Recently there has been increasing development of cell and gene therapy products using CRISPR-Cas9 genome editing technology to treat disease at the genetic level. One of the assays required to determine biodistribution and pharmacodynamic effects of the products in nonclinical development is an amplicon-based next generation sequencing (NGS) assay to evaluate the changes in DNA sequence at the target site across a variety of tissues. These NGS assays are quickly moving into the regulated bioanalytical space for nonclinical development. This presentation will discuss the potential parameters that should be evaluated for an amplicon-based NGS assay to detect gene editing at a target site and some of the strategies used to validate assays for use in regulated bioanalytical laboratories. This includes critical reagents, assay range, precision and accuracy, selectivity, LOD assessment, robustness, and data interpretation. This presentation will discuss some of the assay attributes to consider when setting acceptance criteria to monitor assay performance and the evaluation of the LOD.
Learning Objectives:
Gain an understanding of the context of use for amplicon-based NGS assays
Learn about the assay parameters that should be evaluated during method development and validation for an amplicon-based NGS assay
Understand the unique challenges that NGS assays present in comparison to other bioanalytical assays.