Efficient and cell-specific delivery of DNA is essential for the effective and safe use of gene delivery technologies. Consequently, a large variety of technologies have been developed and applied in a wide range of ex vivo and in vivo applications, including multiple approaches based on viral vectors. However, widespread success of a technology is largely determined by the versatility of the method and the ease of use. The rationally designed adapter technology previously developed redirects widely used human adenovirus serotype 5 (HAdV-C5) to a defined cell population, by binding and blocking the adenoviral knob tropism while simultaneously allowing fusions of an N-terminal retargeting module. Here we expand modularity, and thus applicability of this adapter technology, by extending the nature of the cell-binding portion. We report successful receptor-specific transduction mediated by a retargeting module consisting of either a DARPin, a single-chain variable fragment (scFv) of an antibody, a peptide, or a small molecule ligand. Furthermore, we show that an adapter can be engineered to carry more than one specificity, allowing dual targeting. Specific HAdV-C5 retargeting was thus demonstrated to human epidermal growth factor receptor 2 (HER2), human folate receptor alpha, and neurotensin receptor 1, effective at vector concentrations as low as a multiplicity of infection of 2.5. Therefore, we report a modular design which allows plug-and-play combinations of different binding modules, leading to efficient and specific mono-or dual-targeting while circumventing tedious optimization procedures. This extends the technology to combinational applications of cell-specific binding, supporting research in gene therapy, synthetic biology, and biotechnology.
