Neuron Breakthrough! Furlanis, Hunker, and colleagues develop novel enhancer AAV tools for precise targeting of cortical and striatal interneurons
Release time：2025-07-14 14:59:02
Precise targeting of specific neuron types has long been a key challenge in deciphering complex neural circuit functions. Recently, two studies published in Neuron have developed enhancer AAV tools specifically targeting striatal and cortical interneurons, providing powerful new methods for neural circuit research and potential therapeutic strategies for neurological diseases.
I. Cortical Interneuron-Specific Enhancer AAV Tools
Furlanis and colleagues have developed a toolbox of enhancer AAVs capable of specifically targeting and manipulating major types and subtypes of GABAergic cortical neurons, as well as cholinergic striatal cells, in both mice and non-human primates. Enhancer candidates were identified via single-cell genomic data analysis, and the constructed tools were validated under various conditions using anatomical, morphological, and electrophysiological characterization. These tools enable labeling, activity monitoring, and optogenetic manipulation. Notably, the enhancer sequences are conserved across species, making them powerful tools for studying neural circuits and functions, and for developing precision-targeted therapies.
Using Dlx5a-Cre::INTACT mice, GFP+ nuclei from the cortex and striatum were isolated and analyzed with single-cell ATAC-seq. Candidate cell type–specific enhancers were identified via the COSG algorithm. These enhancer candidates were cloned into rAAV vectors to drive the expression of dTomato, GCaMP6f, or ChR2, and packaged into the PHP.eB serotype to enable blood-brain barrier penetration. The viruses were delivered into mice of different ages via stereotaxic (STX), intracerebroventricular (ICV), or retro-orbital (RO) injection. Three weeks post-injection, specificity and sensitivity were assessed using immunohistochemistry, in situ hybridization, and electrophysiological recordings.
The tools were also applied to non-human primates (rhesus macaques) and human brain slices to assess sequence conservation and functional relevance. In non-human primates, enhancer elements such as BiPVe3 and BiPVe4 drove the labeling of cells with similar morphology and electrophysiological properties to those in mice—for instance, fast-spiking PV+ cells. In human brain slices, the BiVIPe4 enhancer continued to specifically target VIP cells.
Figure 1: Strategy for identifying and characterizing enhancer AAV tools with cell type specificity.
Characterization of 7 Specific Enhancers
II. Striatal Neuron-Specific Enhancer AAV Tools
Hunker, Wirthlin, and colleagues have developed an enhancer AAV toolkit that enables precise targeting of major neuronal populations in the striatum of both rodents and primates. These include medium spiny neurons (MSNs), direct pathway neurons (D1 MSNs), indirect pathway neurons (D2 MSNs), Sst-Chodl, Pvalb-Pthlh, and cholinergic interneurons. The enhancer activities are conserved across rodents and primates, and the tools can be delivered via various viral routes and carry different transgenes, enabling cell-type-specific labeling, optogenetic manipulation, and multiplexed labeling. This provides a powerful resource for studying striatal cell types and circuits, as well as for the development of novel AAV-based gene therapies.
Candidate enhancer sequences were selected from public ATAC-seq datasets, including the human Brain Open Chromatin Atlas (BOCA) and the mouse Cis-element Atlas (CATlas), based on their proximity to marker genes of striatal cell types. These enhancers were cloned into AAV vectors upstream of a minimal promoter driving the SYFP2 fluorescent reporter gene and packaged using the blood-brain barrier-penetrant PHP.eB capsid. Viruses were injected into C57Bl6/J mice via retro-orbital (RO) injection, and expression was analyzed 3–5 weeks later. In total, 48 enhancers with strong activity were identified, covering major striatal neuronal populations:20 pan-MSN enhancers, 5 D1 MSN-specific enhancers, 7 D2 MSN-specific enhancers, 5 Sst-Chodl interneuron enhancers, 7 cholinergic interneuron enhancers, 4 Pvalb-Pthlh interneuron enhancers.
Three viral injection routes—STX, ICV, and RO—were tested to evaluate their impact on enhancer specificity. Results showed RO injection yielded the highest specificity, STX the lowest, and ICV intermediate. For example, the cholinergic enhancer AiE0873m_3xC2 showed 94% specificity with RO injection, 88% with ICV, but dropped to 50% with STX. However, scRNA-seq analysis revealed that certain enhancers, such as the D1 MSN enhancer AiE0779m, maintained 100% D1 MSN specificity across all delivery routes.
Figure 2: Hypothesized enhancer-driven cell-type-specific expression in the striatum.
Characterization of Specific Enhancers
Future Perspectives
These two toolkits overcome the limitations of traditional transgenic models, offering precise tools for deciphering neural circuit functions and developing gene therapies for neurodegenerative diseases. Future research may further explore strategies for combinatorial enhancer use and investigate their therapeutic potential in disease models.
Original article links: Striatal toolkit: https://doi.org/10.1016/j.neuron.2025.04.035 Cortical toolkit: https://doi.org/10.1016/j.neuron.2025.05.002
Brain Case is currently working on updating its library of GABAergic promoters. If you’re interested in promoters targeting GABAergic neurons such as PV, SST, or VIP, feel free to contact us at bd@ebraincase.com
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