Details

<p>List of Contributors xix</p> <p>Preface xxiii</p> <p><b>Section I Introduction 1</b></p> <p><b>1 Introduction to AAV-based in vivo Gene Therapy 3<br /></b><i>Oscar Segurado</i></p> <p>1.1 Introduction 3</p> <p>1.2 Advantages and Disadvantages for AAV in vivo 13</p> <p>1.3 Technology Platforms of AAV-based in vivo Gene Therapy 14</p> <p>1.4 AAV Serotypes and Tissue Affinity 18</p> <p>1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 19</p> <p>1.6 Predictions for Scientific and Medical Progress 22</p> <p>1.7 Predictions for Market Adoption 24</p> <p>1.8 Final Thoughts 26</p> <p><b>2 Recent Development in in vivo Clinical Gene Therapy Platforms 35<br /></b><i>John Murphy and Jane Owens</i></p> <p>2.1 Introduction 35</p> <p><b>Section II Translational Biomarkers for Gene Therapy 61</b></p> <p><b>3 Biomarker and Bioanalytical Readouts for the Development of AAV Gene Therapy 63<br /></b><i>Yanmei Lu and Wibke Lembke</i></p> <p>3.1 Introduction 63</p> <p>3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD) Biomarkers 66</p> <p>3.3 Safety and Monitoring Biomarkers and Readouts 71</p> <p>3.4 Predictive and Diagnostic Biomarkers for Study Enrollment and Patient Stratification 80</p> <p>3.5 Summary 82</p> <p><b>4 Nonclinical and Clinical Study Considerations for Biodistribution, Shedding, and Pharmacokinetics/Pharmacodynamics 87<br /></b><i>Manuela Braun and Kefeng Sun</i></p> <p>4.1 Biodistribution and Viral Shedding 87</p> <p>4.2 Pharmacokinetic/Pharmacodynamic (PK/PD) Modeling and Clinical Dose Selection of Gene Therapy 100</p> <p>4.3 Summary 109</p> <p><b>5 Immunogenicity of AAV Gene Therapy Products 117<br /></b><i>Vibha Jawa and Bonnie Wu</i></p> <p>5.1 Innate and Adaptive Immunity Induced by AAV-Based Gene Therapies 117</p> <p>5.2 Preclinical Immunogenicity Risk Assessment 119</p> <p>5.3 Clinical Manifestation Associated with Immunogenicity 123</p> <p>5.4 Clinical Mitigation Strategy 127</p> <p><b>Section III Bioanalysis for Gene Therapy 135</b></p> <p><b>6 Bioanalytical Methods to Detect Preexisting and Post-administration Humoral Immune Responses Against AAV Capsid Proteins 137<br /></b><i>Christian Vettermann and Boris Gorovits</i></p> <p>6.1 Introduction 137</p> <p>6.2 Considerations for AAV Total Antibody Assays 138</p> <p>6.3 Considerations for Cell-based Transduction Inhibition Assays 145</p> <p><b>7 Bioanalytical Methods to Study Biodistribution and Shedding of AAV-Based Gene Therapy Vectors 163<br /></b><i>Christian Vettermann and Russell Soon</i></p> <p>7.1 Introduction 163</p> <p>7.2 Choice of Platform: qPCR vs. Digital PCR 164</p> <p>7.3 Aspects of Method Development 168</p> <p>7.4 Back-Calculation Formulas and Extraction Efficiency Assessments 172</p> <p>7.5 Sensitivity Requirements 177</p> <p>7.6 Specificity Requirements 179</p> <p>7.7 Standard Curve Performance, Colinearity, Precision, and Accuracy 180</p> <p>7.8 Selectivity Assessment and Matrix Interference 181</p> <p>7.9 Sample Stability Considerations 182</p> <p>7.10 Data Reporting Formats, Acceptance Criteria, and Trending 184</p> <p>7.11 Immunocapture qPCR: An Ultra-Sensitive Method to Detect Intact AAV Capsids 187</p> <p><b>8 Transgene mRNA Expression Analysis 193<br /></b><i>Venkata Vepachedu and Hsing-Yin Liu</i></p> <p>8.1 Purpose of Measuring Transgene mRNA 193</p> <p>8.2 Technologies to Quantify Transgene Expression in Tissues 196</p> <p>8.3 Summary 211</p> <p><b>9 Quantification of Transgene Protein Expression and Biochemical Function 215<br /></b><i>Robert Dodge and Liching Cao</i></p> <p>9.1 Introduction 215</p> <p>9.2 Transgene Protein Concentration Determination 216</p> <p>9.3 Transgene Protein Activity Determination 224</p> <p>9.4 Summary 234</p> <p><b>10 Substrate and Distal Pharmacodynamic Biomarker Measurements for Gene Therapy 239<br /></b><i>Liching Cao, Kai Wang, John Lin, and Venkata Vepachedu</i></p> <p>10.1 Introduction 239</p> <p>10.2 Technologies to Quantify Substrate and Distal PD Biomarker 241</p> <p>10.3 Summary 265</p> <p><b>11 Detection of Cellular Immunity to Viral Capsids and Transgene Proteins 271<br /></b><i>Maurus de la Rosa and Magdalena Tary-Lehmann</i></p> <p>11.1 Introduction 271</p> <p>11.2 Methods for the Detection of Cellular Immune Responses 274</p> <p>11.3 Validation of Cellular Assays Using PBMC (Example ELISPOT) 278</p> <p><b>12 Detection of Humoral Response to Transgene Protein and Gene Editing Reagents 291<br /></b><i>George Buchlis and Boris Gorovits</i></p> <p>12.1 Pre- and Post-dose Humoral Immunity to Transgene-expressed Proteins 291</p> <p>12.2 Relevance of Analytical Protocols Applied in Determining Immune Response to Protein Therapeutics to the Detection of Anti-Transgene Protein Responses 294</p> <p>12.3 Analysis of Immune Response by Binding and Functional Antibody Assay Protocols 295</p> <p>12.4 Comparative Analysis of the Immune Response Evaluation for Transgene Proteins that are Expressed Extracellularly vs. Intracellularly 297</p> <p>12.5 Humoral Immune Response to Gene Editing Reagents 298</p> <p><b>13 rAAV Integration: Detection and Risk Assessment 317<br /></b><i>Jing Yuan, Irene Gil-Farina, Raffaele Fronza, and Laurence O. Whiteley</i></p> <p>13.1 Introduction 317</p> <p>13.2 Review of Regulatory Guidance and Discussion Points that Are Raised on AAV Carcinogenesis 324</p> <p>13.3 Assessing the Biologic Relevance of AAV Integration Profile 335</p> <p>13.4 Conclusion and Future Direction 337</p> <p><b>14 Detection and Quantification of Genome Editing Events in Preclinical and Clinical Studies 347<br /></b><i>Marina Falaleeva, Shengdar Tsai, Kathleen Meyer, and Yanmei Lu</i></p> <p>14.1 Introduction 347</p> <p>14.2 Regulatory Guidance on Engineered Nuclease On- and Off-target Assessment 352</p> <p>14.3 Strategies and Methodologies to Evaluate On-target and Off-target Activities 353</p> <p>14.4 Concluding Remarks 376</p> <p><b>Section IV Companion Diagnostic Development for Gene Therapy 383</b></p> <p><b>15 Introduction to Companion Diagnostics for Gene Therapy 385<br /></b><i>Paul Bartel and Jennifer Granger</i></p> <p>15.1 Introduction to Companion Diagnostics 385</p> <p>15.2 Role in Gene Therapy 386</p> <p>15.3 Overall Strategy 387</p> <p>15.4 Development Process 387</p> <p>15.5 Considerations for Commercialization 390</p> <p>15.6 Conclusion 391</p> <p><b>16 Validation for Gene Therapy Companion Diagnostics 393<br /></b><i>Karen L. Richards and Kennon Daniels</i></p> <p>16.1 Introduction 393</p> <p>16.2 Development of CTAs for Use in GTx Clinical Trials 397</p> <p>16.3 Best Practices for Sample Banking and Consent of Subjects 401</p> <p>16.4 Design Considerations 402</p> <p>16.5 Bridging Studies 404</p> <p>16.6 Commensurate Regulatory Review and Approval of GTx cdx 406</p> <p>16.7 Concluding Sections 406</p> <p><b>17 Regulatory Considerations for Gene Therapy Companion Diagnostics 409<br /></b><i>Mica Elizalde and Paul Bartel</i></p> <p>17.1 Introduction 409</p> <p>17.2 US Fda 409</p> <p>17.3 European Union 416</p> <p>17.4 Other Regulated Markets 420</p> <p>17.5 Development Strategy with the Therapeutic 422</p> <p>17.6 Partner Relationship 424</p> <p>17.7 Commercial and Post-Approval Considerations 425</p> <p>17.8 Final Word 426</p> <p><b>Section V Regulatory Perspectives on Gene Therapy 429</b></p> <p><b>18 Current Regulatory Landscape for Gene Therapy Product Development and the Role of Biomarkers 431<br /></b><i>Laura I. Salazar-Fontana PhD and Mike Havert PhD</i></p> <p>18.1 Introduction 431</p> <p>18.2 What is Gene Therapy? 432</p> <p>18.3 Biomarkers Defined 433</p> <p>18.4 Early Gene Therapy Biomarkers 434</p> <p>18.5 Current Expectations for Gene Therapy Biomarkers 437</p> <p>18.6 Safety Biomarkers for Gene Therapy Products 438</p> <p>18.7 Concluding Remarks 442</p> <p>References 443</p> <p>Index 449</p>

<p><b>Yanmei Lu,</b> 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. <p><b>Boris Gorovits,</b> 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.

<p><b>Industry-centric perspective on translational and bioanalytical challenges and best practices for gene therapies</b> <p><i>Drug Development for Gene Therapy</i> focuses on the translational and bioanalytical challenges and best practices for gene therapy modalities, presenting a significant body of data, including information related to safety and efficacy, necessary to advance through the development pipeline into clinical use. The text covers bioanalytical methods and platforms including patient screening assays, different PCR tests, enzyme activity assays, ELISpot, NGS, LC/MS, and immunoassays, with FDA and EMA guidelines on gene therapy safety and efficacy, along with companion diagnostics regulations from US and EU perspectives. <p>The chapters offer an in-depth discussion of the basics and best practices for translational biomarkers, bioanalysis, and developing companion diagnostics / lab tests for gene therapies in the pharma and biopharma industries. To aid in reader comprehension, the text includes clinical examples of relevant therapies in related chapters. Some of the core topics covered include study design, immunogenicity, various bioanalytical methods and their applications, and global regulatory issues. <p>Written by two highly qualified authors with significant experience in the field, <i>Drug Development for Gene Therapy</i> includes information on: <ul><li>Bioanalytical methods to detect pre-existing antibodies against adeno-associated viruses (AAV) capsids</li> <li>Detection of cellular immunity and humoral response to viral capsids and transgene proteins, and immunogenicity of gene therapy products</li> <li>Nonclinical and clinical study considerations and methods for biodistribution and shedding</li> <li>Quantification of transgene protein expression and biochemical function, and substrate and distal pharmacodynamic biomarker measurements for gene therapy</li> <li>Detection and quantification of rAAV integration and off-target editing</li> <li>Current regulatory landscape for gene therapy product development and the role of biomarkers and general regulatory considerations for gene therapy companion diagnostics</li></ul> <p>With comprehensive coverage of the subject, <i>Drug Development for Gene Therapy</i> is a must-have resource for researchers and developers in the areas of pharmaceuticals, biopharmaceuticals, and contract research organizations (CROs), along with professors, researchers, and advanced students in chemistry, biological, biomedical engineering, pharmaceuticals, and medical sciences.

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