Targeting purinergic receptors in glioblastoma with nanobody-displaying AAV vectors

The standard therapy for glioblastoma, surgery followed by radio- and chemotherapy, results in ATP release from damaged and stressed cells. The GPI-anchored ecto-enzyme CD73 and the ATP-gated P2X7 ion channel are often expressed on glioblastoma cells. CD73 contributes to the cold tumor microenvironment by generating immunosuppressive adenosine. P2X7 promotes tumor growth by enhancing cell proliferation and survival. Here we show that these membrane proteins can serve as entry receptors for recombinant Adeno-associated viruses (AAV). To this end, we genetically inserted CD73-specific or P2X7-specific nanobodies into a surface loop of the AAV9 capsid. These nanobody-displaying AAV greatly enhanced the transduction of CD73- or P2X7-expressing HEK cells in vitro and patient-derived glioblastoma cells ex vivo. Our results pave the way for the therapeutic application of these nanobody-displaying AAV, for example, to express immune-activating cytokines or checkpoint inhibitors in the tumor cells to turn a cold tumor microenvironment hot.