Genetically Engineered Parasites Smuggle Therapeutics into the Brain

The blood-brain barrier is a crucial protective membrane that evolved to shield the brain from pathogens and toxic substances that may be present in the blood. It also excludes the vast majority of potential therapeutics, severely limiting treatment options for brain tumors, neurodegenerative diseases, and genetic disorders of the central nervous system.¹

But some pathogens have evolved mechanisms to sneak past these defenses. Perhaps the most successful invader is Toxoplasma gondii, a single-celled parasite that establishes chronic infections within neurons and inhabits up to 30 percent of the human population.²

In a study recently published in Nature Microbiology, an international team of researchers harnessed these parasitic powers to deliver therapeutic macromolecules not only across the blood-brain barrier, but into neurons as well.³ In mice, they showed that genetically engineered Toxoplasma secreted a functional version of methyl CpG binding protein 2 (MeCP2), a protein in which mutations cause the rare neurological disorder Rett Syndrome.

“I like the paper,” said Jeroen Saeij, a parasitologist at the University of California, Davis who was not involved in the study. “I thought it was a good proof of concept paper. Obviously, it’s [in] the very early stage.” While Saeij noted the safety concerns inherent in using a potential pathogen as a delivery vehicle, he also acknowledged the benefits of this approach. “Crossing the blood-brain barrier is not so easy; many drugs or therapies have trouble actually getting into the brain. And Toxoplasma naturally goes into the brain, which is a big advantage.”

As a graduate student in neuroscience at Tel Aviv University, study coauthor Shahar Bracha, now at the Massachusetts Institute of Technology, learned all about the difficulty of delivering drugs to the brain. So, when she came across research on Toxoplasma, she saw an opportunity where most others had seen only an opportunistic pathogen: Perhaps this brain-targeting parasite could be engineered to produce therapeutics once it arrived at its destination.

“It was a crazy idea in the beginning,” admitted Bracha. But the more she read, the more it started to seem possible. “The idea of trying to solve problems in bioengineering by going into nature and trying to find a place where evolution already developed a solution—instead of trying to engineer things from scratch—that’s an idea that inspired me a lot,” she said.

Toxoplasma seemed to already possess most of the qualities needed for brain-targeting therapeutics: It could evade the host immune system, cross the blood-brain barrier, spread through the brain with minimal damage, enter neurons, and secrete proteins into the host cells. 

First, the researchers searched the literature for proteins linked to human neurological disorders, for which there was preclinical evidence that even partial restoration of the protein was beneficial. They came up with several candidate proteins that could function as potential therapies. However, they needed to figure out how to express these proteins in a Toxoplasma secretory organelle called the dense granule, which is one of three secretion systems present in the parasite. This organelle is responsible for secreting proteins during the chronic infection stage, when Toxoplasma is safely ensconced in a vacuole within the host neuron. The researchers engineered Toxoplasma to produce different candidate proteins, which were each fused to GRA16, a protein that, once secreted by the dense granule, travels to the host cell nucleus. 

Of the many therapeutic proteins they engineered into Toxoplasma, only a few—including MeCP2 and transcription factor EB, which may help counteract neurodegeneration—successfully hitched a ride with GRA16 into the host cell nucleus. However, it was one thing to show that this Frankenstein of a protein could make it to the host nucleus and quite another to show that it would be functional when it arrived. 

Bracha and her colleagues introduced the engineered parasite to human brain cells in vitro and found that the GRA16-MeCP2 protein bound to methylated DNA in a manner similar to normal MeCP2. Furthermore, the fusion protein altered gene expression in human brain organoids, suggesting, although not conclusively proving, that MeCP2 functioned normally even when attached to GRA16. The researchers demonstrated that these genomic adjustments had not interfered with Toxoplasma’s trademark talent: When peripherally injected into mice, parasites carrying the fusion protein were still able to make their way into the brain.

The scientists’ work is only just beginning, however. “It’s important to emphasize that this is a proof of concept for a very new idea,” said Bracha. “Definitely the safety profile of these kinds of vectors needs to be improved by attenuation of the parasite. That’s something that has been done with other biologics. With viral gene therapies there’s been decades of research that was performed to attenuate and optimize them for delivery.”

Saeij noted that Toxoplasma may be especially tricky to attenuate, however. “Most research has shown that Toxoplasma strains that are less virulent…have trouble reaching the brain, because normally they get destroyed by the immune system. So, it’s a little bit of a Catch-22: If you want to make Toxoplasma less virulent and less dangerous, it’s probably also going to be less able to get into the brain.”

Researchers still believe it may be possible to attenuate Toxoplasma in other ways and Bracha said that study coauthor Lilach Sheiner, a parasitologist at the University of Glasgow, is currently exploring possible solutions. One option is engineering auxotrophy so that an organism cannot proliferate in the absence of a compound needed for its growth. They are also exploring ways to design genetic kill switches for on-demand elimination. If researchers are, in fact, able to achieve this attenuation, they will be well on their way to transforming Toxoplasma from a pathogen into an ally in the fight against neurological disease.

Disclosure of conflicts of interest: Shahar Bracha is a co-inventor on a patent application related to this work and a scientific advisor for Epeius Pharma Ltd.