Mapping the Structural Determinants Required for AAVrh.10 Transport across the Blood-Brain Barrier

Abstract

Effective gene delivery to the CNS by intravenously administered adeno-associated virus (AAV) vectors requires crossing the blood-brain barrier (BBB). To achieve therapeutic CNS transgene expression, high systemic vector doses are often required, which poses challenges such as scale-up costs and dose-dependent hepatotoxicity. To improve the specificity and efficiency of CNS gene transfer, a better understanding of the structural features that enable AAV transit across the BBB is needed. We generated a combinatorial domain swap library using AAV1, a serotype that does not traverse the vasculature, and AAVrh.10, which crosses the BBB in mice. We then screened individual variants by phylogenetic and structural analyses and subsequently conducted systemic characterization in mice. Using this approach, we identified key clusters of residues on the AAVrh.10 capsid that enabled transport across the brain vasculature and widespread neuronal transduction in mice. Through rational design, we mapped a minimal footprint from AAVrh.10, which, when grafted onto AAV1, confers the aforementioned CNS phenotype while diminishing vascular and hepatic transduction through an unknown mechanism. Functional mapping of this capsid surface footprint provides a roadmap for engineering synthetic AAV capsids for efficient CNS gene transfer with an improved safety profile.

Structure of the AAVhu.37 capsid by cryoelectron microscopy

“…4a and 4b). Determinants of BBB penetration have been mapped to VR-I by making chimeric capsids between the BBB-penetrating AAVrh.10 and the nonpenetrating AAV1 (Albright et al, 2018). The AAVrh.10 VR-I residues that were identified as sufficient to confer BBB penetration on AAV1 are absolutely conserved in AAVhu.37.…”

Taking a Hint from Structural Biology: To Better Understand AAV Transport across the BBB

“…[1][2][3][4] However, what capsid features distinguish these particular AAV serotypes from the rest regarding the CNS phenotype remains a puzzle. To investigate this issue, Albright et al 5 first generated a library of chimeric capsid variants through DNA shuffling using AAV1 and AAVrh.10 as the parental capsids. These two capsids share 85% amino acid sequence homology but display distinct CNS phenotypes: AAV1 transduces only brain vasculature, whereas AAVrh.10 can cross the BBB and transduce neurons and astrocytes in addition to blood vessels.…”

“…12 This in vitro BBB model revealed insights into the interaction between rAAV and BBB in the process of transendothelial movement, suggesting that the transport of AAV9 vector across the BBB is an active, cell-mediated process, and that it does not disrupt the BBB integrity. 12 The new study by Albright et al 5 is also important for expanding the vector tool box for CNS gene therapy. The engineered AAVR1X capsid can not only cross the BBB and transduce neurons, but also shows markedly reduced transduction in the liver and, therefore, may have a better safety profile.…”

“…3,4 More recently, targeted probes have been developed that utilize antibodies, aptamers, or peptides to bind to cancerous tissue more specifically and further enhance the tumor-to-background ratio (TBR). 5,6 In this issue of Molecular Therapy, Predina et al 7 report on their pilot clinical trial of a near-infrared smallmolecule probe targeting folate receptor-a (FRa) to sensitively detect pulmonary adenocarcinomas (PAs) during surgery. In line with previous clinical trials of FRa-targeted probes in ovarian and colorectal cancers and of fluorescently-labeled anti-EGFR antibodies, such as cetuximab-IRD800, in head and neck cancer, this trial lends further support to the clinical utility of intraoperative molecular imaging (IMI) in identifying lesions that might otherwise be missed.…”

Modulation of Sialic Acid Dependence Influences the Central Nervous System Transduction Profile of Adeno-associated Viruses

“…However, the capsid-glycan interactions that might influence the mechanisms underlying AAV capsid transport across the BBB into the CNS are not well understood. Toward furthering this understanding, our lab recently identified structural determinants on the AAVrh.10 capsid, which when grafted onto the AAV1 capsid (AAV1RX) permits CNS entry (17). The engineered AAV1RX variant demonstrates marked transduction of neurons across multiple brain regions and reduced tropism for glial and vascular endothelial cells in the brain.…”