✍🏼 Raymond Wu, Frank Luh, Soo-Mi Kweon, Yun Yen

🏠 Sino-American Cancer Foundation, Covina, CA, USA.

📑 Molecular Therapy Methods & Clinical Development (2025)

Abstract
Viral copy-number (VCN) assay is a powerful, effective method to quantify toxicity, cellular kinetics, and durability of virus-modified cell therapy products. The qualification and validation of assay requires reference control. Traditionally, plasmids and cell lines are used as reference controls, but development and qualification of those controls require considerable time and resources. We propose a reference synthetic DNA fragment containing amplicons of woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and ribonuclease P protein subunit p30 (RPP30), connected by HindIII restriction enzyme cutting site, as a useful tool to qualify and validate duplex droplet digital PCR (ddPCR) assays for VCN. Using this hybrid amplicon, we qualified the duplex WPRE/RPP30 ddPCR assay by determining range of quantification, precision, bias, and robustness of the assay. The varying amount of input DNA showed upper limit, lower limit, and linearity of the assay. Coefficient of variation (CV) and % recovery showed assay precision and accuracy, respectively. Furthermore, the hybrid amplicon was used to determine assay robustness with potential conditions of variability. The hybrid amplicon was a comparable alternative to cell reference standards for validating VCN assay. In conclusion, WPRE-RPP30 hybrid amplicon can be used as a routine quality control measure to validate digital PCR assays.

How the WOLF is used in this study
The WOLF G2 Cell Sorter was used to isolate single GFP-positive Jurkat cells following lentiviral transduction with a CD19-targeting CAR construct. After transduction, cells expressing GFP were identified and individually dispensed into 96-well plates using the WOLF in single-cell sorting mode. This approach enabled the researchers to generate clonal populations from single transduced cells, which were then expanded and screened for GFP expression. The use of the WOLF ensured gentle handling of the cells during sorting, maintaining cell viability while enabling precise selection of successfully transduced clones for downstream genomic analysis and functional studies.