by Sophianne Loh
Rice University students and faculty are said to possess “unconventional wisdom.” We aren’t afraid to dream of solutions that seem impossible, push past limited frameworks of thought, and leap into uncharted territory. This ideal is perhaps most apparent in the neuroengineering research done at Rice. For years, neurological conditions like Alzheimer’s or epilepsy have been treated in much the same way: surgical procedures, injections, and electrical stimulation of the brain. [1] These treatments aim to “fix” dysfunctional neural circuits, which are often the origin of these neurological and psychological disorders. Unfortunately, not only are these options invasive, but they fail to target specific dysfunctional neurons, leading to adverse effects on the surrounding healthy brain tissues.
As a result, researchers around the world have been hard at work creating smaller implants and less invasive electrodes on the scales of millimeters or even micrometers. [2] For example, researchers at the Korea Advanced Institute of Science and Technology recently designed a quarter-sized wireless implant that successfully modulated mouse brain neurons. [3] However, continuing to travel down the same path of surgical implants eventually leads to the same major issues: unwanted invasiveness, limited specificity of targets, and, most prominently, obtaining sufficient funding. As Dr. Jerzy Szablowski of the Rice Bioengineering Department points out, “If you look at the amount of funding we have for brain disorders - for example, from National Institutes of Health - you'll find out that the amount of money we spend on research that aims to treat brain disorder is very low compared to the burden that they cause” In fact, Dr. Szablowski notes the cost of creating one new drug for each brain disorder can be up to $1 billion. In comparison, the National Institute of Mental Health dedicated only $2.1 billion in 2021 to research on brain disorder treatments. [4] With over 1,000 neurological disorders affecting over 100 million individuals in America, there is an ever-growing need for less costly and more versatile treatments. [5]
In true Rice fashion, Dr. Szablowski approached this need by traveling down a different route. He wanted to create a one-stop, noninvasive, affordable treatment for multiple neurological disorders. In other words, what if a single technology could treat a variety of neurological disorders with elegant simplicity? Dr. Szablowski has found an answer in acoustically targeted chemogenetics, or ATAC. Developed by Szablowski and advanced by his researchers in the Laboratory for Noninvasive Neuroengineering, ATAC is the first-ever fully noninvasive technique to control neurons with spatial, cell-type, and temporal precision. When applied to the treatment of neurological disorders, ATAC could treat a variety of disorders, as long as they rely on abnormal neuronal activity.
Despite the simplicity of the resulting treatment path, ATAC is a complex, multi-step process. It starts with the blood-brain barrier (BBB), a collection of different types of cells tightly joined together that line the blood vessels of the brain. The BBB serves to block certain pathogens or other harmful molecules in the bloodstream from entering the brain. However, this also means that the BBB won’t normally allow the passage of therapeutic molecules into target areas in the brain. [6] This is where the Szablowski lab turned to FUS-BBBO, or focused ultrasound blood-brain barrier opening, in their development of the ATAC method.
FUS-BBBO is a technique that had been well-researched prior to Dr. Szablowski’s development of ATAC. This ability to build upon a foundation of past research while pioneering a new treatment was important to Dr. Szablowski. He explains, “I wanted to find a method that is well-validated and something that would be safe for human use, and the fastest way to do that is to just look at what is available.” FUS-BBBO (Fig. 1) first involves the injection of microbubbles (typically a lipid or protein shell containing perfluoropropane or sulfur hexafluoride gas) into the desired blood vessels. Immediately after, short, low-pressure ultrasound wave pulses are applied to the BBB target site. This causes the microbubbles in the blood vessels to oscillate, a phenomenon termed stable cavitation. The oscillations apply pressure to the surrounding fluid, which opens the tight junctions between the cells of the BBB temporarily and reversibly. As a result, therapeutic molecules in the bloodstream can go past the BBB to reach neurons in the brain. [7]
Figure 1. (A) Schematic depiction of FUS-BBBO procedure. (B) MRI-guided FUS-BBBO procedure. [6]
Where Dr. Szablowski departs completely from previously known procedures is by combining FUS-BBBO with AAVs, or adeno-associated viruses, that encode engineered protein receptors that can control neurons. An AAV is known as a “vector” for gene therapy. This means scientists can insert desired DNA into the AAV allowing it to deliver and express these genes to certain cells in the body. [8] In ATAC, FUS-BBBO first opens specific areas in the BBB for AAV to deliver its genes. In this way, FUS-BBBO essentially “marks” the specific neural circuits to be treated, contributing to the spatial specificity of the ATAC treatment. The AAV vectors contain DNA that codes for engineered proteins called DREADDs, a type of cell receptor. These cell receptors allow a cell to respond to designer drugs, which are drugs created in the lab, that can get into - but do not activate naturally-occurring receptors in - the brain. When the AAV vector delivers the DREADD genes to the specific neurons marked by the FUS-BBBO procedure, the expression of the DREADDs allows the neurons to be activated by designer drugs. The designer drug used in the ATAC procedure is called clozapine-N-oxide (CNO). CNO has the ability to excite or inhibit certain neurons that express DREADDs, allowing for targeted treatment of neurological disorders. Furthermore, it can be administered in a non-invasive way to patients (i.e., as a pill). [9] It is the combination of FUS-BBBO, gene therapy through the AAV vectors, and CNO that makes ATAC so effective at targeting specific neurons. (Fig. 2)
Figure 2. Depiction of the ATAC procedure. [1]
The Szablowski lab chose to test ATAC by observing its effect on the hippocampus of rodents. Dr. Szablowski describes the hippocampus as “a very relevant brain region for many disorders - epilepsy being one of them. ” So, if ATAC is effective on the hippocampus, then it has the potential to treat a multitude of disorders, ranging from epilepsy to PTSD. Specifically, initial tests focused on how ATAC could modulate memory formation, providing a possible treatment for anxiety disorders. To test the efficacy of the treatment, rodents were exposed to a fear-inducing stimulus in a unique experimental setting. Next, both the rodents who had received AAV and DREADDs and those in a control group received either CNO or saline solution. Twenty-four hours after initial exposure to the stimulus, the rodents were again placed in the same experimental setting to test their fear recall.
The results suggest that ATAC applied in the hippocampus successfully reduced memory formation. In a common memory task, ATAC rodents that received CNO showed less than half of the response than rodents in the control group. Furthermore, the results showed ATAC only targeted memory formation and had no effect on exploratory behavior and sensation of stimuli, confirming the high specificity of the treatment. What does this all mean for us? Well, if ATAC becomes clinically relevant, it could enable a minimally invasive, highly effective way of treating neurological or psychiatric disorders including Parkinson’s disease or epilepsy. As Dr. Szablowski describes, “any [neuropsychiatric disorder] that has the defined activity of the [neural] circuit that needs to be modified” could potentially be treated with ATAC.
In practice, Dr. Szablowski envisions that ATAC would first involve an outpatient procedure that opens the BBB and delivers the gene therapy. After waiting about three weeks, the patient would simply take pills regularly to deliver the CNO to the “painted” areas of the brain. There would no longer be a need for implanted devices and multiple intracranial injections. It’s a streamlined, powerful form of treatment that can greatly simplify what a patient must go through while seeking relief from neurological disorders. However, ATAC still faces a long journey toward reaching clinical relevance. Like all biomedical research endeavors, ATAC must overcome many obstacles. For example, Dr. Szablowski recognizes that ATAC would be a very expensive procedure with current technology and techniques. So, his lab is currently developing accessory technology for ATAC, such as “site-specific therapeutics,” a technique that can be used before AAV vectors to temporarily modulate a specific site of the brain. This would allow medical practitioners to test whether or not their chosen target in the brain for ATAC is indeed correct, based on the disorder and the individual. Site-specific therapeutics would help prevent the unfavorable situation in which expensive, irreversible, and potentially risky gene therapy has been applied to the incorrect site in the brain during ATAC. Once ATAC is ready for clinical trials, they’ll start testing this combination of site-specific therapeutics and ATAC for the treatment of Parkinson’s disease.
With careful planning and creative thinking, the Szablowski lab has created a minimally invasive neurological disorder treatment that maximizes effectiveness and simplifies procedures. While ATAC faces and will continue to face obstacles on its path toward implementation in the medical field, this unique solution brings us closer to a future of accessible and optimized biological treatments for complicated disorders.
Works Cited
[1] Szablowski, J. O.; Lee-Gosselin, A.; Lue, B.; Malounda, D.; Shapiro, M. G. Acoustically Targeted Chemogenetics for the Non-Invasive Control of Neural Circuits. Nature Biomedical Engineering 2018, 2 (7), 475–484.
[2] Kozai, T. D. Y. The History and Horizons of Microscale Neural Interfaces. Micromachines 2018, 9 (9), 445.
[3] Kim, C. Y.; Ku, M. J.; Qazi, R.; Nam, H. J.; Park, J. W.; Nam, K. S.; Oh, S.; Kang, I.; Jang, J.-H.; Kim, W. Y.; Kim, J.-H.; Jeong, J.-W. Soft Subdermal Implant Capable of Wireless Battery Charging and Programmable Controls for Applications in Optogenetics. Nature Communications 2021, 12 (1).
[4] 2021 Autumn Inside NIMH. https://www.nimh.nih.gov/research/research-funded-by-nimh/inside-nimh/2021-autumn-inside-nimh (accessed Apr 6, 2022).
[5] Gooch, C. L.; Pracht, E.; Borenstein, A. R. The Burden of Neurological Disease in the United States: A Summary Report and Call to Action. Annals of Neurology 2017, 81 (4), 479–484.
[6] Daneman, R.; Prat, A. The Blood–Brain Barrier. Cold Spring Harbor Perspectives in Biology 2015, 7 (1).
[7] Wang, J. B.; Di Ianni, T.; Vyas, D. B.; Huang, Z.; Park, S.; Hosseini-Nassab, N.; Aryal, M.; Airan, R. D. Focused Ultrasound for Noninvasive, Focal Pharmacologic Neurointervention. Frontiers in Neuroscience 2020, 14.
[8] Naso, M. F.; Tomkowicz, B.; Perry, W. L.; Strohl, W. R. Adeno-Associated Virus (AAV) as a Vector for Gene Therapy. BioDrugs 2017, 31 (4), 317–334.
[9] Burnett, C. J.; Krashes, M. J. Resolving Behavioral Output via Chemogenetic Designer Receptors Exclusively Activated by Designer Drugs. The Journal of Neuroscience2016, 36 (36), 9268–9282.