AAV9 Structural Rearrangements Induced by Endosomal Trafficking pH and Glycan Attachment

Adeno-associated viruses (AAVs) are small non-enveloped ssDNA viruses, that are currently being developed as gene therapy biologics. After cell entry, AAVs traffic to the nucleus using the endo-lysosomal pathway. The subsequent decrease in pH triggers conformational changes to the capsid that enables the externalization of the capsid protein (VP) N-termini, including the unique domain of the minor capsid protein VP1 (VP1u), which permits phospholipase activity required for the capsid lysosomal egress. Here, we report the AAV9 capsid structure, determined at the endosomal pHs (7.4, 6.0, 5.5, and 4.0) and terminal galactose-bound AAV9 capsids at pHs 7.4 and 5.5 using cryo-electron microscopy and three-dimensional image reconstruction. Taken together these studies provide insight into AAV9 capsid conformational changes at the 5-fold pore during endosomal trafficking, both in the presence and absence of its cellular glycan receptor. We visualized, for the first time, that acidification induces the externalization of the VP3 and possibly VP2 N-termini, presumably in prelude to the externalization of VP1u at pH 4.0, that is essential for lysosomal membrane disruption. In addition, the structural study of AAV9-galactose interactions demonstrates AAV9 remains attached to its glycan receptor at the late endosome pH 5.5. This interaction significantly alters the conformational stability of the variable region I of the VPs, as well as the dynamics associated with VP N-terminus externalization. Importance There are 13 distinct Adeno-associated virus (AAV) serotypes that are structurally homologous and whose capsid proteins (VP1-3) are similar in amino acid sequence. However, AAV9 is one of the most commonly studied and used as gene therapy vector. This is part because, AAV9 is capable of crossing the blood brain barrier as well as readily transduces a wide array of tissues, including the central nervous system. In this study we provide AAV9 capsid structural insight during intracellular trafficking. Although the AAV capsid has been shown to externalize the N-termini of its VPs, to enzymatically disrupt the lysosome membrane at low pH, there was no structural evidence to confirm this. By utilizing AAV9 as our model, we provide the first structural evidence that the externalization process occurs at the protein interface at the icosahedral 5-fold symmetry axis and can be triggered by lowering pH.

An engineered serum albumin-binding AAV9 capsid achieves improved liver transduction after intravenous delivery in mice

ABSTRACTRecombinant adeno-associated viral (AAV) vectors are frequently used to deliver nucleic acids for in vivo applications and are currently the leading platform for therapeutic gene delivery in gene therapy clinical trials. Presently, there is a need for improved AAV vectors with optimized transduction efficiency in target tissues. In these studies, an engineered albumin-binding consensus domain (ABDCon) was incorporated into the AAV9 capsid via fusion to the N-terminus of the AAV9 VP2 capsid protein to generate a variant AAV9 capsid with albumin-binding properties. The variant capsid, called AAV9-ABDCon, formed viable genome-containing vector particles and exhibited binding to human serum albumin. The AAV9 capsid, on the other hand, was not found to bind to human serum albumin by the methods used in this study. Following intravenous administration, the modified AAV9-ABDCon vector was found to achieve higher levels of transduction in liver tissue compared to AAV9. These findings suggest that serum albumin-binding may be a potential method to augment AAV-mediated liver-directed gene delivery.