Protein engineering enables better treatment for rare inherited metabolic disorders

GlycoNet researchers develop hybrid enzyme to unlock the potential of enzyme replacement therapy 

By Ali Chou

One in every 5,000 Canadian newborns develops lysosomal storage disorders (LSD), a type of metabolic diseases caused by genetic mutations. Tay-Sachs and Sandhoff diseases are amongst the most severe LSD. Children suffering from these diseases lack an enzyme that can recycle used lipids (gangliosides) in their neurons. Eventually, the un-recycled gangliosides accumulate in the brain, interfering with normal biological processes, leading to brain damage. This is fatal in many cases and there are no known cures for Tay-Sachs or Sandhoff disease. 

For some LSDs, patients can receive intravenous infusion of solutions containing the enzymes they are missing, a procedure called enzyme replacement therapy (ERT). ERT can be compared to patching potholes on the road: the infused, healthy enzymes fill in the gap left by the deficient enzymes. Although effective, there are challenges to this procedure. The infused enzymes have to be stable enough to circulate in the blood stream before reaching the targeted neurons. They also have to cross the blood-brain barrier—a highly-selective border that separates the blood and biomolecules from the brain—to arrive at the neurons. Neither of these challenges have been met clinically for Tay-Sachs and Sandhoff disease.

For years, researchers have been trying to design the “perfect” enzyme, one that can sail steadily through the blood vessels and across the blood-brain barrier. But much like developing a type of asphalt strong enough to withstand Canadian winters, the elusive search for this perfect enzyme continues. GlycoNet researchers however are working on a uniquely promising approach. 

Drs. Brian Mark, Barbara Triggs-Raine and Helene Perreaultfrom the University of Manitoba are forging ahead, determined to find that elusive, perfect enzyme. The team has recently been awarded a CIHR grant to evaluate a stable hybrid enzymethat can be used in ERT for children with Tay-Sachs and Sandhoff diseases. 

“The defective enzyme that causes Tay-Sachs and Sandhoff diseases is a relatively large protein encoded by two separate genes,” says Mark. “In order to make enough functional enzyme for ERT, we extracted the important features of both genes to make a new hybrid gene. I did this in collaboration with Dr. Don Mahuran (Toronto Hospital for Sick Children, retired). The enzyme encoded by the hybrid gene is now much easier to produce, and more stable than the natural enzyme.” 

Mark and his team have moved the goal post ahead several steps by visualizing the enzyme responsible for Tay-Sachs and Sandhoff diseases at a very high resolution. “This gave us insight to how the enzyme clears the metabolites in the brain and how other proteins are involved in the clearing process,” he says. “All this information lays a solid foundation for developing the enzyme for use in clinical testing for ERT.” 

However, one main roadblock remains: after infusion into the blood, the enzyme has to pass through the blood-brain barrier to reach the brain and carry out the desired therapeutic function.

To find the solution, Mark’s team is exploring different “Trojan horse”, molecules that are recognized by the barrier and trigger it to transport cargo through. Think of going through a gate to enter a National Park. The blood-brain barrier is the mechanical arm stopping you from entering. The “Trojan horse” molecule is the car displaying a park pass. The hybrid enzyme is the passengers in the car. In the case of Mark’s research, the fusion of Trojan horse molecules with hybrid enzyme is like a car with a pass in which there are passengers—the vehicle and its occupants can get through the gate and head to their intended destination (i.e. the brain).  

Yet, a successful passage through the blood-brain barrier does not guarantee an uptake of the enzyme by neurons. It needs to be decorated with phosphate groups, the amount and pattern of which dictate the efficiency of the uptake by the cells. To solve this problem, Mark is working with Perrault, a mass spectrometrist, to monitor the enzyme, making sure it has the optimal presentation and attachment of phosphate groups. Triggs-Raine, an expert in CRISPR gene editing technology is generating a mouse model of the disease. It will allow the team to validate if the recombinant enzyme could alleviate the symptoms of the disease. 

Mark thinks this research could also help advance another type of treatment—gene therapy. Unlike ERT, which uses a healthy enzyme to replace a mutated one, gene therapy is a technique that supplements mutated gene (DNA) with healthy DNA. Given the remarkable advances in virus-mediated gene delivery, this healthy DNA could potentially be delivered to patients’ brains to produce functional enzyme within their neurons. 

“One technical hurdle of gene therapy is that there is a limited capacity of genetic material that can be package into a virus for delivery,” says Mark. “By reducing the size of two genes to a compact hybrid, the DNA material is now small enough to address this issue.”

From protein engineering to mass spectrometry and gene editing, Mark and his collaborators are determined to find solutions to treating LSD. “To answer complex questions for diseases like LSDs, a single laboratory simply does not have the capacity or the tools to fully explore the problem,” says Mark. “By combing different skills from collaborators, we created a team with the necessary breadth of knowledge and experience to maximize our potential. Our project contributes to a strategy—crossing the blood-brain barrier—that could be coupled to a whole range of different therapeutics. We hope that the project will serve as a springboard for designing effective therapeutics and drug delivery systems to the brain for a wide range of diseases.”