AAV-mediated Inner ear gene delivery triggers mild host immune responses in the mammalian inner ear
Data files
May 01, 2025 version files 79.46 MB
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Figure_1-7_S1-3.zip
79.45 MB
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README.md
13.10 KB
Abstract
Hearing loss is a common disability affecting the world’s population. However, its treatment options are limited. More recently, adeno-associated virus (AAV)-mediated inner ear gene therapy has shown great promise as a treatment for hereditary hearing loss. However, the host immune responses to AAV-mediated gene therapy in the mammalian inner ear are not well understood. In this study, two serotypes of AAV vectors were injected individually into the mouse inner ear to evaluate the host innate and adaptive immune responses up to one month after inner ear gene delivery. Our results suggest that the host innate and adaptive immune responses to AAV-mediated inner ear gene delivery are limited and mild, which is favorable for its clinical translation.
Dataset DOI: 10.5061/dryad.9p8cz8wth
Description of the data and file structure
This dataset contains raw data collected at the NIDCD/NIH and published under DOI: 10.1016/j.omtm.2025.101456. If you use this dataset, please cite the associated publication as a reference."
The datasets contain 10 folders. The names and descriptions are as follows:
Figure 1: Confocal images for localization of resident macrophages in the mouse cochlea. Cx3cr1GFP/GFP mice are used to localize resident macrophages in the cochlea.
Figure 2: Confocal images for showing chronological changes in macrophage activation after AAV2.7m8-CAG-tdTomato inner ear gene delivery in Cx3cr1GFP/GFP mice
Figure 3: Confocal images for presenting macrophage and T-cell activation after AAV-mediated inner ear gene delivery in C57BL/6 mice
Figure 4: Evaluation of AAV-mediated inner ear gene delivery triggers neutralizing antibody production by using flow cytometry
Figure 5: Evaluating anti-capsid peripheral cellular immune response triggered with AAV-mediated inner ear gene delivery by using ELISpot assay
Figure 6: Assessment of pro-inflammatory cytokine and chemokine activation in response to AAV-mediated inner ear gene delivery by using electrochemiluminescence assay.
Figure 7: examined vector biodistribution in the liver after AAV-mediated inner ear gene delivery by using ddPCR to detect viral DNA
Figure S1: Confocal images for showing chronological changes in macrophage activation after AAV2-CAG-tdTomato inner ear gene delivery in Cx3cr1GFP/GFP mice
Figure S2: Confocal images for localization of resident macrophages in the mouse cochlea. C57BL6 mice are used to localize resident macrophages in the cochlea.
Figure S3: Confocal images for localization of macrophages and T-cells after AAV-mediated gene delivery in C57BL/6 mouse cochlea
Files and variables
Folder: Figure 1
Content: Seven TIFF files and one Imaris file
Description:
Mid-modiolus cross-sectional image of the cochlea from a non-surgery Cx3cr1GFP/GFP mouse shows the localization of macrophages in the adult mouse cochlea. The scale bar represents 200 µm.
- TIFF file of confocal image for cx3cr1_control_whole cochlea cross section
The following six magnified images are from various regions of the cochlea, which show that macrophages reside in the adult mouse cochlea. The scale bar is 20 µm, eGFP (green) shows Cx3cr1, which is used as a macrophage marker, and Hoechst (white) shows the nucleus.
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TIFF file of confocal image for cx3cr1_control_stria vascularis
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TIFF file of confocal image for cx3cr1_control_spiral limbus
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TIFF file of confocal image for cx3cr1_control_Rosenthal’s canal
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TIFF file of confocal image for cx3cr1_control_modiolus
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TIFF file of confocal image for cx3cr1_control_sensory epithelium
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TIFF file of confocal image for cx3cr1_control_spiral prominence
The following Imaris file showed 3D reconstruction images of the cochlear middle turn from a non-surgical Cx3cr1GFP/GFP mouse. This file will be able to open with the free software Imaris Viewer. By 3D reconstruction, macrophages are observed along the basilar membrane to the osseous spiral lamina. The scale bars represent 20 µm. The scale bar is 20 µm, eGFP (green) shows Cx3cr1, which is used as a macrophage marker, and Hoechst (white) shows the nucleus.
- Imaris file of confocal image for cx3cr1_control
Folder: Figure 2
Content: Six high-resolution TIFF files for the middle turn of the whole mount cochlea
Description:
Confocal images of the Cx3cr1GFP/GFP cochlear middle turns are shown. Adult (8- to 10-week-old) Cx3cr1GFP/GFP mice underwent AAV2.7m8-CAG-tdTomato injections via the PSCC approach, and cochleae were processed on POD 1, 3, 7, 14, 21, 28 to show chronological changes after AAV vector injections. along with a non-surgery Cx3cr1GFP/GFP mouse (control) for comparison. The eGFP (green) shows Cx3cr1, which shows macrophages, and CD68 (blue) antibodies are used to cl-label macrophages, tdTomato (magenta) expression indicates viral transduction, and Hoechst and phalloidin stains (white) label the nucleus and F-actin. The scale bar represents 20 µm
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TIFF file of confocal image for control
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TIFF file of confocal image for POD1
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TIFF file of confocal image for POD3
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TIFF file of confocal image for POD7
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TIFF file of confocal image for POD14
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TIFF file of confocal image for POD28
Folder: Figure 3
Content: 12 Excel files.
Description:
C57BL/6 mice were used to localize the macrophages and T cells in the cochlea and utricle after AAV-mediated inner ear gene delivery. Quantification of macrophages (detected by anti-IBA1 antibody) and T cells (detected by anti-CD3 antibody) was done using whole-mount images of the sensory epithelium in apical, middle, and basal turns of the cochlea and lateral wall, as well as the utricle. Quantifications were performed manually with z stuck on POD3 and POD28. Animals that did not undergo surgery were used as non-surgical controls to compare. Quantifications of each group are shared in Excel files as listed below.
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Excel file for POD3 sensory epithelium macrophage
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Excel file for POD3 sensory epithelium T-cells
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Excel file for POD3 lateral wall macrophage
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Excel file for POD3 lateral wall T-cells
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Excel file for POD3 utricle macrophage
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Excel file for POD3 utricle T-cells
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Excel file for POD28 sensory epithelium macrophage
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Excel file for POD28 sensory epithelium T-cells
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Excel file for POD28 lateral wall macrophage
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Excel file for POD28 lateral wall T-cells
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Excel file for POD28 utricle macrophage
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Excel file for POD28 utricle T-cells
Folder: Figure 4
content: One Excel file
Description:
The neutralizing antibody titers against AAV were quantified in mouse serum after AAV2-CAG-eGFP, AAV2.7m8-CAG-eGFP, or vehicle injections. The summary table of viral neutralizing antibody titer of each mouse is shared in this Excel file named summary of neutralizing titers. In the paper, the Kruskal-Wallis test with Dunn’s multiple comparisons was used for statistical analysis. The following 5 groups are included which are AAV2-CAG-eGFP-injected-mouse serum against AAV2 vector, vehicle-injected-mouse serum against AAV2 vector, AAV2.7m8-CAG-eGFP-injected-mouse serum against AAV2.7m8 vector, AAV2-CAG-eGFP-injected-mouse serum against AAV2.7m8 vector, and AAV2.7m8-CAG-eGFP-injected-mouse serum against AAV2 vector.
- Excel file for summary of neutralizing titers
Folder: Figure 5
Content: 2 Excel files
Description:
Splenocytes (POD28) were stimulated in vitro with an overlapping peptide library spanning the VP1 sequence of AAV2.7m8 or AAV2 and analyzed using an IFN-γ ELISpot assay. The individual raw data of AAV2.7m8-CAG-eGFP-injected mice (n = 7) and AAV2-CAG-eGFP injected mice (n = 7) are shared as Excel files as shown below. With our analysis, responses were considered positive when the number of spot-forming colonies (SFCs) per 1 × 106 cells was >50 and at least 3-fold higher than the negative control.
- Excel file of ELLISpot data for the AAV2.7m8-injected group
- Excel file of ELLISpot data for the AAV2-injected group
Folder: Figure 6
content: 1 Excel file
Description:
The secretion of pro-inflammatory cytokines and chemokines in the perilymph and serum of mice injected with AAV2.7m8-CAG-eGFP (1.0 × 1010 GC), AAV2-CAG-eGFP (1.0 × 1010 GC), or vehicle was examined with an electrochemiluminescence approach. Non-surgery mice were used as controls. The pro-inflammatory cytokines and chemokines that were assessed include IFN-γ, interleukin (IL)-1β, IL-2, IL-6, IL-10, tumor necrosis factor (TNF)-α, and CXCL1. The summary table for the raw data of each mouse is shared as an Excel file. In the paper, we used the Kruskal-Wallis test with Dunne’s multiple comparisons for statistical analysis.
- Excel file for the quantification of pro-inflammatory cytokines and chemokines
Folder: Figure 7
Files: 1 Excel file
Description:
To assess vector biodistribution, DNA were extracted from liver of AAV2.7m8-CAG-eGFP-injected mice (POD3, n = 4; POD28, n = 6), AAV2-CAG-eGFP-injected mice (POD3, n = 4; POD28, n = 6), vehicle-injected mice (POD3, n = 4; POD28: n = 6), and non-surgery mice (n = 8) and amplified by digital droplet PCR (ddPCR). Viral genomes were detected using probes targeting the eGFP sequence, and albumin was used as a reference. The viral genome detection of the non-surgery mice was considered as background and used as a positivity threshold. The summary table of raw data for each group of mice is shared as an Excel file.
- Excel file for the summary of digital droplet PCR (ddPCR) raw data
Folder: Figure S1
Files: 4 TIFF files
Description:
Confocal images of the Cx3cr1GFP/GFP cochlear middle turns are shown. Adult (8- to 10-week-old) Cx3cr1GFP/GFP mice underwent AAV2-CAG-tdTomato injections via the PSCC approach, and cochleae were processed on POD 3, 14, 28 to show chronological changes after AAV vector injections along with a non-surgery Cx3cr1GFP/GFP mouse (control) for comparison. High-resolution TIFF files were shared as listed below.
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TIFF file of confocal image for control
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TIFF file of confocal image for POD3
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TIFF file of confocal image for POD14
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TIFF file of confocal image for POD28
The eGFP (green) shows Cx3cr1, which identifies macrophages, and CD68 (blue) antibodies are used to co-label macrophages, tdTomato (magenta) expression indicates viral transduction, and Hoechst and phalloidin stains (white) label the nucleus and F-actin. The scale bar represents 20 µm
Folder: Figure S2
Content: Seven high-resolution TIFF files and one Imaris file
Description:
Mid-modiolus cross-sectional image of the cochlea from a non-surgical C57BL6 mouse shows the localization of macrophages in the adult mouse cochlea. The scale bar represents 200 µm.
- TIFF file of confocal image for bl6_control_wholecochlea cross section
The following six magnified images are from various regions of the cochlea where macrophages and T cells are shown. CD3 (T-cell marker shown in green), Iba1 (macrophage marker shown in magenta), and Hoechst and phalloidin (label the nucleus and F-actin in white). The scale bar is 20 μm.
- TIFF file of confocal image for bl6_control_atria vascularis
- TIFF file of confocal image for bl6_control_spiral limbus
- TIFF file of confocal image for bl6_control_Rosenthal's canal
- TIFF file of confocal image for bl6_control_modiolus
- TIFF file of confocal image for bl6_control_sensory epithelium
- TIFF file of confocal image for bl6_control_spiral prominence
The following Imaris file shows 3D reconstruction images of the cochlear middle turn from a non-surgical C57BL6 mouse. This file will be able to open with the free software Imaris Viewer. By 3D reconstruction, macrophages are observed along the basilar membrane to the osseous spiral lamina, which is a similar result to Figure 1. CD3 (T-cell marker shown in green), IBA1 (macrophage marker shown in magenta), and Hoechst and phalloidin (label the nucleus and F-actin in white). The scale bar is 20 μm.
- Imaris file of confocal image for non-surgery control
Folder: Figure S3
Content: 7 high-resolution TIFF files
Description:
Localization of macrophages and T-cells after AAV-mediated gene delivery in C57BL/6 mouse cochlea was examined. C57BL/6 mice were used to localize the macrophages and T-cells in the cochlea after AAV-mediated inner ear gene delivery. Confocal images of the cochlear middle turn from a non-surgical C57BL/6 mouse are shown. Confocal images of the cochlear middle turns from AAV2.7m8-CAG-eGFP, AAV2-CAG-eGFP, or vehicle-injected mice on POD3 and POD28 are shown with a non-surgery control. High-resolution TIFF files of confocal images for each group are shared as below. The transduction of AAV was shown by eGFP (green). IBA1 (magenta) was used as a macrophage marker. CD3 (blue) was used as a T-cell marker. Hoechst and phalloidin stains (white) label the nucleus and F-actin, respectively. The scale bar represents 20 µm.
- TIFF file of confocal image for AAV2.7m8_POD3
- TIFF file of confocal image for AAV2.7m8_POD28
- TIFF file of confocal image for AAV2_POD3
- TIFF file of confocal image for AAV2_POD28
- TIFF file of confocal image for vehicle_POD3
- TIFF file of confocal image for vehicle_POD28
- TIFF file of confocal image for non-surgery control
Code/software
Imaris Viewer for Mac and Windows is found as follows:
Imaris Viewer - Imaris - Oxford Instruments
Access information
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