AAV6 vectors provide superior gene transfer compared to AAV9 vectors following intramyocardial administration

Figures

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  Figure 1. AAV6 and MyoAAV4A efficiently transduce NRVMs (A) Experimental design. (B) Direct fluorescent images of NRVMs 3 days post-transduction with various AAV vector pseudotypes. Scale bars, 100 μm. (C) Percentage of GFP-expressing cells determined by flow cytometry. (D) Relative expression levels of GFP determined by RT-qPCR. Data are presented as mean ± SEM. Data were compared using one-way ANOVA with post-hoc Fisher’s LSD test. ∗∗p < 0.01; ∗∗∗, ###p < 0.001. ∗ denotes comparison between groups and UT. # denotes comparison between groups and AAV6. UT, untransduced; AAV9_10×, AAV9 at a 10 times higher dose.

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  Figure 2. AAV6 and MyoAAV4A efficiently transduce mouse myocardium (A) Experimental design. (B) Direct fluorescent images of mouse hearts. Scale bars, 2 mm. (C) Immunostaining images of hearts and zoom-in images from the injection sites. Scale bars, 1 mm (upper) or 200 μm (lower). (D) Quantification of the integrated density of direct GFP fluorescence, n = 6. (E) mRNA expression level of GFP in left ventricles, n = 3. (F) AAV vector genome copies in ventricles, n = 3. Data are presented as mean ± SEM. Data were compared using one-way ANOVA with post-hoc Fisher’s LSD test. ∗, #p < 0.05; ∗∗, ##p < 0.01; ∗∗∗, ###p < 0.001. ∗ denotes comparison between groups and PBS. # denotes comparison between groups and AAV6. IM, intramyocardial; AAV9_10×, AAV9 at a 10 times higher dose; vg/dg, vector genomes per diploid genome.

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  Figure 3. Intramyocardial injection of AAV vector does not induce vector-related cardiac fibrosis (A) Example of picrosirius red staining images of hearts injected with PBS or AAV6-cTnT-GFP vector. Black arrows indicate regions of cardiac fibrosis. Scale bars, 1 mm. (B) Quantification of the fibrosis area in hearts injected with PBS or AAV vectors. No significant differences were observed among the groups, n = 3. (C) Picrosirius red (left) and immunofluorescence (right) staining images of a heart injected with AAV6-cTnT-GFP. Stainings were performed using consecutive sections. Scale bars, 500 μm. Data are presented as mean ± SEM. Data were compared using one-way ANOVA with post-hoc Fisher’s LSD test. ns, not significant.

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  Figure 4. AAV6 outperforms AAV9 independently of the expression cassette and mouse background (A) Scheme of the in vitro experiment. (B) Direct fluorescent images of NRVMs 3 days post-transduction with AAV6- and AAV9-pseudotyped vectors. Yellow arrows indicate weakly GFP-expressing cells. Scale bars, 100 μm. (C) Expression level of GFP mRNA determined by RT-qPCR, n = 4. (D) Scheme of the in vivo experiment and representative heart images. Scale bars, 2 mm. (E) Quantification of direct GFP fluorescence, (F) GFP mRNA expression, and (G) AAV vector genome copies in FVB female mice injected with AAV6- or AAV9-CMV-GFP, n = 3. (H) Scheme of the in vivo experiment and representative heart images. Scale bars, 2 mm. (I) Quantification of direct GFP fluorescence, (J) GFP mRNA expression, and (K) AAV vector genome copies in C57BL/6J male mice injected with AAV6- or AAV9-CMV-GFP, n = 4. Data are presented as mean ± SEM. Data were compared using Student’s t test. ∗p < 0.05; ∗∗p < 0.01. ∗ denotes comparison between AAV6 and AAV9. vg/dg, vector genomes per diploid genome.

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  Figure 5. AAV6 outperforms AAV9 in transducing pig myocardium (A) Experimental design. (B) Direct fluorescent macroscopy of pig heart tissues injected with AAV6- or AAV9-CMV-GFP. Sampled tissues are encircled using a white dashed line. Scale bars, 1 cm. (C) Immunostaining images from pig heart tissues injected with 200 μL (high dose) AAV6- or AAV9-CMV-GFP. Scale bars, 200 μm. (D) Quantification of the GFP mRNA expression and (E) AAV vector genome copies in pig heart tissues injected with AAV6- or AAV9-CMV-GFP, n = 3. Data are presented as mean ± SEM. Data were compared using two-way ANOVA with post-hoc Fisher’s LSD test. ∗,#p < 0.05; ##p < 0.01; ∗∗∗p < 0.001. ∗ denotes comparison between AAV serotypes. # denotes comparison between injection volumes. IM, intramyocardial. vg/dg, vector genomes per diploid genome.

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  Figure 6. AAV6 outperforms AAV9 in transducing hiPSC-CMs and human heart slices (A) Scheme of the hiPSC-CM experiment. (B) Quantification of the AAV vector genomes in hiPSC-CMs transduced with AAV6- or AAV9-CMV-GFP, n = 4. (C) Scheme of the human LAAS experiment. (D) Quantification of the AAV vector genomes in human LAASs transduced with AAV6- or AAV9-CMV-GFP, n = 4 donors. Data are presented as mean ± SEM. Data were compared using two-way ANOVA with post-hoc Fisher’s LSD test (B) or Student’s t test (D). ∗p < 0.05; ∗∗∗, ###p < 0.001. ∗ denotes comparison between AAV serotypes. # denotes comparison between multiplicities of infection. MOI, multiplicity of infection. vg/dg, vector genomes per diploid genome.