Targeting AAV vectors to the central nervous system by engineering capsid–receptor interactions that enable crossing of the blood–brain barrier

Abstract

Viruses have evolved the ability to bind and enter cells through interactions with a wide variety of cell macromolecules. We engineered peptide-modified adeno-associated virus (AAV) capsids that transduce the brain through the introduction of de novo interactions with 2 proteins expressed on the mouse blood–brain barrier (BBB), LY6A or LY6C1. The in vivo tropisms of these capsids are predictable as they are dependent on the cell- and strain-specific expression of their target protein. This approach generated hundreds of capsids with dramatically enhanced central nervous system (CNS) tropisms within a single round of screening in vitro and secondary validation in vivo thereby reducing the use of animals in comparison to conventional multi-round in vivo selections. The reproducible and quantitative data derived via this method enabled both saturation mutagenesis and machine learning (ML)-guided exploration of the capsid sequence space. Notably, during our validation process, we determined that nearly all published AAV capsids that were selected for their ability to cross the BBB in mice leverage either the LY6A or LY6C1 protein, which are not present in primates. This work demonstrates that AAV capsids can be directly targeted to specific proteins to generate potent gene delivery vectors with known mechanisms of action and predictable tropisms.

Generation of an enhancer-driven gene expression viral tool specific to dentate granule cell-types through direct hippocampal injection

“…In future studies we aim to use a systemic injection strategy, to attain an overview of the expression pattern driven by the enhancer on a brain wide scale. To this end, the PHP.eB serotype might be a good candidate, as it has been shown to have a highly efficient transduction across the blood brain barrier (BBB) ( Huang et al, 2023 ), more so than its predecessor PHP.B ( Deverman et al, 2016 ). The development of BBB crossing EDGE viruses might also be relevant for future clinical application in gene therapy research.…”

Delivery of DNA-Based Therapeutics for Treatment of Chronic Diseases

“…In addition to the 13 natural serotypes, novel hybrid AAV vectors have been developed and are still being developed [ 30 , 31 ]. Each serotype, regardless of natural or novel identity, has specific receptor and tissue tropisms, as reviewed by Issa et al [ 27 ] ( Table 2 ), with engineered serotypes being generated to modulate such tropisms [ 32 , 33 , 34 ]. With this in mind, serotype selection is critical for maximizing efficiency of the therapy [ 18 , 30 , 35 ].…”

Human cell surface-AAV interactomes identify LRP6 as blood-brain-barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder

“…Using this broad, unbiased screen, we identified several new AAV interactions with implications for the host immune response (human interleukin 3 (IL3) binding to AAV9), enhanced BBB crossing across species (via low-density-lipoprotein-receptor-related-protein 6 (LRP6) binding by AAV9-X1.1 and CAP-Mac), and peripheral tissue tropism (through pancreas-expressed glycoprotein 2 (GP2) binding by AAV9-X1.1 and CAP-Mac). Understanding the mechanism of action of systemic AAVs through methods such as those used here will be critical for successful vector translation and enables design of improved vectors, as well as other therapeutic protein modalities, for specific targets 33,34 .…”