use in GTx Clinical Trials 397
16.3 Best Practices for Sample Banking and Consent of Subjects 401
16.4 Design Considerations 402
16.5 Bridging Studies 404
16.6 Commensurate Regulatory Review and Approval of GTx cdx 406
16.7 Concluding Sections 406 17 Regulatory Considerations for Gene Therapy Companion Diagnostics 409 Mica Elizalde and Paul Bartel
17.1 Introduction 409
17.2 US Fda 409
17.3 European Union 416
17.4 Other Regulated Markets 420
17.5 Development Strategy with the Therapeutic 422
17.6 Partner Relationship 424
17.7 Commercial and Post-Approval Considerations 425
17.8 Final Word 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
18.7 Concluding Remarks 442 References 443 Index 449