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Breaking News | New Recombinant AAV Achieves Highly Efficient and Specific Transduction of Microglia

Time:2024-10-15 16:57:11

Microglia are closely associated with human health and various CNS diseases, making them a focal point in neuroscience research. Typically, research relies on transgenic mouse models, which are time-consuming, expensive, and impractical for therapeutic applications. Recombinant adeno-associated viruses (rAAV) are widely used in neuroscience due to their safety and diverse tissue-targeting properties, but efficient and specific AAVs for microglia are rare.

In July 2024, Xu Fuqiang and Lin Kunzhang's team at CAS published a preprint on BioRxiv titled “Microglia-specific transduction via AAV11 armed with IBA1 promoter and miRNA-9 targeting sequences.” This study evaluated various AAV vectors carrying the mIBA1 promoter and miRNA-9 targeting sequences for transducing microglia in the caudate putamen (CPu) brain region. The findings showed that AAV11 facilitated highly specific and efficient microglial transduction across different brain regions and the spinal cord. Reduced injection doses allowed for precise structural labeling of microglia, making AAV11 a powerful tool for microglia research.


 

Comparison of the characteristics of several serotype AAVs in transducing microglia

Four different AAVs equipped with the mIBA1 promoter and miRNA-9 targeting sequences were evaluated for their specificity and efficiency in transducing microglia in the adult mouse brain. With an injection dose of 2×10^9 VG and volume of 200 nL, brain tissues were collected and immunofluorescent staining performed after three weeks. Co-localization of EGFP and the microglial marker IBA1 was analyzed to determine cell type specificity. AAV11 showed the highest specificity (approximately 98.9%) and efficiency (about 947 cells) in the striatum.

Figure 1. In vivo screening of AAVs for efficient microglia transduction

AAV11 specifically transduces microglia in the DG region of the mouse brain

To verify whether AAV11 can efficiently transduce microglia in the DG region of the brain, AAV11 was injected into the DG region at a dose of 2×10^9 VG (Figure 2A). Three weeks later, brain tissues were collected, sectioned, and subjected to immunofluorescence staining (Figure 2B). AAV11 demonstrated strong transduction capability in microglia within the hippocampal DG region (Figures 2B and 2C). Quantitative analysis of EGFP and IBA1 double-positive cells indicated that AAV11 primarily transduced microglia with a specificity of approximately 97% (Figure 2D).

Figure 2. AAV11 efficiently and specifically transduces microglia in the DG brain region

AAV11 specifically transduces microglia in the SNr region of the mouse brain

To verify the efficiency of AAV11 in transducing microglia in the SNr region, AAV11 was injected into the SNr region of mice at a dose of 2×10^9 VG (Figure 3A). Three weeks later, brain tissues were collected, sectioned, and subjected to immunofluorescence staining (Figure 3B). In the SNr, most microglia were observed to be labeled with green fluorescence (Figures 3B and 3C). Fluorescence colocalization analysis showed that AAV11-mediated gene expression in SNr microglia had about 90% specificity (Figure 3D).

Figure 3. AAV11 efficiently and specifically transduces microglia in the SNr brain region

AAV11 specifically transduces microglia in the spinal cord of mice

Spinal cord microglia play a crucial role in spinal cord injury repair, inflammation regulation, pain generation, and the development of potential therapeutic strategies. Therefore, developing a gene delivery system targeting spinal cord microglia is essential. The authors investigated whether AAV11 could efficiently transduce microglia in the spinal cord. AAV11 was injected into the lumbar segment of the spinal cord at a dose of 5×10^9 VG (Figure 4A). Three weeks later, spinal cord tissues were collected, sectioned, and subjected to immunofluorescence staining (Figure 4B). Quantitative analysis of EGFP and IBA1 double-positive cells indicated that AAV11 had strong transduction efficiency in spinal cord microglia, with about 80% specificity (Figures 4B and 4C).

Figure 4. AAV11 efficiently and specifically transduces microglia in the spinal cord

AAV11 can sparsely and brightly label microglia

The complex morphology of microglia is crucial to their function in the brain. Microglia possess intricate processes and can undergo morphological changes under different physiological or disease conditions. Labeling the fine morphology of microglia is essential for understanding their changes in different states and interactions with other neural cells. However, simple and universal methods for labeling microglia morphology in the mammalian brain are lacking. Here, by reducing the viral injection volume to 50nL at a dose of 5×10^8 VG into the dentate gyrus (DG) of the hippocampus (Figure 5A), the authors found that AAV11 could sparsely label microglia in the DG region three weeks later (Figure 5B). Using laser confocal scanning, the fine structural details of microglia were clearly observed (Figure 5C). Therefore, AAV11 can be used for sparse labeling of microglia to analyze their fine structure.

Figure 5. AAV11 sparsely labels microglia

By directly injecting AAV11 containing the IBA1 promoter and a set of four tandem sequences targeting miR-9, we achieved specific transduction of microglia in specific brain regions of adult mice. Additionally, we demonstrated that AAV11 shows high transduction specificity for microglia in various brain regions and the spinal cord. Furthermore, by reducing the injection dose, AAV11 can be used for sparse labeling of microglia. This work provides a powerful viral tool for studying the structure and function of microglia.

We are very interested in collaborating with your laboratory to expand the applications of our serum type. If you are interested, please feel free to contact us at kz.lin@siat.ac.cn or BD@ebraincase.com


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