Abstract
The field of gene therapy is rapidly growing with a greater number of clinical trials for gene therapeutics every year. Lentiviral vectors provide a valuable method for permanently integrating transgene sequences into the genome of infected cells. In order to use this powerful tool as gene therapy, reliable quantitative methods must exist to correctly measure the amount of transgene and the functional titer of these vectors. We show the development of two PCR based transgene titration methods using qPCR and ddPCR with an inter-assay precision of 9.3% CV and 8.6%CV respectively. Quantitation of functional titer was done using a lentiviral vector containing a eGFP transgene and flow cytometry. This method had an inter-assay precision of 27.3% CV. We also developed ddPCR based integrated copy number assay that can be modified to characterize lentiviral vectors with any transgene. These methods should be able to be adapted to other lentiviral based gene therapeutics by changing the transgene primer/probe sequences to match the target transgene and perform with similar precision.