{"product_id":"drug-development-for-gene-therapy-9781119852780","title":"Drug Development for Gene Therapy","description":"\u003cb\u003eBook Synopsis\u003c\/b\u003e\u003cbr\u003e\u003cbr\u003e\u003cbr\u003e\u003cb\u003eTable of Contents\u003c\/b\u003e\u003cbr\u003e\u003cp\u003eList of Contributors xix\u003c\/p\u003e \u003cp\u003ePreface xxiii\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection I Introduction 1\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e\u003cb\u003e1 Introduction to AAV-based in vivo Gene Therapy 3\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eOscar Segurado\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e1.1 Introduction 3\u003c\/p\u003e \u003cp\u003e1.1.1 History of Gene Therapy 3\u003c\/p\u003e \u003cp\u003e1.1.2 AAV-based in vivo Gene Therapy: A Revolution in Medicine 4\u003c\/p\u003e \u003cp\u003e1.1.3 The AAV Vector Structure 11\u003c\/p\u003e \u003cp\u003e1.1.4 Cell Entry and Transduction Pathway 12\u003c\/p\u003e \u003cp\u003e1.2 Advantages and Disadvantages for AAV in vivo 13\u003c\/p\u003e \u003cp\u003e1.2.1 Effectiveness and Advantages of AAV Vectors for in vivo Gene Therapy 13\u003c\/p\u003e \u003cp\u003e1.2.2 Challenges of AAV Vectors for in vivo Gene Therapy 14\u003c\/p\u003e \u003cp\u003e1.3 Technology Platforms of AAV-based in vivo Gene Therapy 14\u003c\/p\u003e \u003cp\u003e1.3.1 cDNA Replacement 15\u003c\/p\u003e \u003cp\u003e1.3.2 Genome Editing 15\u003c\/p\u003e \u003cp\u003e1.3.2.1 Zfn 16\u003c\/p\u003e \u003cp\u003e1.3.2.2 TALENs 16\u003c\/p\u003e \u003cp\u003e1.3.2.3 CRISPR\/Cas 9 16\u003c\/p\u003e \u003cp\u003e1.3.3 Base Editing and Prime Editing 17\u003c\/p\u003e \u003cp\u003e1.3.4 RNAi Gene Silencing 17\u003c\/p\u003e \u003cp\u003e1.3.5 Gene Addition 18\u003c\/p\u003e \u003cp\u003e1.4 AAV Serotypes and Tissue Affinity 18\u003c\/p\u003e \u003cp\u003e1.4.1 The Liver as a Biofactory 19\u003c\/p\u003e \u003cp\u003e1.4.2 The CNS as a Biofactory 19\u003c\/p\u003e \u003cp\u003e1.4.3 The Muscle as a Biofactory 19\u003c\/p\u003e \u003cp\u003e1.5 Precision Medicine: Screening and Monitoring Biomarkers, Companion Diagnostics 19\u003c\/p\u003e \u003cp\u003e1.5.1 Gene Therapy Clinical Trials: Spotlight on Hemophilia A 20\u003c\/p\u003e \u003cp\u003e1.6 Predictions for Scientific and Medical Progress 22\u003c\/p\u003e \u003cp\u003e1.6.1 Predictions for Challenges in the Field 22\u003c\/p\u003e \u003cp\u003e1.6.2 Addressing Durability 23\u003c\/p\u003e \u003cp\u003e1.6.3 Addressing Immunogenicity 24\u003c\/p\u003e \u003cp\u003e1.6.4 Addressing Malignancy 24\u003c\/p\u003e \u003cp\u003e1.7 Predictions for Market Adoption 24\u003c\/p\u003e \u003cp\u003e1.7.1 Patients and Patient Advocacy Groups 25\u003c\/p\u003e \u003cp\u003e1.7.2 Physicians, Clinical Guidelines, Regulatory Agencies 25\u003c\/p\u003e \u003cp\u003e1.7.3 Payers 26\u003c\/p\u003e \u003cp\u003e1.8 Final Thoughts 26\u003c\/p\u003e \u003cp\u003e1.8.1 Can We Afford in vivo Gene Therapies? 26\u003c\/p\u003e \u003cp\u003e1.8.2 Can in vivo Gene Editing Replace Gene Therapy? 27\u003c\/p\u003e \u003cp\u003eReferences 28\u003c\/p\u003e \u003cp\u003e\u003cb\u003e2 Recent Development in in vivo Clinical Gene Therapy Platforms 35\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eJohn Murphy and Jane Owens\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e2.1 Introduction 35\u003c\/p\u003e \u003cp\u003e2.1.1 rAAV-cDNA Replacement Therapies 35\u003c\/p\u003e \u003cp\u003e2.1.1.1 Introduction: Approved rAAV-cDNA Replacement Therapies 36\u003c\/p\u003e \u003cp\u003e2.1.1.2 Glybera (alipogene tiparvovec), Marketed by uniQure 36\u003c\/p\u003e \u003cp\u003e2.1.1.3 Luxturna (voretigene neparvovec-rzyl), Marketed by Spark Therapeutics 38\u003c\/p\u003e \u003cp\u003e2.1.1.4 Zolgensma (onasemnogene abeparvovec), Marketed by Novartis 40\u003c\/p\u003e \u003cp\u003e2.1.2 Introduction: rAAV-cDNA (gene) Therapy Candidates in Clinical Development 46\u003c\/p\u003e \u003cp\u003e2.1.2.1 AAV-Gene Replacement Clinical Trials for the Eye 47\u003c\/p\u003e \u003cp\u003e2.1.2.2 Clinical Trials for Heart Disease 47\u003c\/p\u003e \u003cp\u003e2.1.2.3 Clinical Trials for Hematologic and Metabolic Disease (Targeting the Liver) 48\u003c\/p\u003e \u003cp\u003e2.1.2.4 Clinical Trials for Skeletal Muscle 48\u003c\/p\u003e \u003cp\u003e2.1.3 Introduction: rAAV-as a Vehicle for in vivo Gene Editing 48\u003c\/p\u003e \u003cp\u003e2.1.3.1 Non-nuclease Mediated Methods 48\u003c\/p\u003e \u003cp\u003e2.1.3.2 Nuclease-mediated Homology Directed Repair 52\u003c\/p\u003e \u003cp\u003e2.1.4 Nuclease-mediated Gene Disruption following AAV Delivery 54\u003c\/p\u003e \u003cp\u003e2.1.5 Challenges and Opportunities with AAV as a Delivery Vehicle for Nuclease-Mediated Gene Editing 56\u003c\/p\u003e \u003cp\u003eReferences 56\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection II Translational Biomarkers for Gene Therapy 61\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e\u003cb\u003e3 Biomarker and Bioanalytical Readouts for the Development of AAV Gene Therapy 63\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eYanmei Lu and Wibke Lembke\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e3.1 Introduction 63\u003c\/p\u003e \u003cp\u003e3.1.1 AAV-Mediated in vivo Gene Therapy 63\u003c\/p\u003e \u003cp\u003e3.1.2 Biomarker Category and Utility 65\u003c\/p\u003e \u003cp\u003e3.2 Pharmacokinetic (PK) and Pharmacodynamic (PD) Biomarkers 66\u003c\/p\u003e \u003cp\u003e3.2.1 Viral Biodistribution and Shedding 66\u003c\/p\u003e \u003cp\u003e3.2.2 Transgene mRNA Expression 68\u003c\/p\u003e \u003cp\u003e3.2.3 Transgene and Target Protein Activity and Concentration 68\u003c\/p\u003e \u003cp\u003e3.2.4 Substrate and Other Distal PD Biomarkers 70\u003c\/p\u003e \u003cp\u003e3.3 Safety and Monitoring Biomarkers and Readouts 71\u003c\/p\u003e \u003cp\u003e3.3.1 Assessment of genotoxicity 72\u003c\/p\u003e \u003cp\u003e3.3.1.1 AAV Integration\/Insertional Mutagenesis Risk 72\u003c\/p\u003e \u003cp\u003e3.3.1.2 AAV Germline Transmission Risk 73\u003c\/p\u003e \u003cp\u003e3.3.1.3 Off-Target Gene Editing 73\u003c\/p\u003e \u003cp\u003e3.3.2 Biomarkers for Immune-Mediated Toxicity 74\u003c\/p\u003e \u003cp\u003e3.3.2.1 Hepatotoxicity 74\u003c\/p\u003e \u003cp\u003e3.3.2.2 Thrombotic Microangiopathy 76\u003c\/p\u003e \u003cp\u003e3.3.2.3 Muscle Toxicity 77\u003c\/p\u003e \u003cp\u003e3.3.2.4 Immunogenicity Assessment for rAAV Gene Therapy 77\u003c\/p\u003e \u003cp\u003e3.3.3 Safety Biomarkers for Nonimmune Organ-Specific Toxicity 78\u003c\/p\u003e \u003cp\u003e3.3.3.1 Dorsal Root Ganglia Toxicity 78\u003c\/p\u003e \u003cp\u003e3.3.3.2 Other Target Organ Toxicity Biomarkers 79\u003c\/p\u003e \u003cp\u003e3.4 Predictive and Diagnostic Biomarkers for Study Enrollment and Patient Stratification 80\u003c\/p\u003e \u003cp\u003e3.4.1 Preexisting Anti-Capsid Antibody 80\u003c\/p\u003e \u003cp\u003e3.4.1.1 Companion Diagnostic 81\u003c\/p\u003e \u003cp\u003e3.4.2 Preexisting Anti-Transgene Protein Antibody 81\u003c\/p\u003e \u003cp\u003e3.5 Summary 82\u003c\/p\u003e \u003cp\u003eReferences 82\u003c\/p\u003e \u003cp\u003e\u003cb\u003e4 Nonclinical and Clinical Study Considerations for Biodistribution, Shedding, and Pharmacokinetics\/Pharmacodynamics 87\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eManuela Braun and Kefeng Sun\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e4.1 Biodistribution and Viral Shedding 87\u003c\/p\u003e \u003cp\u003e4.1.1 Introduction to Biodistribution and Viral Shedding 87\u003c\/p\u003e \u003cp\u003e4.1.1.1 Definition and Terminology for Biodistribution and Shedding 88\u003c\/p\u003e \u003cp\u003e4.1.1.2 Global Regulatory Guidance on Conducting Biodistribution and Shedding Studies 88\u003c\/p\u003e \u003cp\u003e4.1.2 Nonclinical Biodistribution and Shedding Studies for AAV Vectors 89\u003c\/p\u003e \u003cp\u003e4.1.2.1 Design, Execution, and Reporting 90\u003c\/p\u003e \u003cp\u003e4.1.2.2 Examples 95\u003c\/p\u003e \u003cp\u003e4.1.3 Clinical Biodistribution and Shedding Studies for AAV Vectors 96\u003c\/p\u003e \u003cp\u003e4.1.3.1 General Considerations in Viral Shedding Studies in the Clinical Setting 97\u003c\/p\u003e \u003cp\u003e4.1.3.2 Biodistribution Characterization in Human: Necessity and Concerns 98\u003c\/p\u003e \u003cp\u003e4.1.3.3 Examples 98\u003c\/p\u003e \u003cp\u003e4.1.4 Gaps and Challenges on Biodistribution and Shedding Characterization 99\u003c\/p\u003e \u003cp\u003e4.2 Pharmacokinetic\/Pharmacodynamic (PK\/PD) Modeling and Clinical Dose Selection of Gene Therapy 100\u003c\/p\u003e \u003cp\u003e4.2.1 Overview on PK\/PD and Dose Selection Strategies for Gene Therapy 100\u003c\/p\u003e \u003cp\u003e4.2.1.1 AAV Dosing Regimen – Safety Relationship and Safety-based Clinical Dose Projection 101\u003c\/p\u003e \u003cp\u003e4.2.1.2 AAV Dose – Pharmacodynamics\/Efficacy Relationship and Projection of Pharmacologically-Active Dose (PAD) 102\u003c\/p\u003e \u003cp\u003e4.2.2 Dose Scaling Approaches: Allometric and Activity-Based Methods 102\u003c\/p\u003e \u003cp\u003e4.2.3 Mechanistic Approaches to Modeling Gene Therapy 105\u003c\/p\u003e \u003cp\u003e4.2.3.1 Modeling and Simulation of AAV Biodistribution 106\u003c\/p\u003e \u003cp\u003e4.2.3.2 Modeling Transgene Product PK and PD of the Transgene Product 106\u003c\/p\u003e \u003cp\u003e4.2.4 Clinical Pharmacology Considerations for Gene Therapy 106\u003c\/p\u003e \u003cp\u003e4.2.4.1 Variability in Transgene Product Levels and\/or Treatment Response 106\u003c\/p\u003e \u003cp\u003e4.2.4.2 Durability of Transgene Expression and\/or Treatment Response 107\u003c\/p\u003e \u003cp\u003e4.2.5 Gaps and Challenges on PK\/PD and Clinical Dose Selection 108\u003c\/p\u003e \u003cp\u003e4.2.5.1 Interspecies difference in AAV Transduction and Immunogenicity 108\u003c\/p\u003e \u003cp\u003e4.2.5.2 Availability of Clinical Samples and Bioanalytical Assays 109\u003c\/p\u003e \u003cp\u003e4.2.5.3 Availability of Long-Term Follow-Up Data 109\u003c\/p\u003e \u003cp\u003e4.3 Summary 109\u003c\/p\u003e \u003cp\u003eReferences 110\u003c\/p\u003e \u003cp\u003e\u003cb\u003e5 Immunogenicity of AAV Gene Therapy Products 117\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eVibha Jawa and Bonnie Wu\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e5.1 Innate and Adaptive Immunity Induced by AAV-Based Gene Therapies 117\u003c\/p\u003e \u003cp\u003e5.1.1 Innate Immune Response 117\u003c\/p\u003e \u003cp\u003e5.1.2 Adaptive Immune Response 119\u003c\/p\u003e \u003cp\u003e5.2 Preclinical Immunogenicity Risk Assessment 119\u003c\/p\u003e \u003cp\u003e5.2.1 Product-related Risk Factors 120\u003c\/p\u003e \u003cp\u003e5.2.2 Process and Manufacturing-Related Risk Factors 120\u003c\/p\u003e \u003cp\u003e5.2.3 Patient-Related Risk Factors 121\u003c\/p\u003e \u003cp\u003e5.2.4 Nonclinical Assessment of Immunogenicity 121\u003c\/p\u003e \u003cp\u003e5.2.5 Animal Models for Assessing Innate Immunity 122\u003c\/p\u003e \u003cp\u003e5.2.6 Animal Models for Assessing Adaptive Immunity 122\u003c\/p\u003e \u003cp\u003e5.2.7 Impact of Immunogenicity on Animal Selection and Interpretation of Study Results 123\u003c\/p\u003e \u003cp\u003e5.3 Clinical Manifestation Associated with Immunogenicity 123\u003c\/p\u003e \u003cp\u003e5.3.1 Pre-existing Immunity Against AAV Vector May Compromise Therapeutic Efficacy and Patient Safety 124\u003c\/p\u003e \u003cp\u003e5.3.2 Treatment Induced Anti-AAV Capsid Antibodies may Prevent Re-dosing 124\u003c\/p\u003e \u003cp\u003e5.3.3 Antibody Specific to Transgene Protein could lead to Toxicity or Unwanted Immunity 125\u003c\/p\u003e \u003cp\u003e5.3.4 Risk of Immunogenicity Associated with Different Administration Routes 125\u003c\/p\u003e \u003cp\u003e5.3.4.1 Gene Delivery to the Eye or Central Nervous System 126\u003c\/p\u003e \u003cp\u003e5.3.4.2 Gene Delivery to Liver 126\u003c\/p\u003e \u003cp\u003e5.3.4.3 Gene Delivery to Muscle 126\u003c\/p\u003e \u003cp\u003e5.3.5 Product- and Process-related Impurity Related Immunogenicity 127\u003c\/p\u003e \u003cp\u003e5.4 Clinical Mitigation Strategy 127\u003c\/p\u003e \u003cp\u003eReferences 129\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection III Bioanalysis for Gene Therapy 135\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e\u003cb\u003e6 Bioanalytical Methods to Detect Preexisting and Post-administration Humoral Immune Responses Against AAV Capsid Proteins 137\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eChristian Vettermann and Boris Gorovits\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e6.1 Introduction 137\u003c\/p\u003e \u003cp\u003e6.2 Considerations for AAV Total Antibody Assays 138\u003c\/p\u003e \u003cp\u003e6.2.1 Nature of AAV TAb Assay Analyte 138\u003c\/p\u003e \u003cp\u003e6.2.2 Primary Analytical Methodologies applied for AAV TAb Detection 139\u003c\/p\u003e \u003cp\u003e6.2.3 Tab Assay Critical Reagent Considerations 140\u003c\/p\u003e \u003cp\u003e6.2.3.1 Positive and Negative Control Selection 140\u003c\/p\u003e \u003cp\u003e6.2.3.2 Capture and Detection Reagents 141\u003c\/p\u003e \u003cp\u003e6.2.3.3 Sample Testing Strategy 142\u003c\/p\u003e \u003cp\u003e6.2.4 Key Assay Qualification\/Validation Parameters 142\u003c\/p\u003e \u003cp\u003e6.2.4.1 Assay Sensitivity 142\u003c\/p\u003e \u003cp\u003e6.2.4.2 Serotype Specificity 142\u003c\/p\u003e \u003cp\u003e6.2.4.3 Precision 143\u003c\/p\u003e \u003cp\u003e6.2.4.4 Matrix Interference and Selectivity 143\u003c\/p\u003e \u003cp\u003e6.2.4.5 Assay Cut-Point 143\u003c\/p\u003e \u003cp\u003e6.2.5 TAb Assay Data Interpretation 144\u003c\/p\u003e \u003cp\u003e6.3 Considerations for Cell-based Transduction Inhibition Assays 145\u003c\/p\u003e \u003cp\u003e6.3.1 Principle and Methodology of Cell-based AAV TI Assays 145\u003c\/p\u003e \u003cp\u003e6.3.2 AAV TI Assay Development: Designing for Clinical Relevance 146\u003c\/p\u003e \u003cp\u003e6.3.3 Key Assay Validation Parameters 147\u003c\/p\u003e \u003cp\u003e6.3.3.1 Screening and Titer Cut-Points 147\u003c\/p\u003e \u003cp\u003e6.3.3.2 Limit of Detection 148\u003c\/p\u003e \u003cp\u003e6.3.3.3 Precision 150\u003c\/p\u003e \u003cp\u003e6.3.3.4 Specificity 150\u003c\/p\u003e \u003cp\u003e6.3.3.5 Confirmatory Steps to Ensure Specific Detection of Neutralizing AAV Antibodies 150\u003c\/p\u003e \u003cp\u003e6.3.3.6 Selectivity\/Matrix Interference 151\u003c\/p\u003e \u003cp\u003e6.3.3.7 Stability 151\u003c\/p\u003e \u003cp\u003e6.3.4 Sample Testing Strategy and Monitoring Assay Performance 152\u003c\/p\u003e \u003cp\u003e6.3.5 Data Interpretation: Preexisting TI Titer and Clinical Efficacy 152\u003c\/p\u003e \u003cp\u003e6.3.6 Value and Challenges of Standardizing TAb and TI Assays 156\u003c\/p\u003e \u003cp\u003eReferences 157\u003c\/p\u003e \u003cp\u003e\u003cb\u003e7 Bioanalytical Methods to Study Biodistribution and Shedding of AAV-Based Gene Therapy Vectors 163\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eChristian Vettermann and Russell Soon\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e7.1 Introduction 163\u003c\/p\u003e \u003cp\u003e7.2 Choice of Platform: qPCR vs. Digital PCR 164\u003c\/p\u003e \u003cp\u003e7.3 Aspects of Method Development 168\u003c\/p\u003e \u003cp\u003e7.4 Back-Calculation Formulas and Extraction Efficiency Assessments 172\u003c\/p\u003e \u003cp\u003e7.5 Sensitivity Requirements 177\u003c\/p\u003e \u003cp\u003e7.6 Specificity Requirements 179\u003c\/p\u003e \u003cp\u003e7.7 Standard Curve Performance, Colinearity, Precision, and Accuracy 180\u003c\/p\u003e \u003cp\u003e7.8 Selectivity Assessment and Matrix Interference 181\u003c\/p\u003e \u003cp\u003e7.9 Sample Stability Considerations 182\u003c\/p\u003e \u003cp\u003e7.10 Data Reporting Formats, Acceptance Criteria, and Trending 184\u003c\/p\u003e \u003cp\u003e7.11 Immunocapture qPCR: An Ultra-Sensitive Method to Detect Intact AAV Capsids 187\u003c\/p\u003e \u003cp\u003eReferences 189\u003c\/p\u003e \u003cp\u003e\u003cb\u003e8 Transgene mRNA Expression Analysis 193\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eVenkata Vepachedu and Hsing-Yin Liu\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e8.1 Purpose of Measuring Transgene mRNA 193\u003c\/p\u003e \u003cp\u003e8.1.1 Transgene Encodes Therapeutic Protein Entity 194\u003c\/p\u003e \u003cp\u003e8.1.2 Transgene Encodes Other Entities 196\u003c\/p\u003e \u003cp\u003e8.2 Technologies to Quantify Transgene Expression in Tissues 196\u003c\/p\u003e \u003cp\u003e8.2.1 RT-qPCR or RT-dPCR 196\u003c\/p\u003e \u003cp\u003e8.2.1.1 RNA Extraction (Separate vs. DNA\/RNA Co-extraction), Quality Testing, and Quantification 197\u003c\/p\u003e \u003cp\u003e8.2.1.2 Co-extraction of DNA and RNA from same Sample 199\u003c\/p\u003e \u003cp\u003e8.2.1.3 Quantification and Quality Testing of total RNA in Purified Extracts 200\u003c\/p\u003e \u003cp\u003e8.2.1.4 Quantification Using DNA vs. RNA Standards 201\u003c\/p\u003e \u003cp\u003e8.2.1.5 Assay Qualification\/Validation and Report 201\u003c\/p\u003e \u003cp\u003e8.2.1.6 Reporting 205\u003c\/p\u003e \u003cp\u003e8.2.2 In Situ Hybridization (ISH) 206\u003c\/p\u003e \u003cp\u003e8.2.2.1 Values of ISH for Discovery Studies 207\u003c\/p\u003e \u003cp\u003e8.2.2.2 Semi-quantitative, Tissue Fixation, Probe to Reference Classic Procedure 208\u003c\/p\u003e \u003cp\u003e8.3 Summary 211\u003c\/p\u003e \u003cp\u003eReferences 211\u003c\/p\u003e \u003cp\u003e\u003cb\u003e9 Quantification of Transgene Protein Expression and Biochemical Function 215\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eRobert Dodge and Liching Cao\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e9.1 Introduction 215\u003c\/p\u003e \u003cp\u003e9.2 Transgene Protein Concentration Determination 216\u003c\/p\u003e \u003cp\u003e9.2.1 Human Transgene in Preclinical Species 216\u003c\/p\u003e \u003cp\u003e9.2.2 Human Transgene Assessment for Intracellular Proteins 216\u003c\/p\u003e \u003cp\u003e9.2.3 Human Transgene Protein Assessment for Non-secreted Proteins 218\u003c\/p\u003e \u003cp\u003e9.2.4 Human Transgene Protein Assessment for Secreted Proteins 220\u003c\/p\u003e \u003cp\u003e9.2.5 Human Transgene Protein Assessment for Expressed Therapeutics 221\u003c\/p\u003e \u003cp\u003e9.2.6 Transgene Protein Assay Format Considerations 221\u003c\/p\u003e \u003cp\u003e9.2.6.1 Immunoassays 222\u003c\/p\u003e \u003cp\u003e9.2.6.2 Mass Spectrometry Assays 222\u003c\/p\u003e \u003cp\u003e9.2.6.3 Semiquantitative Assay Formats 223\u003c\/p\u003e \u003cp\u003e9.3 Transgene Protein Activity Determination 224\u003c\/p\u003e \u003cp\u003e9.3.1 Method Development Considerations 224\u003c\/p\u003e \u003cp\u003e9.3.1.1 Enzyme Kinetics, the Initial Rate of Reaction, and Substrate Concentration 224\u003c\/p\u003e \u003cp\u003e9.3.1.2 Reference Standard 226\u003c\/p\u003e \u003cp\u003e9.3.1.3 Sample Processing 228\u003c\/p\u003e \u003cp\u003e9.3.1.4 Buffers and Incubation Temperature 230\u003c\/p\u003e \u003cp\u003e9.3.1.5 Assay Dynamic Range, Minimum Required Dilution, Matrix Interference, and Parallelism 230\u003c\/p\u003e \u003cp\u003e9.3.1.6 Specificity and Selectivity 231\u003c\/p\u003e \u003cp\u003e9.3.1.7 Quality Controls (QCs) 232\u003c\/p\u003e \u003cp\u003e9.3.2 Method Validation 234\u003c\/p\u003e \u003cp\u003e9.4 Summary 234\u003c\/p\u003e \u003cp\u003eReferences 235\u003c\/p\u003e \u003cp\u003e\u003cb\u003e10 Substrate and Distal Pharmacodynamic Biomarker Measurements for Gene Therapy 239\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eLiching Cao, Kai Wang, John Lin, and Venkata Vepachedu\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e10.1 Introduction 239\u003c\/p\u003e \u003cp\u003e10.2 Technologies to Quantify Substrate and Distal PD Biomarker 241\u003c\/p\u003e \u003cp\u003e10.2.1 Liquid Chromatography\/Tandem Mass Spectrometry (lc-ms\/ms) 241\u003c\/p\u003e \u003cp\u003e10.2.1.1 Method Development Challenges and Resolutions 241\u003c\/p\u003e \u003cp\u003e10.2.1.2 Method Validation by LC-MS\/MS 245\u003c\/p\u003e \u003cp\u003e10.2.2 Histology 246\u003c\/p\u003e \u003cp\u003e10.2.3 Functional Activity and Immunoassays 248\u003c\/p\u003e \u003cp\u003e10.2.3.1 Method Validation of Immunoassay 249\u003c\/p\u003e \u003cp\u003e10.2.4 mRNA Detection of Downstream Target Expression as a PD Biomarker 253\u003c\/p\u003e \u003cp\u003e10.2.4.1 RT-qPCR for Relative Gene Expression Analysis 254\u003c\/p\u003e \u003cp\u003e10.2.4.2 RNA-seq 259\u003c\/p\u003e \u003cp\u003e10.2.4.3 Nanostring Technology 260\u003c\/p\u003e \u003cp\u003e10.2.4.4 Regulatory Considerations for RNA Quantitation in GLP Studies 261\u003c\/p\u003e \u003cp\u003e10.2.5 Single-cell Analysis 263\u003c\/p\u003e \u003cp\u003e10.3 Summary 265\u003c\/p\u003e \u003cp\u003eReferences 266\u003c\/p\u003e \u003cp\u003e\u003cb\u003e11 Detection of Cellular Immunity to Viral Capsids and Transgene Proteins 271\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eMaurus de la Rosa and Magdalena Tary-Lehmann\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e11.1 Introduction 271\u003c\/p\u003e \u003cp\u003e11.1.1 Humoral and Cellular Immune Responses to Gene Therapy 271\u003c\/p\u003e \u003cp\u003e11.1.2 Selected Clinical Observations Showing the Lack of Understanding About T-Cell-Mediated Immune Responses and the Need for Sensitive T-Cell Analytics 272\u003c\/p\u003e \u003cp\u003e11.2 Methods for the Detection of Cellular Immune Responses 274\u003c\/p\u003e \u003cp\u003e11.2.1 Methods to Detect T-Cell Responses in Clinical Trials 274\u003c\/p\u003e \u003cp\u003e11.2.1.1 Enzyme-Linked Immunosorbent Spot Assay 274\u003c\/p\u003e \u003cp\u003e11.2.1.2 Intracellular Cytokine Staining 276\u003c\/p\u003e \u003cp\u003e11.2.1.3 Tetramer Staining 276\u003c\/p\u003e \u003cp\u003e11.2.1.4 Proliferation Assays 276\u003c\/p\u003e \u003cp\u003e11.2.1.5 Cytokine Bead Array 276\u003c\/p\u003e \u003cp\u003e11.2.1.6 Gene Expression Profiling 277\u003c\/p\u003e \u003cp\u003e11.2.1.7 Multiplexed Epitope Mapping 277\u003c\/p\u003e \u003cp\u003e11.2.1.8 Conclusion 277\u003c\/p\u003e \u003cp\u003e11.2.2 Technical Challenges of Detecting Cellular Immune Responses 277\u003c\/p\u003e \u003cp\u003e11.3 Validation of Cellular Assays Using PBMC (Example ELISPOT) 278\u003c\/p\u003e \u003cp\u003e11.3.1 Validation Strategies 278\u003c\/p\u003e \u003cp\u003e11.3.1.1 Precision 279\u003c\/p\u003e \u003cp\u003e11.3.1.2 Specificity 279\u003c\/p\u003e \u003cp\u003e11.3.1.3 Limit of Detection and Range 280\u003c\/p\u003e \u003cp\u003e11.3.1.4 Common Exceptions for ELISPOT Validation: Accuracy, Linearity, and Reproducibility 281\u003c\/p\u003e \u003cp\u003e11.3.2 Parameters Affecting ELISPOT Assay Performance 282\u003c\/p\u003e \u003cp\u003e11.3.2.1 PBMC Sample Handling: Temperature, Resting, and Serum 282\u003c\/p\u003e \u003cp\u003e11.3.2.2 Antigen Concentration and Number of Replicates 285\u003c\/p\u003e \u003cp\u003eReferences 286\u003c\/p\u003e \u003cp\u003e\u003cb\u003e12 Detection of Humoral Response to Transgene Protein and Gene Editing Reagents 291\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eGeorge Buchlis and Boris Gorovits\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e12.1 Pre- and Post-dose Humoral Immunity to Transgene-expressed Proteins 291\u003c\/p\u003e \u003cp\u003e12.1.1 Risk-based Analysis of Response Probability and Impact 291\u003c\/p\u003e \u003cp\u003e12.1.1.1 Route of Administration 291\u003c\/p\u003e \u003cp\u003e12.1.1.2 Biodistribution of Vector, Vector Serotype, Dose, and Expression Level 293\u003c\/p\u003e \u003cp\u003e12.1.1.3 Patient Immune Status: Age, Prior Exposure, No Endogenous Production, Immunosuppression, and Autoimmunity 293\u003c\/p\u003e \u003cp\u003e12.1.1.4 Response Induction vs. Response Boosting 294\u003c\/p\u003e \u003cp\u003e12.2 Relevance of Analytical Protocols Applied in Determining Immune Response to Protein Therapeutics to the Detection of Anti-Transgene Protein Responses 294\u003c\/p\u003e \u003cp\u003e12.3 Analysis of Immune Response by Binding and Functional Antibody Assay Protocols 295\u003c\/p\u003e \u003cp\u003e12.4 Comparative Analysis of the Immune Response Evaluation for Transgene Proteins that are Expressed Extracellularly vs. Intracellularly 297\u003c\/p\u003e \u003cp\u003e12.5 Humoral Immune Response to Gene Editing Reagents 298\u003c\/p\u003e \u003cp\u003e12.5.1 Diversity of Gene Editing Systems 298\u003c\/p\u003e \u003cp\u003e12.5.2 Immunological Potential of CRISPR-Cas System 299\u003c\/p\u003e \u003cp\u003e12.5.3 Detection of Anti-Cas9 Protein Immunity in Animal and Human Matrix 301\u003c\/p\u003e \u003cp\u003e12.5.4 Strategies Proposed to Mitigate Anti-Cas9 Immunity 304\u003c\/p\u003e \u003cp\u003eReferences 304\u003c\/p\u003e \u003cp\u003e\u003cb\u003e13 rAAV Integration: Detection and Risk Assessment 317\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eJing Yuan, Irene Gil-Farina, Raffaele Fronza, and Laurence O. Whiteley\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e13.1 Introduction 317\u003c\/p\u003e \u003cp\u003e13.1.1 Biology of AAV Vectors as it Relates to Mechanisms of AAV Integration 318\u003c\/p\u003e \u003cp\u003e13.1.2 Literature Review of AAV Studies in Relation to Neoplasia Development 318\u003c\/p\u003e \u003cp\u003e13.2 Review of Regulatory Guidance and Discussion Points that Are Raised on AAV Carcinogenesis 324\u003c\/p\u003e \u003cp\u003e13.2.1 Factors to Consider in the Design of Nonclinical Studies Evaluating AAV Integration 325\u003c\/p\u003e \u003cp\u003e13.2.2 Methods for rAAV Integration Analysis 326\u003c\/p\u003e \u003cp\u003e13.2.3 AAV Data Analysis Methods 328\u003c\/p\u003e \u003cp\u003e13.2.3.1 AAV Primary Analysis 331\u003c\/p\u003e \u003cp\u003e13.2.3.2 Impurity Analysis 332\u003c\/p\u003e \u003cp\u003e13.2.3.3 AAV Genome Rearrangements 332\u003c\/p\u003e \u003cp\u003e13.2.3.4 Integration Site Analysis 332\u003c\/p\u003e \u003cp\u003e13.2.3.5 Clonality Analysis 333\u003c\/p\u003e \u003cp\u003e13.2.3.6 Genotoxic Integrations 334\u003c\/p\u003e \u003cp\u003e13.3 Assessing the Biologic Relevance of AAV Integration Profile 335\u003c\/p\u003e \u003cp\u003e13.4 Conclusion and Future Direction 337\u003c\/p\u003e \u003cp\u003eReferences 338\u003c\/p\u003e \u003cp\u003e\u003cb\u003e14 Detection and Quantification of Genome Editing Events in Preclinical and Clinical Studies 347\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eMarina Falaleeva, Shengdar Tsai, Kathleen Meyer, and Yanmei Lu\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e14.1 Introduction 347\u003c\/p\u003e \u003cp\u003e14.1.1 Genome Editing Modalities and Molecular Outcomes 348\u003c\/p\u003e \u003cp\u003e14.1.2 Clinical Trials Using Genome Editing Technologies 350\u003c\/p\u003e \u003cp\u003e14.2 Regulatory Guidance on Engineered Nuclease On- and Off-target Assessment 352\u003c\/p\u003e \u003cp\u003e14.3 Strategies and Methodologies to Evaluate On-target and Off-target Activities 353\u003c\/p\u003e \u003cp\u003e14.3.1 Strategies to Evaluate Off-target Sites in Preclinical and Clinical Studies 353\u003c\/p\u003e \u003cp\u003e14.3.2 Techniques to Identify Genome Wide Off-target Sites 355\u003c\/p\u003e \u003cp\u003e14.3.3 Targeted Approaches to Measure Short Insertions and Deletions 356\u003c\/p\u003e \u003cp\u003e14.3.3.1 Droplet Digital™ PCR 365\u003c\/p\u003e \u003cp\u003e14.3.3.2 Endonuclease Mismatch Cleavage Assays 366\u003c\/p\u003e \u003cp\u003e14.3.3.3 Sanger Sequencing Combined with Sequence Trace Decomposition 368\u003c\/p\u003e \u003cp\u003e14.3.3.4 Indel Detection by Amplicon Analysis (IDAA) 369\u003c\/p\u003e \u003cp\u003e14.3.4 Technologies to Measure Large Genomic Rearrangements 369\u003c\/p\u003e \u003cp\u003e14.3.5 Discussion 374\u003c\/p\u003e \u003cp\u003e14.4 Concluding Remarks 376\u003c\/p\u003e \u003cp\u003eReferences 376\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection IV Companion Diagnostic Development for Gene Therapy 383\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e\u003cb\u003e15 Introduction to Companion Diagnostics for Gene Therapy 385\u003cbr\u003e \u003c\/b\u003e\u003ci\u003ePaul Bartel and Jennifer Granger\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e15.1 Introduction to Companion Diagnostics 385\u003c\/p\u003e \u003cp\u003e15.2 Role in Gene Therapy 386\u003c\/p\u003e \u003cp\u003e15.3 Overall Strategy 387\u003c\/p\u003e \u003cp\u003e15.4 Development Process 387\u003c\/p\u003e \u003cp\u003e15.5 Considerations for Commercialization 390\u003c\/p\u003e \u003cp\u003e15.6 Conclusion 391\u003c\/p\u003e \u003cp\u003eReferences 391\u003c\/p\u003e \u003cp\u003e\u003cb\u003e16 Validation for Gene Therapy Companion Diagnostics 393\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eKaren L. Richards and Kennon Daniels\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e16.1 Introduction 393\u003c\/p\u003e \u003cp\u003e16.1.1 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\u003c\/p\u003e \u003cp\u003e16.1.2 Summary of Validation Requirements for Gene Therapy Companion Diagnostics (GTx CDx) 395\u003c\/p\u003e \u003cp\u003e16.1.3 Role of CDx in Therapeutic Development and Unique Challenges to Validating GTx CDx 395\u003c\/p\u003e \u003cp\u003e16.1.4 Key Considerations for Developing GTx cdx 396\u003c\/p\u003e \u003cp\u003e16.2 Development of CTAs for Use in GTx Clinical Trials 397\u003c\/p\u003e \u003cp\u003e16.2.1 Stratification vs. Selection 397\u003c\/p\u003e \u003cp\u003e16.2.2 Regulatory Risk Determination: Significant or Nonsignificant? 398\u003c\/p\u003e \u003cp\u003e16.2.3 CTA Design Considerations 400\u003c\/p\u003e \u003cp\u003e16.2.4 CTA Validation Requirements 401\u003c\/p\u003e \u003cp\u003e16.3 Best Practices for Sample Banking and Consent of Subjects 401\u003c\/p\u003e \u003cp\u003e16.3.1 Validation Strategies for CDxs for Commercial Use 401\u003c\/p\u003e \u003cp\u003e16.4 Design Considerations 402\u003c\/p\u003e \u003cp\u003e16.4.1 Single-site vs. Distributable Kit 402\u003c\/p\u003e \u003cp\u003e16.4.2 Validation Requirements 402\u003c\/p\u003e \u003cp\u003e16.5 Bridging Studies 404\u003c\/p\u003e \u003cp\u003e16.6 Commensurate Regulatory Review and Approval of GTx cdx 406\u003c\/p\u003e \u003cp\u003e16.7 Concluding Sections 406\u003c\/p\u003e \u003cp\u003e16.7.1 Summary of Validation Considerations for CTAs\/CDx in GTx Clinical Trials 406\u003c\/p\u003e \u003cp\u003e16.7.2 Summary of Validation Considerations for CTAs\/CDx to Enable GTx Marketing 407\u003c\/p\u003e \u003cp\u003eReferences 407\u003c\/p\u003e \u003cp\u003e\u003cb\u003e17 Regulatory Considerations for Gene Therapy Companion Diagnostics 409\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eMica Elizalde and Paul Bartel\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e17.1 Introduction 409\u003c\/p\u003e \u003cp\u003e17.2 US Fda 409\u003c\/p\u003e \u003cp\u003e17.2.1 Clinical Trials for Investigational Device Exemption 410\u003c\/p\u003e \u003cp\u003e17.2.2 US FDA Marketing Authorization Pathways 413\u003c\/p\u003e \u003cp\u003e17.2.2.1 510(k) process 413\u003c\/p\u003e \u003cp\u003e17.2.2.2 PMA Process 414\u003c\/p\u003e \u003cp\u003e17.2.2.3 HDE Process 414\u003c\/p\u003e \u003cp\u003e17.2.2.4 Differences Between 510(k) and PMA 415\u003c\/p\u003e \u003cp\u003e17.2.3 US FDA Pre-submission Feedback 416\u003c\/p\u003e \u003cp\u003e17.3 European Union 416\u003c\/p\u003e \u003cp\u003e17.3.1 European Union Clinical Trials 416\u003c\/p\u003e \u003cp\u003e17.3.2 European Union Marketing Authorization Pathways 418\u003c\/p\u003e \u003cp\u003e17.4 Other Regulated Markets 420\u003c\/p\u003e \u003cp\u003e17.4.1 Global Regulatory Strategy 421\u003c\/p\u003e \u003cp\u003e17.5 Development Strategy with the Therapeutic 422\u003c\/p\u003e \u003cp\u003e17.5.1 Considerations for Rare Disease Indications 423\u003c\/p\u003e \u003cp\u003e17.6 Partner Relationship 424\u003c\/p\u003e \u003cp\u003e17.6.1 Importance of the Partner Relationship 424\u003c\/p\u003e \u003cp\u003e17.7 Commercial and Post-Approval Considerations 425\u003c\/p\u003e \u003cp\u003e17.7.1 Future Proofing the Companion Diagnostic 425\u003c\/p\u003e \u003cp\u003e17.7.2 Modifications of the Companion Diagnostic 426\u003c\/p\u003e \u003cp\u003e17.8 Final Word 426\u003c\/p\u003e \u003cp\u003eReferences 426\u003c\/p\u003e \u003cp\u003e\u003cb\u003eSection V Regulatory Perspectives on Gene Therapy 429\u003c\/b\u003e\u003c\/p\u003e \u003cp\u003e\u003cb\u003e18 Current Regulatory Landscape for Gene Therapy Product Development and the Role of Biomarkers 431\u003cbr\u003e \u003c\/b\u003e\u003ci\u003eLaura I. Salazar-Fontana PhD and Mike Havert PhD\u003c\/i\u003e\u003c\/p\u003e \u003cp\u003e18.1 Introduction 431\u003c\/p\u003e \u003cp\u003e18.2 What is Gene Therapy? 432\u003c\/p\u003e \u003cp\u003e18.3 Biomarkers Defined 433\u003c\/p\u003e \u003cp\u003e18.4 Early Gene Therapy Biomarkers 434\u003c\/p\u003e \u003cp\u003e18.5 Current Expectations for Gene Therapy Biomarkers 437\u003c\/p\u003e \u003cp\u003e18.6 Safety Biomarkers for Gene Therapy Products 438\u003c\/p\u003e \u003cp\u003e18.6.1 Immune Toxicities to in vivo gene therapy 438\u003c\/p\u003e \u003cp\u003e18.6.2 Immune Toxicities to Ex Vivo GT 441\u003c\/p\u003e \u003cp\u003e18.6.3 Long-Term Risks 442\u003c\/p\u003e \u003cp\u003e18.7 Concluding Remarks 442\u003c\/p\u003e \u003cp\u003eReferences 443\u003c\/p\u003e \u003cp\u003eIndex 449\u003c\/p\u003e","brand":"John Wiley \u0026 Sons Inc","offers":[{"title":"Default Title","offer_id":48738370748759,"sku":"9781119852780","price":153.0,"currency_code":"GBP","in_stock":true}],"thumbnail_url":"\/\/cdn.shopify.com\/s\/files\/1\/0817\/1739\/5799\/files\/9781119852780.jpg?v=1723811986","url":"https:\/\/bookcurl.com\/products\/drug-development-for-gene-therapy-9781119852780","provider":"Book Curl","version":"1.0","type":"link"}