Caltech’s Enzyme Discovery Enables New Mechanism for Crossing the Blood–Brain Barrier

Caltech researchers found an enzyme that permits viral vectors to cross the blood-brain barrier, doubtlessly aiding mind dysfunction drug improvement and analysis.

The blood–mind barrier (BBB) is a stringent, almost impenetrable layer of cells that guards the mind, defending the very important organ from hazards within the bloodstream equivalent to toxins or micro organism and permitting solely a really restricted set of small molecules, equivalent to vitamins, to cross via. This layer of safety, nevertheless, makes it troublesome for researchers to review the mind and to design medicine that may deal with mind issues.

Now, a brand new examine from Caltech has recognized a beforehand unknown mechanism by which sure viral vectors—protein shells engineered to hold varied desired cargo—can cross via the BBB. This mechanistic perception might present a brand new method to designing viral vectors for analysis and therapeutic purposes. Understanding this and different new mechanisms may additionally give perception into how the mind’s defenses could also be exploited by emergent pathogens, enabling researchers to arrange strategies to dam them.

A well timed method to discovering putative BBB transporters: (1) Directed evolution yields numerous AAVs with enhanced mind efficiency. (2) BBB-specific membrane proteins are recognized and screened in vitro for his or her capacity to spice up AAV efficiency. (3) Computational strategies allow high-throughput goal screening and reverse engineering of novel viral, protein, and chemical instruments. Credit: Tim Shay and Gradinaru Lab at Caltech

The analysis was carried out within the laboratory of Viviana Gradinaru (Caltech BS ’05), the Lois and Victor Troendle Professor of Neuroscience and Biological Engineering and director of the Center for Molecular and Cellular Neuroscience, a part of the Tianqiao and Chrissy Chen Institute for Neuroscience at Caltech, and seems within the journal Science Advances on April 19. The study’s first authors are Timothy Shay (PhD ’15), the scientific director of Caltech’s Beckman Institute CLOVER Center; bioengineering graduate Xiaozhe Ding (PhD ’23); and CLOVER research associate Erin Sullivan.

Though the BBB serves as the brain’s formidable defense, certain viruses have naturally evolved the ability to bypass it. For decades, researchers have studied how to use these viruses as a kind of BBB-crossing Trojan Horse; to do so, researchers scrape out the original viral cargo carried by the viruses and then use their hollow shell to ferry beneficial therapeutics or tools for research. Viral vectors with the ability to cross the BBB can deliver desired genes to the brain through a simple injection into the bloodstream and thus do not need to be invasively injected into the brain. Unfortunately, most vectors derived from naturally evolved viruses are very inefficient at crossing the BBB, and so they must be administered at high doses, increasing the risk of side effects.

Carbonic anhydrase IV (CA-IV) enables enhanced brain access from the bloodstream. Fluorescent image of CA-IV protein expression on the mouse blood-brain-barrier (BBB) and AlphaFold2-generated structural model of CA-IV bound to the engineered loop of a BBB-crossing viral vector. Credit: Erin Sullivan and Xiaozhe Ding, Gradinaru Lab at Caltech

Inspired by nature, Gradinaru lab has over the past decade used the process of directed evolution—a technique pioneered at Caltech by Nobel Laureate Frances Arnold—to guide the evolution of vectors and enhance their ability to cross the BBB. Over the years, the group has generated dozens of vectors with different abilities to cross the BBB and target various tissues and cell types in a variety of species. In the process, they noticed that distinct vectors can behave differently across model organisms, suggesting that these vectors may each have identified distinct and efficient paths from the bloodstream to the brain.

However, although researchers knew that these vectors could cross, it was still unclear how they were crossing. Where are the entry points in the fortified wall of the BBB?

In this new study, the team led by Shay, Sullivan, and Ding aimed to identify these mechanisms using a multidisciplinary approach that combines the researchers’ expertise in techniques of protein chemistry, molecular biology, and data science, respectively. First, Shay and Sullivan developed a cell-culture screen to quickly test the ability of scores of diverse proteins found on the surface of the BBB to enhance the infectivity of vectors in a dish. Ding then used an advanced computational model (based on a complex artificial intelligence program called AlphaFold) to simulate how vectors interact with the different proteins, revealing the geometries of the interactions uncovered in the screen. Next, a kind of “March Madness” competition process—which is the subject of an upcoming paper—determined which vectors interacted best with which proteins, and recapitulated the experimental results of the screen.

Outlook: AAVs engineered to reach the brain from the bloodstream enable mechanistic insights into blood-brain-barrier biology, including identification of novel receptors, that can help design next-generation viral and non-viral delivery vectors for the brain and maybe also anticipate and fight emergent pathogens. Credit: Catherine Oikonomou and Viviana Gradinaru, Caltech

The team discovered a particular enzyme, called carbonic anhydrase IV (CA-IV), that enables a few different viral vectors to cross the BBB. Interestingly, CA-IV is an ancient enzyme that is found on the BBBs of many diverse species, including humans; it was not previously known to facilitate any kind of BBB-crossing process. In the future, this combined experimental and computational approach may accelerate the discovery of additional solutions to BBB crossing and the team is excited about the possibilities to apply these molecular gateways to the delivery of brain therapeutics.

“Blood-brain-barrier crossing is a key biological puzzle,” says Gradinaru. “To say that an enzyme that regulates blood pH and lets us taste the fizz in soda, is an unintuitive target for helping viruses through the BBB would be an understatement. Now we can leverage CA-IV, and other exciting targets that continue to emerge from our approach rooted in identifying the mechanisms of BBB-crossing viral vectors, to help us design next-generation viral and non-viral delivery vectors for the brain. And maybe, it will also help us build resilience against emergent pathogens that could hijack the same routes for brain entry.”

Understanding the range of mechanisms by which viral vectors cross into the brain is critical for enabling personalized treatments across diverse human populations. Brains, and their BBBs, vary widely across species and even among humans. In fact, an individual’s BBB can vary over their own lifetime. By revealing new BBB-crossing mechanisms, a wider range of neuropharmaceutical delivery options can be tailored to individuals with diverse biological profiles.

Reference: “Primate-conserved carbonic anhydrase IV and murine-restricted LY6C1 enable blood–brain barrier crossing by engineered viral vectors” by Timothy F. Shay, Erin E. Sullivan, Xiaozhe Ding, Xinhong Chen, Sripriya Ravindra Kumar, David Goertsen, David Brown, Anaya Crosby, Jost Vielmetter, Máté Borsos, Damien A. Wolfe, Annie W. Lam and Viviana Gradinaru,
19 April 2023, Science Advances.
DOI: 10.1126/sciadv.adg6618

Funding was provided by the National Institutes of Health and Caltech’s Beckman Institute for CLARITY, Optogenetics and Vector Engineering Research (CLOVER).