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

• Published in: Nature communications 2024-09, Vol.15 (1), p.7853-15, Article 7853
• Main Authors: Shay, Timothy F., Jang, Seongmin, Brittain, Tyler J., Chen, Xinhong, Walker, Beth, Tebbutt, Claire, Fan, Yujie, Wolfe, Damien A., Arokiaraj, Cynthia M., Sullivan, Erin E., Ding, Xiaozhe, Wang, Ting-Yu, Lei, Yaping, Chuapoco, Miguel R., Chou, Tsui-Fen, Gradinaru, Viviana
• Format: Article
• Language: English
• Subjects: 42/47; 631/1647/2163; 631/1647/2258/1258/1260; 631/378/1341; 631/61/2300/1514; Animals; Binding sites; Blood-brain barrier; Blood-Brain Barrier - metabolism; Brain - metabolism; Capsid - metabolism; Capsid Proteins - genetics; Capsid Proteins - immunology; Capsid Proteins - metabolism; Capsids; Cell surface; Dependovirus - genetics; Dependovirus - metabolism; Directed evolution; DNA microarrays; Expression vectors; Gene therapy; Gene transfer; Genetic Vectors - administration & dosage; Genetic Vectors - genetics; HEK293 Cells; Humanities and Social Sciences; Humans; Immune system; Immunomodulation; Interleukin 3; Interleukin-3 - metabolism; Intravenous administration; Low Density Lipoprotein Receptor-Related Protein-6 - metabolism; multidisciplinary; Protein Binding; Receptor density; Receptors; Science; Science (multidisciplinary); Side effects; Transcytosis; Tropism; Viruses
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-024-52149-0

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 adapt an unbiased human cell microarray platform to determine the extracellular and cell surface interactomes of natural and engineered AAVs. We identify 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 with enhanced blood-brain barrier crossing in non-human primates after intravenous administration. The unbiased cell microarray screening approach also allows us to identify off-target tissue binding interactions of engineered brain-enriched AAV capsids that may inform vectors’ peripheral organ tropism and side effects. Our cryo-electron tomography and AlphaFold modeling of capsid-interactor complexes reveal LRP6 and IL3 binding sites. 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. Engineered adeno-associated viruses (AAVs) aim to improve safety and potency for use in gene therapy but mechanisms underlying these features are poorly understood. Here, authors use unbiased screens to identify an interaction with the human immune system and a determinant of enhanced brain potency.