Promoter And PRE Triage For Gene Expression And Function Optimization

By Shen Lu, Christopher B. Driscoll, Kelsey Dupont, Emmanuel Nekongo, Alexander Boucher, Andrew Basinski, Elise Kim, Ana Maria Chadbourne, Cherylene A. Plewa, and Stacie L Seidel

Optimizing lentiviral vector designs to increase titer, decrease production costs, and increase potency is an active area of exploration especially for vectors entering large scale manufacture. Here, we tested the effect of different promoters and PREs (Post-Regulatory Element) on multiple CAR and transgenic TCR constructs on titer, integration, expression, and in vitro function. To achieve this, we developed a scaled down mid-throughput lentiviral vector production platform to produce multiple constructs in tandem with sufficient titers for functional screening. Experiments were separated into two stages. In the 1^st stage we triaged three unique MND promoters, and four types of PRE elements. The lead constructs were moved to the 2^nd stage triage comparing EF1a, MSCV, MND, and SFFV promoters, for titer, transduction, expression and function using 4 genes of interest (GOI).