AAV capsids target muscle-resident cells with different efficiencies—A comparative study between AAV8, AAVMYO, and AAVMYO2

Figures

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  Figure 1. Transduction of mononuclear cells in the TA after i.v. injection of AAV8, AAVMYO, and AAVMYO2 (A) Schematic representation of the experimental procedure. 8- to 10-week-old male adult C57BL/6 were injected i.v. with PBS, AAV8, AAVMYO, or AAVMYO2 at the titer of 3.0 × 1012 or 3.0 × 1013 vg/kg (n = 4 mice per condition). All viruses contained a tdTomato transgene driven by a CMV promoter to label transduced cells. Three weeks post-injection, TAs were collected and digested to generate a single-cell suspension that was stained for CD31, CD45, Integrin-α7, and Sca-1. Flow cytometry was then used to characterize the proportion of tdTomato-expressing cells and the median tdTomato signal for each mouse. (B, C, and D) AAV-specific quantifications of the proportion of tdTomato+ cells (upper panels) and median tdTomato signal intensity (lower panels) in mice treated with AAV8 (B), AAVMYO (C), and AAVMYO2 (D) at 3.0 × 1012 or 3.0 × 1013 vg per kg. na = not applicable, when <5 tdTomato+ cells were detected in the cell population the median tdTomato signal was not measured. Data are shown as means ± SD. In (B), (C), and (D), statistics were evaluated using two-way ANOVAs with Tukey’s multiple…

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  Figure 2. Transduction of mononuclear cells in the TA after IM injection of AAV8, AAVMYO and AAVMYO2 (A) Schematic representation of the experimental procedure. Ten-week-old male adult C57BL/6 mice were injected IM with PBS, AAV8, AAVMYO, or AAVMYO2 at the titer of 1.0 × 1010 (equivalent to approximately 3.3 × 1011 vg/kg) or 1.0 × 1011 vg per TA (equivalent to approximately 3.3 × 1012 vg/kg) (n = 4 mice per condition). All viruses contained a tdTomato transgene driven by a CMV promoter to allow the labeling of transduced cells with tdTomato. Three weeks post-injection, TAs were collected and digested to generate a single-cell suspension that was stained for CD31, CD45, Integrin-α7, and Sca-1. Flow cytometry was then used to characterize the proportion of tdTomato-expressing cells and the median tdTomato signal for each mouse. (B, C, and D) AAV-specific quantifications of the proportion of tdTomato+ cells and median tdTomato signal intensity in mice treated with AAV8 (B), AAVMYO (C), and AAVMYO2 (D) at 1.0 × 1010 or 1.0 × 1011 vg per TA. (E) Mid-belly cross-sections of TAs from mice injected IM with PBS or 1.0 × 1011 vg (equivalent to approximately 3.3 × 1012 vg/kg) of AAV8, AAVMYO, or…

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  Figure 3. Quantification of AAV8, AAVMYO, and AAVMYO2 transduction levels at different time points after IM administration (A) Schematic representation of the experimental procedure. Ten-week-old male adult C57BL/6 mice were injected IM with PBS, AAV8, AAVMYO, or AAVMYO2 at the titer of 1.0 × 1011 vg per TA (equivalent to approximately 3.3 × 1012 vg/kg) (n = 4 mice per condition). All viruses contained a tdTomato transgene driven by a CMV promoter to allow the labeling of transduced cells with tdTomato. Three and 6 weeks post-injection, TAs were collected and digested to generate a single-cell suspension that was stained for CD31, CD45, Integrin-α7, and Sca-1. Flow cytometry was then used to characterize the proportion of tdTomato-expressing cells and the median tdTomato signal for each mouse. (B, C, and D) AAV-specific quantifications of the proportion of tdTomato+ cells and median tdTomato signal intensity in mice treated with AAV8 (B), AAVMYO (C), and AAVMYO2 (D) at 1.0 × 1011 vg per TA and collected 3 or 6 weeks post-administration. (E) Quantification of the proportion of CD31+, CD45+, Integrin-α7+, and Sca-1+ cells detected during flow cytometry. n = 4. Data are shown as means ± SD.…

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  Figure 4. AAVMYO can deliver transgenes to muscle-residing mononuclear cells in the dyW/dyW mouse model of LAMA2 MD (A) Schematic representation of the experimental procedure. 5-week-old dyW/dyW mice and their wild-type littermates were injected IM with AAVMYO at the titer of 1.0 × 1010 vg per TA (equivalent to approximately 5.0 × 1011 vg/kg for wild-type mice and approximately 1.25 × 1012 vg/kg in dyW/dyW mice). The virus contained a tdTomato transgene driven by a CMV promoter. Three weeks post-injection, TAs were collected and smFISH was performed to detect AAV DNA and cell-specific markers. (B) Mid-belly cross-sections of wild-type and dyW/dyW TAs after IM injection of 1.0 × 1010 vg of AAVMYO. Upper panels, left: overview of staining for tdTomato (red) and Laminin-β1-γ1 (white). Upper panels, right: smFISH to detect tdTomato antisense strand (AAV DNA, magenta) and immunostaining for Laminin-β1-γ1 (white) and DAPI (blue). Note the presence of many AAV DNA-positive nuclei. Lower panels (cell type): smFISH to detect tdTomato antisense strand (AAV DNA) and mRNA of cell markers for endothelial cells (Pecam1), immune cells (Ptprc), MuSCs (Pax7), and FAPs (Pdgfra). DAPI (blue) was used to…