iGEM: How Stuyvesant Students Are Changing Stroke Therapy

Every year, starting in 2004, high school teams from around the world compete in the iGEM competition, which centers around the development of synthetic biology projects across many different fields that are then presented in an exposition. This exposition, called “The iGEM Jamboree,” was held in Paris for the first time. EmpireGene, a team of students from Stuyvesant and other high schools in New York, placed in the top ten of this year’s iGEM competition. Seniors Hailey Seltzer, Yeju Moon, Ryan Lee, Joseph Jeon, and Brigid Allen, and juniors Matthew Huang, Andrew Park, and Vanessa Chen helped create the NeuroTrojan project.

EmpireGene started developing its project in the winter of 2022. They decided on stroke recovery as the topic of their research because several of the team members had family members who had suffered strokes. Strokes occur when blood vessels are unable to deliver oxygen to the brain. Ischemic strokes—the most common type—are caused by blood clots, while hemorrhagic strokes happen if an artery in the brain leaks or bursts. This deprivation of oxygen in the brain leads to neural damage, which in turn causes a wide range of long-term effects such as aphasia, numbness or paralysis, and memory loss. Recovery varies from person to person, with some quickly returning to full functioning and others being left with a lifelong disability. Because of this, EmpireGene wanted to develop a method of post-stroke neural repair.

The team looked to introduce two neurotrophins: Neurotrophin-3 (NT-3) and Fibroblast Growth Factor 2 (FGF-2). Neurotrophins regulate the development and function of the nervous system, and the two that EmpireGene used in their experiment are proven to stimulate tissue repair and neural growth. However, there was a major challenge in getting these neurotrophins into the brain—the blood-brain barrier (BBB). The BBB restricts the flow of molecules into the brain, preventing toxic substances from entering but allowing for the simple diffusion of molecules such as oxygen and glucose. Some substances cross, thanks to transport systems on the membrane, but this requires the molecules to be compatible with receptors. Neither NT-3 nor FGF-2 would be able to cross the blood-brain barrier on their own. The EmpireGene team looked to develop a way to make this delivery of neurotrophins possible.

To do this, they used Human Insulin Receptor Monoclonal Antibody (HIRMAb), which they referred to as a “biological Trojan horse.” HIRMAb binds to insulin receptors on the cells that line the BBB. After binding, HIRMAb undergoes receptor-mediated transcytosis, the process in which a molecule bound to a receptor is transferred through the cell and across the BBB. EmpireGene’s idea was that if NT-3 and FGF-2 were bound to HIRMAb, they could hitch a ride across the barrier and then be released for the neurons to uptake.

To create these fused proteins, EmpireGene designed plasmids, circular pieces of DNA, which contained the genetic code for HIRMAb’s two subunits as well as one of the two neurotrophins. Many gel electrophoresis tests were performed to ensure that the gene editing was successful. The next test was to see if this genetic code led to the production of the correct proteins. Thus, the plasmids were added to a dish of E. coli bacteria. The bacteria uptook the plasmids and were then found to produce and secrete the fused proteins that EmpireGene had intended them to. Next, these plasmids were transfected into the mammalian cell line known as CHO-K1 to avoid the use of animal testing in the lab.

The next step was to test if CHO-K1 cells could produce the intended proteins, and that these proteins would pass through the BBB. They added the media that the transfected CHO-K1 cells were grown in to the upper compartment of a transwell. In theory, the cells should have secreted the “Trojan horse” fused proteins into this media. This was separated from the lower compartment by a model of the BBB. If the Trojan horse approach to transporting the neurotrophins was effective, then the fused proteins should have been found in the lower compartment, having successfully passed across the membrane.

However, the presence of NT-3 and FGF-2 was not detected in the lower compartment. Further study allowed the EmpireGene team to conclude that the problem likely lay in a mutation that caused the generation of the fused proteins in the CHO-K1 cells to terminate early. Gel electrophoresis alone was used to confirm that plasmids appeared the way they should. This would not necessarily detect a small mutation creating an early stop codon in the DNA sequence, as this would only be made clear through genetic sequencing.

Due to the timeline of the competition, the experiment could not be redone in time for the presentation in Paris. Experimentation lasted the entire summer and well into October. Even though this was not the desired result, it did not disprove the team’s hypothesis that the fused Trojan horse protein would be able to pass through the BBB. The results from the E. coli bacteria’s production of the intended proteins along with the ability of these proteins to bind to the insulin receptor indicate that the potential for this new therapy remains, though further testing is required.

EmpireGene is confident that their results are a major step forward in the scientific research field of stroke recovery. “With research comes complications and trial and error, so I was a bit upset that the experiment didn’t fully work out, but overall really happy because that’s a huge part [of the process],” said team member Hailey Seltzer. Seltzer’s passion for science began as a freshman taking Modern Biology, and says that participating in iGEM allowed her to find her love of research. She encourages other students to explore opportunities in the lab: “Find a professor that conducts research or a competition similar to [iGEM] and just go for it. If you end up liking research, there’s a whole host of opportunities waiting for you.”