Drug Target Strategies
Mammalian cell-based libraries offer an affordable and physiologically relevant context for high throughput screening for applied biomedical research. Current technologies to develop mammalian cell display libraries either result in multiple transduced cells, or are inefficient to prepare large library screens and may carry vector backbone. Here, we introduce the TARGATT™ site-specific, integrase-mediated gene integration technology which provides an efficient strategy for integrating a gene of interest into a preselected, transcriptionally active locus. The TARGATT™ system provides a 1:1 variant-to-cell ratio with a single cell containing a single copy of transgene inserted at a single docking site. This provides a very efficient, uniform transgene expression, and reproducibility for constructing large isogenic cell libraries for rapid, high throughput screening. We engineered a TARGATT™-HEK293 Master Cell line containing an integrase recognition landing pad in the Hipp11 safe harbor locus in HEK293 cells. In combination with an HSV-TK/GCV negative selection or promoter-less reporter genes to enrich for cells that have library plasmid integrated, this master cell line showed an > 20% knock-in efficiency without selection. With selection, virtually 100% efficiency can be achieved. The TARGATT™-HEK293 Master Cell Line thus enables stable cell line generation, and 4-8x larger library sizes than currently available technologies. The TARGATT™ technology is an efficient system for creating large cell-based library screens for applications in directed evolution (such as vaccine development, drug screening), genome wide screening, and biotherapeutic drug discovery and biomanufacturing.