Epilepsy affects over 50 million people worldwide, making it one of the most common neurological disorders. Although medication helps many patients achieve seizure control, approximately one-third continue to experience seizures despite treatment. Seizures often arise when the brain’s excitation–inhibition (E/I) balance breaks down. In healthy conditions, specialized inhibitory neurons act as a natural braking system, releasing a neurotransmitter called gamma-aminobutyric acid (GABA) that helps prevent excessive electrical activity. When this inhibitory control is weakened, abnormal bursts of activity can spread through the brain and trigger seizures.
For years, researchers have viewed inhibitory neurons as an attractive target for gene therapy, which aims to treat diseases by introducing modified genetic material into cells. However, delivering therapeutic genes specifically to these neurons has proven difficult. The most widely used delivery vehicles for gene therapy are engineered viruses called adeno-associated vectors (AAVs). While certainly versatile, AAVs can only carry a limited amount of genetic cargo, roughly around 4.7 kilobases. Currently available genetic switches (or promoters, DNA sequences that control when and where genes are expressed) that specifically target inhibitory neurons are too large, taking more than half of that space. They are also too weak to drive a significant therapeutic effect, especially when delivered through the bloodstream.
Against this backdrop, a research team led by Professor Hirokazu Hirai, Director of the Viral Vector Core Center, Gunma University Initiative for Advanced Research, Japan, set out to find an effective solution. In their latest study, which was made available online in the journal Molecular Therapy on June 25, 2026, the researchers describe a compact genetic switch called compact mouse glutamic acid decarboxylase (cmGAD67) promoter that selectively targets inhibitory neurons while leaving enough room inside AAV vectors for therapeutic genes. The researchers used this novel strategy to develop and test a gene therapy-based approach designed to suppress seizures in multiple mouse models of epilepsy. The paper was actively co-authored by Assistant Professor Yuuki Fukai of the Viral Vector Core Center, Gunma University Initiative for Advanced Research, and Dr. Ayumu Konno of the Department of Neurophysiology & Neural Repair, Gunma University Graduate School of Medicine, Japan.
Sharing the novelty of their findings, Prof. Hirai explains, “ Because cmGAD67 is highly compact, it also helps overcome one of the major technical limitations of AAV vectors, which is their restricted cargo capacity. Therefore, our work expands opportunities for therapeutic gene delivery and innovation in the gene therapy industry. ”
The team discovered cmGAD67 by analyzing DNA sequences that naturally regulate the activity of the GAD67 gene, which is expressed in inhibitory neurons and plays a key role in GABA production. The developed promoter is only 410 base pairs long, making it substantially smaller than available alternatives. Despite its compact size, experiments in mice showed that it can drive strong and highly selective gene expression in inhibitory neurons in multiple brain regions. Notably, the promoter showed particularly strong expression in parvalbumin-positive inhibitory neurons, a subtype that plays a central role in controlling excessive brain activity.
The cmGAD67 promoter was used in an AAV vector to deliver the gene for GAD65, another enzyme involved in the production of GABA. The researchers reasoned that increasing GABA production in inhibitory neurons could strengthen the brain’s natural ability to suppress runaway electrical activity, thus preventing seizures. This cmGAD67-based construct, called AAV-GAD65, was tested in two complementary seizure models in mice.
In a ‘chemical kindling’ model, where repeated drug injections progressively sensitized the brain to seizures, systemic administration of AAV-GAD65 reduced abnormal electrical discharges, suppressed the slow-wave oscillations associated with overexcitability, restored brain network activity toward normal levels, increased GABA levels in the brain, and normalized anxiety-like behavior. In an even more severe version of the model, the treated mice exhibited markedly better survival rates. Meanwhile, in a focal seizure model, where a specific brain region is artificially driven to become hyperactive, local delivery of AAV-GAD65 to the affected brain region greatly reduced seizure severity, with three of the five treated mice showing no observable seizures.
Taken together, these findings suggest that enhancing inhibitory neuron function can restore the E/I balance across different severities of epilepsy. Notably, the researchers believe this technology could have broader applications beyond epilepsy. By enabling selective genetic modulation of inhibitory neurons, cmGAD67 may provide a versatile platform for developing gene therapies for neurological disorders associated with disrupted E/I balance.
Though further work will be needed to validate this approach in chronic epilepsy models and assess long-term safety, the present results are very encouraging. “ My laboratory originally developed viral vector technologies to manipulate E/I balance in the brain and study its influence in brain development, learning, and memory. It is particularly rewarding to see a tool originally developed for basic neuroscience research evolve into a potential therapeutic strategy for patients with drug-resistant epilepsy ,” concludes Prof. Hirai.
***
Reference
DOI: 10.1016/j.ymthe.2026.06.007
About Gunma University, Japan
Gunma University is a national university located in Gunma Prefecture, Japan, with campuses in Maebashi and Kiryu. Established in 1949 through the integration of several historic educational institutions, the university offers a broad range of programs in education, informatics, social and information studies, medicine, health sciences, and science and technology. Guided by its vision of fostering intellectual creativity rooted in regional engagement, Gunma University is committed to advancing education, research, and innovation that address both local and global challenges. The university emphasizes interdisciplinary collaboration, digital transformation, data science, and sustainable development, while cultivating graduates with strong professional expertise, ethical awareness, and international perspectives.
Website: https://www.gunma-u.ac.jp/english
About Professor Hirokazu Hirai from Gunma University, Japan
Professor Hirokazu Hirai, M.D., Ph.D., is a neuroscientist and Head of the Department of Neurophysiology and Neural Repair at Gunma University Graduate School of Medicine, Japan, and Director of the GIAR Viral Vector Core Center and Biosignal Genome Resource Center at Gunma University. His research spans both basic and translational neuroscience, with a focus on understanding neural circuit function and developing viral vector-based technologies for studying and treating neurological disorders. He has published over 200 research papers in reputed journals, accumulating over 10,000 citations. Additionally, Prof. Hirai serves as President of the 50 th Annual Meeting of the Japan Neuroscience Society and the 4 th China–Japan–Korea Neuroscience Meeting (2027).
Funding information
This work was supported by grants from the Program for Brain Mapping by Integrated Neurotechnologies for Disease Studies (Brain/MINDS; JP20dm0207057/JP21dm0207111 to Hirokazu Hirai) and Multidisciplinary Frontier Brain and Neuroscience Discoveries (Brain/MINDS 2.0; JP24wm0625103 to Hirokazu Hirai) from the Japan Agency for Medical Research and Development (AMED); and by MEXT/JSPS KAKENHI (20K06906/24K10022 to Nobutake Hosoi, 22K06454/24H01221 to Ayumu Konno, and 23H02791 to Hirokazu Hirai), Specific Research Grant by Takeda Science Foundation (2022088468 to Hirokazu Hirai) and Next-GIP (JPMJSP2146 to Yuuki Fukai).
Molecular Therapy
Experimental study
Animals
A compact GAD67 promoter enables inhibitory neuron-targeted AAV gene therapy for seizure suppression
25-Jun-2026
Gunma University, with Hirokazu Hirai, Ayumu Konno, and Yuuki Fukai listed as inventors, has filed patent applications in the EU (23803614.9), China (202380040106.1), the US (18/865107), and Japan (2024-520489) for the inhibitory neuron-specific promoter described in this study.