DOI: 10.5061/dryad.9p8cz8wth
title: AAV-mediated Inner ear gene delivery triggers mild host immune responses in the mammalian inner ear

description: 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.
description: # AAV-mediated Inner ear gene delivery triggers mild host immune responses
 in the mammalian inner ear Dataset DOI:
 [10.5061/dryad.9p8cz8wth](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. *Cx3cr1^GFP/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 *Cx3cr1^GFP/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 *Cx3cr1^GFP/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 *Cx3cr1^GFP/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. * TIFF
 file of confocal image for cx3cr1_control_stria vascularis * TIFF file of
 confocal image for cx3cr1_control_spiral limbus * TIFF file of confocal
 image for cx3cr1_control_Rosenthal’s canal * TIFF file of confocal image
 for cx3cr1_control_modiolus * TIFF file of confocal image for
 cx3cr1_control_sensory epithelium * 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
 *Cx3cr1^GFP/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 *Cx3cr1^GFP/GFP^*
 cochlear middle turns are shown. Adult (8- to 10-week-old)
 *Cx3cr1^GFP/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 *Cx3cr1^GFP/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 * TIFF file
 of confocal image for control * TIFF file of confocal image for POD1 *
 TIFF file of confocal image for POD3 * TIFF file of confocal image for
 POD7 * TIFF file of confocal image for POD14 * 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. * Excel file for
 POD3 sensory epithelium macrophage * Excel file for POD3 sensory
 epithelium T-cells * Excel file for POD3 lateral wall macrophage * Excel
 file for POD3 lateral wall T-cells * Excel file for POD3 utricle
 macrophage * Excel file for POD3 utricle T-cells * Excel file for POD28
 sensory epithelium macrophage * Excel file for POD28 sensory epithelium
 T-cells * Excel file for POD28 lateral wall macrophage * Excel file for
 POD28 lateral wall T-cells * Excel file for POD28 utricle macrophage *
 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 × 10^6^ cells was &gt;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 × 10^10^ GC), AAV2-CAG-eGFP (1.0 × 10^10^ 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 *Cx3cr1^GFP/GFP^* cochlear middle turns are shown. Adult (8-
 to 10-week-old) *Cx3cr1^GFP/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 *Cx3cr1^GFP/GFP^* mouse (control) for comparison.
 High-resolution TIFF files were shared as listed below. * TIFF file of
 confocal image for control  * TIFF file of confocal image for POD3 * TIFF
 file of confocal image for POD14 * 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](https://imaris.oxinst.com/imaris-viewer) ## Access
 information NA