Q&A with Charles River
When developing a cellular or gene therapy product, one of the most sensitive tools related to these products for biodistribution and shedding evaluations is quantitative polymerase chain reaction also known as qPCR. Biodistribution studies are part of the non-clinical regulatory package to assess where in an organism these products distribute in target and non-target tissues and how long they persist. qPCR can also be used for the evaluation of gene therapy vector shedding and provides critical information about the likelihood of transmission to untreated individuals. Despite its valuable use, the development and validation of this preferred technique comes with a unique set of challenges given the high level of analytical method customization required and the absence of specific regulatory guidance. At Charles River we have developed strategies to help tackle these obstacles as well as for how to implement these assays for successful conduct and completion of nonclinical and/or clinical sample analysis.
Dr. Ancian has spent the last 20 years in the pharma, biotech and CRO industries where he has implemented and monitored biomarker analyses at the discovery, preclinical and clinical stages with expertise in specialized genomics and proteomics platforms. He holds a PhD in molecular and cellular biology from Nice University as well as an engineering degree in chemistry from the National School of Chemistry of Montpellier, France. Philippe is passionate about advanced therapies, in particular cell and gene therapies.
Milena specializes in applications of quantitative polymerase chain reaction (qPCR) such as biodistribution, shedding, and biomarker and gene expression (PD) investigations for the development of advanced therapies. Milena plays a crucial role in guiding clients’ analytical assay design and validation from a scientific and regulatory perspective.
Philippe Ancian
Safety risks for cell and gene therapies depend on the therapeutic agent, route of administration, and target population. The common safety risks for both product types are linked to the pharmacological activity of the therapeutic agent in target or non-target organs. However there are risks specific to each. For gene therapies, the most efficient vectors are viral vectors and one of the specific safety concerns is linked to the intrinsic properties of the virus used. This includes inflammation and immune response against the vector or the inserted gene which in return can be detrimental to the transduced cells or tissues. Another risk associated with certain viral vectors such as retroviruses is the integration of the genetic material in patient DNA, which may occur next to a gene involved in the control of cell growth. Finally, the horizontal transmission of the vector through the vector shedding in biological fluids is a safety risk for patient’s relations and healthcare workers. For cell therapies, safety risks are linked to the intrinsic properties of the product used. One of the most important is linked to uncontrolled proliferation of genetically modified cells, leading potentially to the formation of tumors in target and non-target organs. For both therapeutic agent types, it is essential to follow the product fate in organs using very sensitive techniques to correlate a potential toxic effect with the presence of the investigational product during preclinical studies.
For both cell and gene therapy products, qPCR is an essential part of a biosafety profile characterization. For cell therapy specifically, it is used to evaluate cell fate and retention in animal models and for gene therapies it is a valuable tool to identify target and non-target organs.
Milena Blaga
For gene and cell therapies, the persistence of the therapeutic is critical as the longer the persistence, the higher the duration and risk of delayed adverse findings. Persistence is assessed through biodistribution studies in one or two animal species, and through shedding and blood analyses in clinical studies. Additional general endpoints to assess the safety profile of cell and gene therapeutics include body weights, clinical observations, gross organ examinations and histopathology. Where toxicity is not expected, other biologic outcomes such as transgene expression, optimal biological dose and immune reactions, whether local at the site of administration, or systemic, are alternatives for primary endpoints. In cases where the gene or cell therapy products have the ability to modify the host genome, or to persist for the life span of the recipient, long term follow up is typically advised.
Other safety evaluations are described in regulatory guidances, and correlate with the characteristics of the gene or cell product: the ability to integrate at non-specific sites in the genome raises the risk of inadvertent gene disruption; genome editing technologies may result in off-target events which can similarly disrupt critical parts of the genome; long-term persistence, especially in cases where the gene therapy product has immunological functions, can lead to immune reactions; latent or replication competent products can cause infections. Given the biologic nature of cell and gene therapies, manufacturing is a critical endpoint in safety evaluations, given that any changes in the manufacturing process could affect the safety profile of the final product. All of these risks typically need to be evaluated and characterized to appropriately understand the biological activity of the gene and cell therapy.
Biodistribution and shedding must be evaluated using the most sensitive techniques, often based on DNA exponential amplification by Polymerase Chain Reaction (qPCR). The first consideration is the selection of species for the safety studies, the number of groups and animals per group, the doses of the therapeutic agent, and the general safety endpoints assessed. Secondly, the selection of the tissues and the fluid to be collected is crucial and should be adapted to the intrinsic properties of the therapeutic product and its route of administration.
In a laboratory setting, the main challenges when analyzing samples by qPCR lie in having access to the appropriate infrastructure and workflows to support these. For example, separate labs should be used for isolation and qPCR, and additional divisions between processing of negative and positive samples may also be considered to minimize the risk of contamination. Furthermore, biodistribution and clinical studies often result in large number of samples which need to be stored typically deep-frozen, for very long periods of time due to the duration of such studies. In addition to the original biological samples, daughter samples of DNA, RNA, diluted samples and aliquots are typically prepared during processing which require appropriate labelling, storage, and overall management. Increasing throughput in such molecular biology labs therefore requires appropriate knowledge of the workflows, planning, management and training.
Currently there are no regulatory guidelines for validating qPCR- based assays, so the method development and validation must be based on a science-driven review of the scope of the assay in order to determine the suitability of the method for its intended purpose.
There are various challenges associated with this technology, from the extensive sample processing until a result is reached, including isolation, quantification, quality assessment, to the difficulty in setting acceptance criteria that cover the whole process.
Method validation parameters and method applications have generally been extrapolated from ligand-binding assay regulatory guidances, and typically involve calibration standards linearity, sensitivity, specificity and selectivity assessments, intra- and inter-assay accuracy and precision assessments, all of which are relatively straightforward plate-based endpoints. A major challenge in the method validation is establishing recovery and stability of the therapeutic item or reference, from samples, due to the difficulties in mimicking the biological activity during spiking experiments. Spiking experiments do not result in the vector or plasmid being taken up into the cells, which leaves plasmids susceptible to the harsh extracellular environment and leads to degradation and high variability in recovery and stability. Additionally, biodistribution studies involve the analysis of all major organs, which results in a large panel of tissues, or matrices, of which often only a selected number are validated. While test item or reference standard stability in matrices should be assessed, another difficulty with qPCR-based assays is that matrices can be considered both the nucleic acid (DNA/RNA) samples which are directly analysed, and the biological samples from which these are isolated. As such, stability experiments could be performed in either or both types of samples, which further increases the scope of the validation.
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