Training Session F2 - Friday April 5, 2019, 7am to 5:30pm

"The Gene Therapy Bioanalytical Challenges: PK/Biodistribution, Immunogenicity and Shedding Assays, qPCR, ELISPOT, Flow Cytometry in Bioanalytical labs, and Regulatory Perspectives"

What you need to know to develop bioanalytical assays for Gene Therapy and how Gene Therapy is impacting Bioanalysis in both Pharma and CRO - Multiple platforms & multiple approaches to deal with unsolved bioanalytical issues

(You can click on each blue title below to see details, or simply scroll down to see details)


Part 1: Introduction
Part 2: Biodistribution and Infectivity Assays
Part 3: Immunogenicity
Part 4: Emerging Therapies
Part 5: 2019 White Paper on Gene Therapy
  • Consensus and Conclusions on Gene Therapy for 2019 White Paper

  • Panel Discussion with the Regulators:

    Address your questions on Gene Therapies and interact directly with US FDA CBER


DETAILS of Training Course F-2

Part 1: Introduction
  • Lesson 1
    Evolution of WRIB Recommendations on Gene Therapy from 2017 WP to 2018 WP: Where are we now and what next?
    • How the 2017-2018 White Paper in Bioanalysis Recommendations on Gene Therapy have impacted the current industry practice and regulations
    • Understanding where we started in 2017 and why and where are we now
      • Progresses based on Industry/Regulators’ experience at WRIB
    • Challenges addressed & unanswered concerns
      • Specificity & tissue targeting
      • Specific tissue/cell tropism
      • Selection of viral vectors
      • Immunogenicity issues
      • Understanding pharmacology & safety
      • Regulatory expectations
    • Current Industry standards
    • The latest insights & regulatory developments on this fast-evolving field
      • Focus on what bioanalysis should measure for assessing drug exposure, responses and immunogenicity

  • Lesson 2
    PK Perspective on Biodistribution and Immunogenicity Assessments to Support Gene Therapies Clinical Trials
    • Gene Therapy is showing to be a very targeted treatment for genetic and acquired conditions such as
      • Hemophilia, cancer, muscular dystrophy, cardiovascular diseases
    • Current development to bring a gene of interest to the target cells or tissues
    • Biodistribution and Immunogenicity are crucial assessments to understand Gene Therapy
      • Ability to reach a variety of different cell types and tissue-specific targets
      • Efficiency of transfection in the targeted cells
    • Biodistribution and Immunogenicity assessments for both safety and efficacy in Gene Therapy
      • Why do we conduct biodistribution studies in Gene Therapy?
      • What is the state of the art for biodistribution studies and what may the future look like?
      • How are immunogenicity assessments used to support Gene Therapy studies?
      • What are the challenges in interpreting immunogenicity data from Gene Therapy Clinical Trials?


Part 2: Biodistribution and Infectivity Assays
  • Lesson 3
    Drug Distribution Analysis in the Preclinical Development of Gene and Cell Therapy Products
    • Unique characteristic of Gene Therapy vs other conventional drugs
      • Vector and transgene
      • Importance of drug distribution to understand the PK of both vector and the expressed gene moieties
      • Understanding pharmacology and safety
    • Bioanalytical methods used in Gene Therapy
      • Bridging systemic and tissue exposure
      • Complexity of the bioanalytical support for Gene Therapies
    • Case studies on preclinical development for a number of Gene Therapy programs
      • Discussions on challenges & considerations

  • Lesson 4
    Biodistribution - Recent Development in Transgene Expression Quantification by Hybrid LBA/LCMS
    • Thorough evaluation of biodistribution in Gene Therapy
      • Evaluation of Transgene by LCMS and Hybrid LBA/LCMS
    • Current Hybrid LBA/LCMS well-characterized methods to determine biodistribution
      • Application in Gene Therapy of the Hybrid LBA/LCMS as a technique increasingly employed for the quantification of protein targets in translational and early clinical research of biologics
    • Advanced applications for quantification of expression of the protein product
      • Tissue targeting: Liver, Spleen, Lungs, Kidney, Heart, Brain, Muscle & Skin
      • Current workflow consisting of protein precipitation, pellet digestion and anti-peptide antibody enrichment prior to LCMS based quantification of a surrogate peptide
    • Case studies on Hybrid LBA/LCMS approach for the measurement of structural protein and the expression of a related transgene as a result of gene therapy
      • Novel strategies for quantitative transgene expression analysis catering to an unmet need in the emerging area of gene therapy

  • Lesson 5
    qPCR as integral part of a Bioanalytical Lab for Gene/Nucleic Acids, CAR-T, Oncolytic Virus, Gene Editing and other Emerging Therapies: Method development & Validation Strategies
    • Importance of standardizing qPCR methodologies in the Bioanalytical laboratories
      • qPCR as fundamental “must have” instrument for Gene Therapy
      • Current lack of experience and harmonized industry standards on qPCR for Regulated Bioanalysis
    • qPCR Assessment of Exposure Based on Vector Levels
      • qPCR in support in vivo characterization of CAR-T and Gene Therapy products
    • Current Recommendations on BMV by qPCR
      • What regulatory guidance to use for bioanalytical validation of qPCR methods?
      • Is the 2018 FDA BMV Guidance applicable to qPCR?
    • 2006 FDA Guidance on Gene Therapy Clinical Trials
      • qPCR Assay sensitivity for tissues
      • Use of positive and negative controls
      • Validation tests for qPCR: assay sensitivity, specificity and reproducibility
    • What is critical to qPCR method validation?
      • Drafting together the 2019 Recommendation on qPCR BMV

  • Lesson 6
    Gene Therapy: Utilization of molecular vs flow cytometry methods for PK assessments and MRD analysis, analytical considerations and challenges
    • Current approaches for measuring transgene levels during clinical development in Gene Therapy
    • Applications qPCR (molecular) and Flow Cytometry
      • Pros/Cons, Industry experience, analytical differences and similarities
      • Flow versus Molecular Assays for measurements impacting clinical development in gene therapy
    • Minimal Residual Disease (MRD)
      • Recent developments and technology advancements in MRD monitoring
      • MRD as a surrogate endpoint during Gene Therapy
      • Existing challenges with analytical tools in evolving landscape of cellular and gene therapies

  • Lesson 7
    Viral Vector Shedding Assays in Gene Therapy Development: Quantitative & Infectivity Assays
    • When to conduct Viral Vector Shedding Assays in Gene Therapy
      • Are they always required for AAV?
      • When to differentiate between intact and degraded virus
      • Quantitative assays requirements if vector is detected
      • Pros & cons of qPCR vs Infectivity Assays
      • Regulatory position on Shedding Assays
    • Shedding Assays and assay performance expectations
      • Evaluation of release of vector genome in patient
        • Matrix selection for analysis based in tropism & route of administration
        • Analysis of feces, urine, saliva, nasopharyngeal fluid, pustules, sores, wounds
    • Environmental Assessment (EA)
      • Evaluation of transmission of vector genome from treated to untreated individual
    • Challenges with Shedding evaluation and method development:
      • Replication competence
      • Immunogenicity response
      • Persistence and latency
      • Tropism &Route of administration
      • Long term follow up & monitoring


Part 3: Immunogenicity
  • Lesson 8
    Immunogenicity Strategies against Capsid and Transgene product in support of Viral Vector based Gene Therapy
    • Monitoring of vector and transgene specific immunity is crucial to drive Gene Therapy clinical development
    • Approaches for viral and transgene specific immune responses
      • Pre-treatment / Pre-existing Abs
      • Post-Dose
      • Cellular Immune Responses
      • Humoral Immune Responses
    • Understanding the relevance and need for pre/post treatment immunogenicity measurements
      • Specific considerations on over-expressed proteins from Gene Therapy
    • Immune responses to Transgene Protein
      • Potential risk factors
      • Patient Cross-Reactive Immunologic Material (CRIM) status
      • Host immune system status
      • T-cell responses to transgene
    • Immune responses to Vector
      • Delivery vector (AdV vs. AAV) dependent
      • Route and dose of administration

  • Lesson 9
    Crucial Importance of a Thorough and In-depth Evaluation of Pre-existing Abs to the Viral Capsid during Immunogenicity Assessment of Viral Vectors based Gene Therapy
    • Focus on pre-existing Ab in Gene therapy
      • Case Studies in Immunogenicity Testing for Gene Therapy
      • Screening and enrollment criteria - Lessons Learned
      • Monitoring of antibody and T cell responses
      • Impact of AAV serotype cross-reactivity on assays
      • Setting of Cut Points in the context of pre-existing antibodies to GT capsid
    • Adeno-associated viral vectors (AAV) as preferred vector for gene delivery
    • NAb responses to AAV and impact on patients’ re-dosing
      • Exposure to AAV vectors and pre-existing Nab
      • Pre-existing Ab to the viral capsid and effects on Gene Therapy safety and efficacy
      • Nab method development challenges for pre-existing Ab to AAV
      • Actual data from clinical trials to assess immune responses to AAV vector
    • Reliable bioanalytical determination of
      • Pre-treatment Nabs levels
      • Pre-treatment cytotoxic T cell responses

  • Lesson 10
    ELISPOT as the Gold Standard in Cellular Immunity Evaluation in AAV based Gene Therapy: Method Development and Validation Strategies
    • Enzyme-linked Immunospot (ELISPOT) assay characteristics
      • Highly sensitive technique for the detection, measurement, and functional analysis of immune cells
      • In vitro cellular assays for detecting and enumerating individual immune cells that secrete soluble mediators in response to a stimulus
      • Ability to detect up to 1 in 300,000 low frequency cytokine-secreting cells
      • Ex vivo measurement of low-frequency T cell responses
    • ELISPOT Assay expectations & performance
      • Cell recovery and viability
      • Critical steps in standardizing an ELISPOT assay
      • Overcoming ELISPOT assay Variability
      • State-of-the art tools to control assay performance over time or across laboratories
    • Cellular Immune Responses by ELISPOT
      • Anti-capsid and transgene
      • Pre- and Post-dose possible and potentially impactful
    • Use of ELISPOT for peripheral blood mononuclear cells (PBMCs) assessment
      • Using cryopreserve high quality PBMCs for ensuring optimal performance in functional ELISPOT assays
    • Challenges in engineered reference material for assessing Cellular Immune Response
      • Development of samples that contain a defined number of antigen-specific T cells
    • Central role of Cellular Immune Responses in understanding of the host reaction to Gene Therapy
      • New progresses in monitoring of T cells
      • Development of sensitive and robust assays
      • Critical assays variable in cellular immune assessments and their impact on results
      • Evolution of cellular immune assessment from supporting early discovery decisions to late stage clinical development

  • Lesson 11
    Challenges in LBA vs Cell-Based Nabs Assay development for Gene Therapy: Case Studies
    • Current methodologies for detecting pre-existing capsid immunity
      • LBA-based detection of antibodies
      • Cell based NAb assays
      • in vivo (mouse-based) transduction inhibition assays
    • A novel systematic study of the impact of pre-existing immunity
      • Comprehensive detection by these various methods
        • Results comparison
      • Effect on efficacy and transgene pharmacodynamics (PD)
    • Case study of transgene PD in NHP with pre-existing AAV capsid immunity, detected by both a cell-based NAb assay (transduction inhibition or TI) and ECL based total antibody assay
      • Evaluation of the biological threshold for tolerable AAV antibody levels
      • Selection of best assay for selection patients who are likely to response to gene therapy


Part 4: Emerging Therapies
  • Lesson 12
    Recent Advancements in CRISPR Gene Editing Therapeutic: PK/PD and Clinical Biomarkers
    • On-going revolution in Gene Therapy by using Genome Editing based on Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Associated 9 (Cas9) technology
      • Recent development of Genome editing
      • CRISPR-Cas9 and Applications of Genome Editing
      • Main targeted diseases with CRISPR-Cas9 gene editing approaches
    • Rise of an entire new class of therapeutics
      • PK/PD implications
      • Development of predictive biomarkers and in Gene Therapy for patient identification
    • Recent applications of CRISPR Cas9 genome editing
      • Transformative gene-based medicines for serious diseases
      • CRISPR in Clinical studies
        • CTX001 in β-thalassemia and sickle cell disease
      • Next-generation gene-edited allogeneic CAR-T
      • Regenerative medicine
        • CRISPR/Cas9-edited allogeneic stem cells

  • Lesson 13
    CRISPR and Applications of Genome Editing: Bioanalytical Strategies & Challenges
    • CRISPR-Cas9 technology characterization & bioanalytical assays needed for
      • Efficacy assessment
      • Safety considerations
      • Specificity evaluation
      • Delivery to the target cells
    • Current industry standards in Bioanalytical evaluation of endogenous vs newly expressed or reduced expression of proteins
      • Detection & quantification of response differentiation
      • Immunogenicity assessment
        • Systemic delivery of Viral vector-CRISPR
        • Newly expressed proteins







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