Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

Adeno-associated viruses (AAVs) are foundational gene delivery tools for basic science and clinical therapeutics. However, lack of mechanistic insight, especially for engineered vectors created by directed evolution, can hamper their application. Here, we adapted an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identified a naturally-evolved and serotype-specific interaction between the AAV9 capsid and human interleukin 3 (IL3), with possible roles in host immune modulation, as well as lab-evolved low-density-lipoprotein-receptor-related-protein 6 (LRP6) interactions specific to engineered capsids that cross the blood-brain barrier in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allowed us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vector peripheral organ tropism and side effects. These results allow confident application of engineered AAVs in diverse organisms and unlock future target-informed engineering of improved viral and non-viral vectors for non-invasive therapeutic delivery to the brain. ### Competing Interest Statement The California Institute of Technology has filed a provisional patent on this work with T.F.S, X.C., S.J., and V.G. listed as inventors. V.G. is a co-founder and board of directors member of Capsida Therapeutics, a fully integrated AAV engineering and gene therapy company. B.W. and C.T. are employees of Charles River Laboratories. The remaining authors declare no competing interests.