New AAV9 engineered variants with enhanced neurotropism and reduced liver off-targeting in mice and marmosets.

Although adeno-associated virus 9 (AAV9) has been highly exploited as delivery platform for gene-based therapies, its efficacy is hampered by low efficiency in crossing the adult blood-brain barrier (BBB) and pronounced targeting to the liver upon intravenous delivery. We generated a new galactose binding-deficient AAV9 peptide display library and selected two new AAV9 engineered capsids with enhanced targeting in mouse and marmoset brains after intravenous delivery. Interestingly, the loss of galactose binding greatly reduced undesired targeting to peripheral organs, particularly the liver, while not compromising transduction of the brain vasculature. However, the galactose binding was necessary to efficiently infect non-endothelial brain cells. Thus, the combinatorial actions of the galactose-binding domain and the incorporated displayed peptide are crucial to enhance BBB crossing along with brain cell transduction. This study describes two novel capsids with high brain endothelial infectivity and extremely low liver targeting based on manipulating the AAV9 galactose-binding domain.