Preclinical Development. Biodistribution Studies in Gene Therapy

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1 ASGCT/ESGCT Early Phase Clinical Trials Training Course Preclinical Development Biodistribution Studies in Gene Therapy Helene Chautard, PhD Versailles. October 25 th, 2012

2 A unique Contract Research Organization Quality Safety - Efficacy Evaluation of Gene and Cell Therapy Products Research Preclinical Clinical Conception, development of analytical methods Sample analysis GLP compliance

3 Preclinical evaluation of GTMPs Provide sufficient information for an appropriate risk assessment GTMPs pose specific safety issues that need to be addressed before clinical use On a case-by-case basis Scientific guidance for GTMPs available from EMA, FDA and ICH Data obtained with other similar products supportive, but not sufficient to warrant first clinical use Preclinical studies aim at establishing proof of concept in non-clinical model(s) biodistribution and persistence recommendation on initial dose and dose escalation scheme identification of potential target organs: biological activity and toxicity identification of indices to be monitored in the clinical trial, and specific patient eligibility criteria Specific considerations for GTMPs : Integration studies, germline transmission, evaluation of immunogenicity and immunotoxicity, oncogenicity /tumorigenicity

4 Biodistribution and shedding studies of GTMPs Biodistribution = pharmacokinetics of a GTMP Preclinical animal study designed to: determine the distribution of the vector to target and non-target sites assess the risk of persistence of the GTMPs that heightens the risk of adverse events Allows determination of expression profile of the transgene can be performed either as a separate study or as a component of a toxicology study (use of same animals in both studies if applicable). Detection of vectors in fluids (shedding) also contribute to environmental risk assessment (ERA) GTMPs include a large variety of products, the design of the biodistribution study depends on the specificity of the product to be evaluated

5 Design (1) Product: as close to clinical batch as possible - Use the same vector - Use the same production - purification processes - Use the same final formulation Animal model - 2 mammal species: one rodents + one non rodents (large animals) - Rodent: at least 5 animals per gender per group per sacrifice time point - Non rodents:3-5 animals per gender per group per sacrifice time point - Use both genders or justify Route of administration and devices - Use the intended clinical route of vector administration and intended delivery devices if possible Associated treatments -They should be addressed as they may impact the vector distribution

6 Design (2) Duration: 2 or 3 sampling time points a short time point: expected time of peak vector detection 1 or 2 two later time points to evaluate clearance of vector in non targeted organs Doses: Most commonly 3 doses Control (vehicle), dose equivalent to the supposed clinical dose ( 1x ) maximum feasible dose (commonly 5X or 10x ). Organs, tissues, fluids (between 20 and 35 different tissues) Recommended list: - peripheral blood, gonads, injection site - highly perfused organs (brain, liver, lungs, heart, spleen ) Other tissues based on toxicity / pathology and route of administration (ex: draining lymph nodes ) Choose a method for tissue collection that avoids potential contamination among different tissue samples.

7 Points to consider Gene expression in target tissues - In addition to biodistribution data, studies to confirm the specificity and duration of gene expression in target tissues are required (especially if target tissue selectivity of the GTMP is expected) Germline transmission issue - If no positive signal in gonads detected in biodistribution study no need for further non-clinical germline transmission studies. -If a positive signal is observed in gonadal tissues at long time point, additional testing will be needed.

8 Preliminary studies: development of specific assays A 3 steps approach to develop assay allowing specific detection of the vector sequence 1- Development - Specific development and optimization of the qpcr conditions - Check the ability to amplify the vector sequence + a sequence specific to animal species in a multiplex qpcr 2- Analytical validation of the qpcr (ICH Q2) - Assess linearity, LOD, LOQ, precision, accuracy and interval of measurement - LOQ of 50 copies of vector/1 µg genomic DNA 3- Test of the validated conditions on organs and fluids samples - Performed on the various specimens to be analyzed during the biodistribution study

9 Biodistribution Study Molecular analysis - Performed according to Good Laboratory Practice (GLP) Grinding of specimens DNA extraction and purification DNA quantification qpcr (Triplicate analysis) Data analysis Study report and documentation Persistence is indicated by : - detectable levels of vector sequences - absence of an apparent downward trend over time Gene expression assays (RT-qPCR) are performed in tissues where vector persistence is observed

10 Conclusion Many variables may affect the design and the outcome of the biodistribution and vector persistence study. Early in the process: Review and learn from similar cases Meet and discuss with regulatory agencies Include partners in your project - CROs, vector producer (CMO), clinicians