Novel Molecular Shuttle Could Deliver Targeted, Diverse Therapies to the Brain

The blood–brain barrier (BBB) is a protective filter that lines the brain's blood vessels and is notoriously hard to penetrate, making the treatment of brain disorders very difficult. Strategies designed to breach the BBB include using brain surgery and focused ultrasound waves to create physical openings for the transportation of medicines and employing engineered adeno-associated viruses (AAVs) to ferry genetic payloads into desired locations, but these methods have limitations. Now, researchers at Caltech have developed a new, more effective way to transport potentially life-changing therapeutics across the BBB. "Despite the economic and social toll of billions of patients suffering from neurological and neuropsychiatric disorders, a major bottleneck remains in successfully delivering therapeutics to the brain to address these conditions," says Viviana Gradinaru (BS '05), the Troendle Professor of Neuroscience and Biological Engineering at Caltech, director and Davis Leadership Chair of the Merkin Institute for Translational Research at Caltech, and a Howard Hughes Medical Institute Investigator. "Our work provides a new and powerful solution for delivery of both preexisting therapeutics that could, in principle, work, but they don't reach the brain, and new therapeutics that haven't been considered yet." A paper describing the team's new method, called BrainCAB (Brain access through Carbonic Anhydrase-binder Bioconjugation), was published on August 26 in Nature Chemical Biology. Two of the study's co-first authors are Xiaozhe Ding (PhD '23) and Xinhong Chen (PhD '23), who conducted the work first as graduate students in the Gradinaru lab and then as staff of the Beckman Institute CLOVER Center directed by Timothy Shay (PhD '15). Gradinaru's research group has been investigating delivery systems across the BBB—which shields our brains from bacteria and viruses but also makes it hard to access when help is needed to fight disease—for more than a decade. One line of previous work focused on engineered AAVs, which can carry therapeutic genetic material across the BBB and into cells. Although AAVs are powerful tools for gene delivery, they are not a general means of directly transporting therapeutic biomolecules such as antibodies, proteins, or oligonucleotides into the brain. In 2023, a team from Gradinaru's lab and the CLOVER Center discovered that a particular enzyme, called carbonic anhydrase IV (CA-IV), sits on the surface of brain blood vessels and acts as a receptor that can enable a few different engineered AAVs to cross the BBB through a natural process called receptor-mediated transcytosis. They started to wonder if CA-IV could mediate nonviral delivery of larger molecules as well. "We thought we should evolve beyond viral delivery and try to engage these new pathways on the blood–brain barrier for medicine delivery," says Gradinaru, who is also an affiliated faculty member of Caltech's Tianqiao and Chrissy Chen Institute for Neuroscience. "That's where it became obvious that we cannot do it by ourselves because we had the path and the biological discovery, but what we needed was a solution that works to basically dock onto these receptors and drag in cargo." Building on Ding's structure modeling work showing that brinzolamide, a drug commonly used as a prescription eye drop to treat glaucoma, could bind to CA-IV at a similar site as the AAV capsids, Gradinaru and Ding recognized that the compound might be adapted to become a molecular shuttle. So, they enlisted the help of Sarah Reisman, Bren Professor of Chemistry and Norman Davidson Leadership Chair of the Division of Chemistry and Chemical Engineering, and members of her lab—including former postdoctoral scholars and co-first authors of the paper Philip Boehm and Alba Carretero-Cerdán—to use chemical synthesis to modify brinzolamide in a way that it could still engage the receptor while carrying medications, acting as a "molecular shuttle" across the BBB. "The fun thing about chemical synthesis is that it's very modular," says Reisman, who is also the Norman Davidson Leadership Chair of the Division of Chemistry and Chemical Engineering. "Once you have your idea, you can make changes to the molecule and ask how it affects the activity. This project was a great collaboration where we'd make compounds, hand them off to Viviana's group, and they would evaluate them, and there was this sort of feedback loop." Together they designed and tested a modified version of brinzolamide that can bind to CA-IV while carrying therapeutics. The team tested the binding of the new molecules to CA-IV from multiple species and then demonstrated that they crossed the BBB both in rodents and nonhuman primates, important preclinical models. As proof of concept, the team tried to deliver atezolizumab—an immunotherapy used in the treatment of cancers such as lung and liver that can metastasize to the brain—using the BrainCAB shuttle and found that it substantially increased delivery of the antibody to the brain. "We already have these drugs available, but if you detect the cancer too late, and it metastasizes, they don't really reach the brain and will not have an impact," Gradinaru explains. "Now, we show that these molecular shuttles increase not only how much gets to the brain but, importantly, how persistent the effect is compared to current state-of-the-art delivery methods, such as transferrin receptor-based shuttles." She notes that compared to protein-based shuttles, BrainCAB's compact small-molecule format may be less likely to trigger an unwanted immune response and may be less disruptive of the physical properties of the attached therapeutic. Its modular chemistry may also simplify manufacturing at scale and make the platform easy to adapt beyond antibodies to a broad range of therapeutic modalities, including RNA medicines. However, Gradinaru cautions that the team's findings are preliminary, and the method needs further refinement and testing with various drugs. But Caltech has filed a patent application covering the technology, which has been licensed to a Caltech startup, Receptive Bio. "One of the missions of Caltech's Merkin Institute for Translational Research is to deploy technologies and try to bridge from the basic science that we're excellent at to translational impact, including impact for health care," Gradinaru says. "This interdisciplinary collaboration between chemistry and neurobiology would have not been possible without the support of the Merkin Institute, whose funding also allowed us to start the first experiment, which was then amplified and brought to the finish line by the National Institutes of Health funding through the NIH BRAIN Initiative." Ding and Chen, who completed their doctoral degrees under Gradinaru, went on to co-found Receptive Bio, the Caltech startup that licensed the BrainCAB technology. They are continuing its preclinical development and testing with the goal of advancing the technology toward clinical studies in humans. Reisman, Shay, and Gradinaru are also continuing to work together to turn their molecular shuttle from a vehicle that can only cross the BBB into one that could also carry multiple functionalities at once, for example, additional therapeutics or receptor-binding molecules for targeting specific cell types and brain areas. "If you have two different types of cargo attached, they can each have a different function that could be cooperative in a beneficial way. The design is modular, and there are a lot of exciting possibilities for the next steps," Reisman says. The CA-IV receptor is specifically enriched in endothelial cells of the BBB, unlike other potential receptors that are widely expressed on other organs and cell types. Therefore, "continued development of multivalent CA-IV-directed small-molecule shuttles could also unlock brain cell-type-specific therapeutic delivery, a fundamental need for neurological disorders caused in concert by dysfunction in multiple cell types," Gradinaru concludes. The Nature Chemical Biology paper is called "CA-IV-directed small-molecule shuttle enables targeted brain delivery of biologics." A full list of authors can be found there. The work was mainly supported by funding from the National Institutes of Health (NIH), the NIH BRAIN Initiative, Caltech's Merkin Institute for Translational Research, and Caltech's Beckman Institute. Gradinaru is a Howard Hughes Medical Institute Investigator and Xinhong Chen was an inaugural Chen Graduate Fellow.