Precise delivery of therapeutic genes is one of the major requirements for in vivo gene therapy, with vector particles selectively transducing their target cells as essential tool. While current receptor-targeted vectors selectively enter cells via a single surface marker, many therapeutically relevant cell types, such as HIV reservoir cells, are defined by multiple cell surface markers. In the HIV context, elimination of proviral genomes in reservoir cells presents an attractive therapeutic strategy. Towards reaching this challenge, we present AAV vectors exhibiting an AND-gated binding behavior for the surface markers CD4 and CD32a.

A CD4- and a CD32a-specific DARPin were inserted into surface-exposed loops of the AAV2 capsid maintaining its structural integrity as determined by high-resolution cryo-electron microscopy. Remarkably, in contrast to their monospecific counterparts, these bispecific vectors exhibited a clear preference for CD4/CD32a double-positive cells. This preference was consistently observed in cell mixtures as well as in vivo after systemic vector administration into a mouse model harboring double-positive cells in bone marrow. When equipped with Cas9 nucleases targeted to the HIV provirus, these vectors inhibited HIV replication in vitro. Double-positive cells mixed into healthy donor blood were reproducibly hit by these vectors, even when making up only a small fraction of cells. Further investigations of the system demonstrated its flexibility in target receptor selection and the potential for improvements in selectivity.

These results suggest a novel avenue for receptor targeting by enabling gene transfer into cell subtypes defined by two cell surface markers.