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

Abstract

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 1012 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.

Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors

“…Her2-AAV vectors obviously can only be applied in Her2/neu-positive cancer. However, AAV vectors targeted against other tumor antigens including EpCAM (25), CD30 (41), and CD105 (42) have been described. A high-throughput based screening system for the identification of novel targeting ligands is available (42).…”

Surface-Engineered Lentiviral Vectors for Selective Gene Transfer into Subtypes of Lymphocytes

“…Recently, the DARPin screening process and a novel DARPin library were adapted to the specific requirements of viral vectors. 80 In this process, the extracellular part of the target receptor of choice is expressed in 293T cells and then provided as purified bait for selection. By choosing particular domains of the receptor, it is possible to control the exact binding site of the LV particle on the target receptor.…”

“…An impressive example is the identification of a DARPin that can be used to target the glutamate receptor subunit 4 (GluA4), without recognition of the closely related GluA1–3 receptors. 80 …”

“… 100 With NKp46, a first marker on these cells was recently targeted. 80 Apart from immunotherapy, T cell-targeted LVs may be able to target the small reservoir of HIV latently infected cells. 101 Receptor-targeted LVs recognizing this reservoir may have the potential to solve this delivery problem.…”

Baculovirus as Versatile Vectors for Protein Display and Biotechnological Applications

“…Surface display of foreign proteins Display of foreign proteins or peptides on the surface of a cell or virus, which we refer to as 'surface-display', is one of the most valuable techniques for protein engineering. Surface display has been used for basic research for the characterization of protein function and identification of protein counterparts (Hartmann et al, 2018;Nguyen et al, 2018), in addition to applied research such as for the establishment of diagnostic tools for infectious disease and development of gene therapies (Rothe et al, 2006;Sergeeva et al, 2006;Brown, 2010;Aghebati-Maleki et al, 2016;Goulart and Santos, 2016;Lee et al, 2017). Thus far, the most widely applied surface-display technique is phage display (Smith et al, 2015;Yang et al, 2017), whereby a library of billions of bacterial phages displaying foreign peptides can be used for screening (Liu et al, 2017a).…”