New Uses for Botox to Edit the Proteome

Researchers at Harvard University and Broad Institute have shown that botulinum toxin proteins can be engineered to enable new medical applications. They can promote neuro-regeneration, regulate growth hormones, calm rampant inflammation, or dampen cytokine storm. The latter is a life-threatening immune response associated to COVID-19 and related diseases.

Botulinum toxin is sold with the trade name botox, among others. Although it can be a deadly poison, many studies have shown that it is useful for cosmetic procedures. For example, botox injections can relax fascial muscles that cause wrinkles and frown lines. They do this by inhibiting release of acetylcholine, which would otherwise cause the muscles to contract.

Beyond cosmetics, the Food and Drug Administration (FDA) approved botox treatments for conditions such as:

• excessive sweating
• chronic migraines
• overactive bladder
• eyelid spasms
• crossed eyes

Botox is also approved as a treatment for medical conditions like cervical dystonia, to relax the muscles that cause pain. When expertly applied, treatments are generally safe. And possible side effects tend to remain near the site of injection.

Botox for Proteomics

Now, the range of potential botulinum toxin applications and possible new uses for botox has been spectacularly expanded. In a study published in Science, the researchers describe how they successfully reprogrammed botulinum toxin proteins called proteases.

Proteases cut proteins to either activate or deactivate them. The reprogrammed proteases are able to cut entirely new protein targets. Some have little or no similarity to the native targets of the starting proteases. And they can simultaneously avoid cutting or otherwise engaging their original targets.

The researchers also started to design treatments that can cross into a cell. Unlike most large proteins, botulinum toxin proteases can enter neurons in large numbers. This gives them a wider reach that makes them all the more appealing as potential therapeutics. Now, there’s a clear path toward the ability to evolve custom proteases with tailor-made instructions for which protein to cut.

"Such a capability could make 'editing the proteome' feasible," said professor David Liu, "in ways that complement the recent development of technologies to edit the genome."

The proteome is the set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a given time, under defined conditions.

Proteases can find and attach to any number of proteins. And, once bound, they can do more than just destroy their target. They could, for example, reactivate dormant proteins. Some diseases, like neurological damage following a stroke, could be successfully treated with protease-based therapies.

But so far, said Liu, protease-based therapeutics have been limited by "the lack of a technology to generate proteases that cleave protein targets of our choosing."

"At the outset," said Dr. Travis Blum, first author of the study, "we didn't know if it was even feasible to take this unique class of proteases and evolve them or teach them to cleave something new because that had never been done before." But using the PACE (Phage-Assisted Continuous Evolution) platform developed bi Liu’s team, the researchers evolved four proteases from three families of botulinum toxin. All four had no detected activity on their original targets and cut their new targets with a high level of specificity.

"In theory, there is a really high ceiling for the number and type of conditions where you could intervene," concluded Blum. "We're still trying to understand the system's limitations, but in an ideal world, we can think about using these toxins to theoretically cleave any protein of interest."