Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood-brain barrier crossing by engineered viral vectors

Abstract

The blood-brain barrier (BBB) presents a major challenge for delivering large molecules to study and treat the central nervous system. This is due in part to the scarcity of targets known to mediate BBB crossing. To identify novel targets, we leverage a panel of adeno-associated viruses (AAVs) previously identified through mechanism-agnostic directed evolution for improved BBB transcytosis. Screening potential cognate receptors for enhanced BBB crossing, we identify two targets: murine-restricted LY6C1 and widely conserved carbonic anhydrase IV (CA-IV). We apply AlphaFold-based in silico methods to generate capsid-receptor binding models to predict the affinity of AAVs for these identified receptors. Demonstrating how these tools can unlock target-focused engineering strategies, we create an enhanced LY6C1-binding vector, AAV-PHP.eC, that, unlike our prior PHP.eB, also works in Ly6a -deficient mouse strains such as BALB/cJ. Combined with structural insights from computational modeling, the identification of primate-conserved CA-IV enables the design of more specific and potent human brain–penetrant chemicals and biologicals, including gene delivery vectors.

Natural Adeno-Associated Virus Serotypes and Engineered Adeno-Associated Virus Capsid Variants: Tropism Differences and Mechanistic Insights

“…In addition, pull-down assays demonstrated that P5*-F6*-K7* alone are sufficient for binding to Ly6A in vitro and constitute the minimal motif for this interaction. In agreement, a newly developed integrative structure computational modeling pipeline used to model the AAV-PHP.eB–Ly6A interaction pointed at P5*-F6* as being essential for binding, showing that Ly6A interacts with one capsid monomer and that additional interactions induce steric clashes [ 259 ].…”

“…After intracranial injection in rat or mice, it transduced preferentially NSCs with a higher efficiency than the parental serotype [ 258 ]. Finally, a recent study engineered AAV-PHP.eC , a novel variant derived from AAV-PHP.C1 which outperformed AAV-PHP.C2 in CNS targeting in multiple mouse strains after IV delivery, transducing neurons and astrocytes [ 259 ].…”

“…Interestingly, whilst AAV-PHP.B showed a dramatic increase in CNS tropism in C57BL/6 mice and other strains, it showed low transduction in the CNS in strains such as BALB/c mice [ 240 , 241 , 242 ]. By comparing membrane protein expression and performing whole-genome SNP and indel analysis between permissive and non-permissive mouse strains for CNS transduction as well as transcriptomic profiling of brain microcapillary endothelia, the receptor responsible for allowing AAV-PHP.B variants to cross the BBB via transcytosis was identified as glycosylphosphatidylinositol (GPI)-anchored protein lymphocyte activation protein-6A (Ly6A)/stem cell antigen-1 (Sca-1) [ 240 , 259 , 308 , 309 ]. This protein is located on membrane lipid rafts and expressed mainly in the endothelial cells in the CNS, albeit also in neurons and glia at lower levels [ 308 , 310 ], but its physiological functions are still unclear, and no ligand has been identified to date [ 311 , 312 ].…”

“…This protein is located on membrane lipid rafts and expressed mainly in the endothelial cells in the CNS, albeit also in neurons and glia at lower levels [ 308 , 310 ], but its physiological functions are still unclear, and no ligand has been identified to date [ 311 , 312 ]. ELISA, surface plasmon resonance (SPR), antibody competition experiments, and the use of Ly6a CRISPR knocked-out BMVECs cells demonstrated that AAV-PHP.B binds to Ly6A on Ly6A-expressing cells, such as primary BMVECs, and uses it for transduction [ 254 , 259 , 308 , 309 ]. The assessment of the binding and transduction of Pro5 CHO cells and derivatives expressing different levels of galactose demonstrated that Ly6A serves as an attachment factor for AAV-PHP.eB independently of galactose and plays a role in the internalization and/or trafficking of virions [ 308 ].…”

An AAV capsid reprogrammed to bind human Transferrin Receptor mediates brain-wide gene delivery

“…As compared to natural AAV capsids such as AAV9 and AAV2 that are used in approved gene therapies, BI-hTFR1 demonstrated the remarkable ability to be actively transported across a human brain endothelial cell layer and to cross the BBB and transduce cells throughout the brains and spinal cords of TFRC KI mice after intravenous delivery. BI-hTFR1 exhibited a broad CNS tropism enhancement similar to those observed with engineered mouse BBB-crossing capsids, e.g., AAV-PHP.B, BI28, AAVF, and 9P31 that bind to mouse GPIanchored proteins LY6A, LY6C1, or Car4 (25,59,60). By engaging highly abundant CNS endothelial cell surface proteins as new receptors, these capsids are able to efficiently cross the BBB and gain access to parenchymal cells.…”