Objective

Gene modified T-Cell immunotherapies are increasingly being used to treat clinically challenging disease such as haematological cancers. These therapies are often targeted to specific cancers using gene modified T-Cell receptors (TCR) or chimeric antigen receptors (CAR) that are transduced into the T-cells using retroviral or lentiviral vectors. Ensuring consistency during the transduction process through controlled delivery and integration of the viral vectors is critical to controlling product quality. However, current methods used to determine the viral copy number (VCN) in transduced cells involve the analysis of viral sequences from a pool of cells.  While this gives an estimate of the VCN within a given population it does not account for cell to cell variation. To address this we have developed a single cell capture, processing and analysis approach to measure viral copy number integration in a human T-cell immunotherapy model. Our approach uses the microfluidic C1 automated capture system from Fludigm combined with high content screening to isolate, identify and phenotype individual cells. Genomic DNA from selected cells is then isolated on the microfluidic chip and prepared for analysis using qPCR. By using specially designed primers targeting viral WPRE and RRE sequences we have been able to successfully detect lentiviral integrations which can be compared to a reference RNase P sequence to accurately quantify the VCN at a single cell resolution. This new characterisation approach offers the potential for increased control over the transduction process, improving a critical step in the immunotherapy manufacture process.