Development and validation of a droplet digital PCR method for quantifying lentiviral vector infectious titer
Xueling Wu,
Xiaoya Zhou,
Yueming Wang,
Jian Wu,
Qian Liang,
Xu Yang,
Kehua Zhang,
Shufang Meng
Affiliations
Xueling Wu
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China
Xiaoya Zhou
Beijing Minhai Biotechnology Co., Ltd., Beijing, 102600, China
Yueming Wang
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China
Jian Wu
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China
Qian Liang
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China
Xu Yang
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China
Kehua Zhang
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China; Corresponding author. National Institutes for Food and Drug Control, Beijing, 100050, China.
Shufang Meng
National Institutes for Food and Drug Control, Beijing, 100050, China; State Key Laboratory of Drug Regulatory Science, Beijing, 100050, China; Corresponding author. National Institutes for Food and Drug Control, Beijing, 100050, China.
Lentiviruses, with their high transduction efficiency and gene expression levels, are widely used as gene delivery vectors in the development of chimeric antigen receptor T cells (CAR-T) and other genetically modified cell therapies. Accurate determination of the lentiviral vector infectious titer is essential to ensure effective transduction and product consistency. In this study, we developed an efficient method for lentiviral vector titration based on digital droplet polymerase chain reaction (ddPCR) technology, enabling absolute quantification of the target gene. Benzonase treatment of non-transduced plasmids substantially shortened the experimental period, reducing cell culture duration from 10-14 days–3 days. The method was rigorously validated by assessing specificity, working range, limit of quantification, precision, accuracy, and robustness. This study demonstrates the feasibility of combining enzymatic digestion with ddPCR to quantify lentiviral vector infectious titer and provides a detailed and readily adaptable methodology for the scientific community.
