In 2019, Deverman’s group identified the protein that serves as a receptor for AAV-PHP.B, a capsid that is efficiently transported across the blood-brain barrier. That made them wonder whether they could develop a screening approach that would find more AAVs that interacted with proteins they knew were expressed on specific cells.

As a proof of concept, the researchers aimed to deliver AAVs to the mouse brain by targeting LY6A and LY6C1, proteins on the surface of cells that line blood vessels in the brain and make up part of the blood-brain barrier in mice.

In a test tube, Huang and her colleagues mixed a pooled library of AAV capsids with the proteins they wanted to target. They then looked for capsids that bound to the target proteins and identified patterns in the capsids’ amino acid sequences. This allowed them to group similar capsids together. 

Next, they tested representative capsids from top performing groups in mice to see which ones reached the brain. They identified hundreds of capsids that bound to LY6A or LY6C1 and delivered their cargo to the central nervous system in mice more efficiently than AAV9, a capsid that crosses the blood-brain barrier and is used in an FDA-approved gene therapy, but is inefficient at delivering to the brain.

“We’re going back to first principles — rather than screening a random library in vivo, we focus on the cell type we want to engage and targeting specific proteins present on the surface of those cells,” Deverman said. “The nice thing about this approach is that we find so many examples in a test tube that we can test in mice. That allows us to take a lot more shots on goal than with in vivo screening.”

For the second round of capsid screening, the researchers added to the library 26 capsids that they and others had previously found bypassed the blood-brain barrier in animal models. The researchers found that 24 of these 26 capsids targeted either LY6A or LY6C1, suggesting that these two proteins are indeed the key targets of the AAVs that are identified through conventional library selections in mice. These proteins, however, do not have human equivalents, but the researchers say that their screening approach will help scientists engineer AAVs to target human proteins much more efficiently. 

Deverman, Huang, and others in the lab have already begun using their screening approach to home in on AAVs that bind to human proteins present on the blood-brain barrier. Over time, the team aims to use their experimental results to identify rules that help predict which proteins have the right characteristics to function as receptors for AAVs and guide the design of AAVs for new applications.