669. Standard Free Droplet Digital PCR as a New Tool for More Precise and Reliable Quality Control of Gene Therapy Vectors

In case using viral vectors as therapeutics in context of gene therapy small changes in vector dose can have fatal outcome for the patient. It has been shown in earlier clinical studies that there is a non-linear dose response for most of the vectors, so the therpeutical window is narrow. Precise vector titration is of extraordinary high importance. The widely distributed standard real time PCR (qPCR) may not provide this in a satisfactory way. In this study we were aiming to address this challenge by implementing a more reliable and accurate strategy for vector quantification. We implemented a new digital PCR (ddPCR) strategy based on droplet formation as tool for vector quantification. By forming around 20.000 individual PCR reactions using sample partitioning in a water oil emulsion the system is able to detect after end point PCR if every single droplet is negative or positive for the target DNA. Importantly, providing a standard is not necessary since quantification is absolute whereas normal qPCR provides standard dependent relative quantification. By choosing primers in the region of adenoviral ITR and fiber, we were able to quantify in one single reaction high capacity adenoviral vector (HCAdV) and the helper virus, which is needed during production of the viral gene deleted HCAdV. We applied this new strategy to quantify HCAdV produced with a shortened protocol using column purification instead of traditional caesium chloride centrifugation. The virus was purified from the same characterized stock using different columns, which were also compared in this study. Once the virus was purified we infected various cell lines with different volumes of the final preparation, after 3h total DNA was isolated and different PCR strategies were performed. First our data showed that this protocol leads to sufficient HCAdV for preclinical in vitro studies. Besides the transducing units calculated from ddPCR vs. qPCR data (≈10^6 transducing units per ml), this was also confirmed by EGFP expression analysis and the number of physical particles measured via UV-vis. Also the helper virus concentration was within an acceptable range (<10%). Second we could demonstrate that our ddPCR was less time consuming, less expensive and more reliable compared to qPCR. Representative primary data is shown in figure 1a. View Large Image | Download PowerPoint SlideChannel 1 represents the droplets, which were positive for the fiber gene whereas channel 2 represents the adenoviral ITR. Figure 1b shows the control plasmid where we subcloned both loci as control. We think by applying ddPCR for gene therapy, patient safety can be increased in clinical trials while scientific results become more reliable.