Bioanalytics – Biomolecular
2019 PharmSci 360
Genome editing offers a modality for gene therapy that is advancing rapidly toward clinical utility. Assessing the efficiency of such treatments in patients requires accurate and sensitive measurements of the proportion of cells that acquire short insertions and deletions (indels) at the intended site, which is monitored using Next Generation Sequencing (NGS). We will present qualification data of quantitative assays using the Illumina MiSeq platform and an in-house bioinformatics pipeline to identify and score indels.
Calibrators with known levels of indels were produced by mixing genomic DNA from edited cell lines that contain 100% indels with DNA from un-edited cells. Amplicons surrounding the sequence targeted for treatment were produced using two sequential PCR amplifications followed by deep sequencing using Miseq. Reproducibility and accuracy were assessed at different indel levels. The results indicate that a quantitative indel assay can be developed using MiSeq analysis for patient monitoring post gene editing.