Yanmei Lu, PhD is currently the Vice President of Biomarker and BioAnalytical Sciences at Sangamo Therapeutics and previously worked at Genentech. Dr. Lu has a PhD in Biochemistry and Molecular Biology and has published 50+ peer-reviewed articles and book chapters.

Boris Gorovits, PhD is currently the Principal at Gorovits BioSolutions, LLC and previously worked at Sana Biotherapeutics, Pfizer and Wyeth Research. Dr. Gorovits has published 80+ journal articles and book chapters.

List of Contributors xix Preface xxiii Section I Introduction 1 1 Introduction to AAV-based in vivo Gene Therapy 3Oscar 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 35John 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 63Yanmei 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 87Manuela 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 117Vibha 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 137Christian 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 163Christian 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 193Venkata 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 215Robert 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 239Liching 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 271Maurus 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 291George 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 317Jing 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 347Marina 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 385Paul 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 393Karen 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 409Mica Elizalde and Paul Bartel 17.1 Introduction 409 17.2 US Fda 409 [...]opean 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 431Laura 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