A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors

Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 10(12) variants. This way, DARPins specific for the glutamate receptor subunit GluA4, the endothelial surface marker CD105, and the natural killer cell marker NKp46 were generated. The identification of DARPins best suited for gene delivery was achieved by screening small-scale vector productions. Both LV and AAV particles displaying the selected DARPins transduced only cells expressing the corresponding target receptor. The data confirm that a straightforward process for the generation of receptor-targeted viral vectors has been established. Moreover, biochemical analysis of a panel of DARPins revealed that their functional cell-surface expression as fusion proteins is more relevant for efficient gene delivery by LV particles than functional binding affinity.