The delivery of drugs across barriers, including the notoriously challenging blood-brain barrier, remains one of the biggest hurdles in drug discovery.
“One of the biggest challenges in treating neurological diseases is getting through the blood-brain barrier (BBB),” explained Oded Rechavi, PhD, professor at Tel Aviv University. “It is very difficult to deliver drugs to the brain via the bloodstream, and this is especially true for large molecules such as proteins, the critical ‘machines’ that carry out many important functions inside the cell.”
And although novel approaches have been tried, and some have made progress, drug delivery to neurons remains an unmet need. A new solution utilizes the unicellular parasite Toxoplasma gondii, which can infect a vast variety of organisms, but reproduces only in the guts of cats. The parasite is very effective in infecting humans, with an estimated third of the global population infected at some point in their lives. In this new study, T. gondii was engineered to deliver drugs to the human brain.
The results were published in Nature Microbiology in the paper, “Engineering Toxoplasma gondii secretion systems for intracellular delivery of multiple large therapeutic proteins to neurons.”
Regarding T. gondii, Rechavi explained, “Most people don’t even feel the infection or only experience mild flu-like symptoms. The parasite is, however, dangerous for people with immune failure due to conditions like AIDS, and for fetuses whose immune system has not yet developed. This is why pregnant women are advised not to eat raw meat which might contain the parasite, and to stay away from cats, who might deliver it through their feces. While ridding the body of the parasite, a healthy immune system has only limited access to the brain, and the parasite remains in the brain throughout the carrier’s lifetime.”
The parasite’s ability to penetrate the human brain and survive there in a dormant state, without reproducing, made it a perfect candidate for the researchers’ novel approach: to genetically engineer T. gondii to secrete therapeutic proteins. In addition, previous work has demonstrated that T. gondii can deliver proteins to host cells.
In this new study, the team used transgenic model animals that were injected with parasites genetically engineered to produce and secrete proteins that travel into cell nuclei. More specifically, they “engineered T. gondii’s endogenous secretion systems, the rhoptries and dense granules, to deliver multiple large (>100 kDa) therapeutic proteins into neurons via translational fusions to toxofilin and GRA16.”
Several lines of evidence proved that the proteins had been delivered to the target area and remained active in the neurons’ nuclei including demonstrated delivery in cultured cells, brain organoids, and in vivo, and probe protein activity using imaging, pull-down assays, scRNA-seq, and fluorescent reporters.
In addition, the researchers demonstrated robust delivery after intraperitoneal administration in mice. As a proof of concept, they demonstrated GRA16-mediated brain delivery of the MeCP2 protein, whose deficiency is associated with Rett syndrome.
“This is a deadly syndrome caused by a deficiency in a single gene called MePC2 in brain cells, and our engineered Toxoplasma gondii was able to deliver it to the target cells,” said Rechavi. “But this is just one example. There are many other diseases caused by deficiency or abnormal expression of a certain protein.”
To ensure the method’s safe and effective therapeutic implementation, for both drug delivery and genetic editing, the company Epeius was established in collaboration with Ramot—the technology transfer company of Tel Aviv University, and with the University of Glasgow’s research and innovation services.
