Unbiased human cell microarray screening identified LRP6 as a blood-brain barrier transcytosis receptor for engineered AAVs and human interleukin 3 (IL3) as a binding partner for AAV9.
Identification of LRP6 and IL3 as AAV binding partners provides mechanistic insight into blood-brain barrier transcytosis and immune modulation, enabling improved engineering of viral vectors for central nervous system delivery.
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.
Shay et al. (2024) studied AAV gene therapy vectors. Engineered AAV capsids (AAV9-X1.1, AAV.CAP-Mac) vs. Natural AAV9 was evaluated on Identification of AAV binding partners. Unbiased human cell microarray screening identified LRP6 as a blood-brain barrier transcytosis receptor for engineered AAVs and human interleukin 3 (IL3) as a binding partner for AAV9.