High-throughput evaluation of cardiac-specific promoters for adeno-associated virus mediated cardiac gene therapy

Figures

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  Fig. 1. Competitive transduction assay to compare efficiency of barcoded rAAV promoters in vitro. A hiPSC-CM, B NRVM and C HuH7 were competitively transduced with a rAAV promoter kit packaged in AAV2, 6 and 9 at MOI 1000 (n = 4 hiPSC-CM, n = 5 NRVM, n = 3 Huh7). For each capsid and cell type combination, results on the left are displayed as boxplots comparing performance of ubiquitous promoter CMV, cardiac-specific promoters (CSP) and liver-specific promoter (LSP), and on the right as boxplots comparing performance of cardiac-specific promoters only. Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn’s post-hoc test with Bonferroni correction (*p < 0.05).

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  Fig. 2. Competitive transduction assay to compare efficiency of barcoded rAAV promoters in mice. C57Bl6 mice (6–8 week male) were injected with a rAAV promoter kit packaged in AAV6 (n = 8) and AAV9 (n = 5) at 1×1011 vg via the tail vein. Four weeks post transduction, mice were sacrificed. A Heart and B liver tissues were collected for DNA/RNA extraction and NGS analysis. For each capsid and tissue combination, results on the left are displayed as boxplots comparing performance of ubiquitous promoter CMV, liver-specific promoter (LSP) and cardiac-specific promoters (CSP), and on the right as boxplots comparing performance of cardiac-specific promoters only. Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn’s post-hoc test with Bonferroni correction (*p < 0.05).

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  Fig. 3. Competitive transduction assay to compare efficiency of barcoded rAAV promoters in rats. SD rats (6–8 week male) were injected with a rAAV promoter kit packaged in AAV6 (n = 5) and AAV9 (n = 4) at 5 × 1011 vg via the tail vein. Four weeks post transduction, rats were sacrificed. A Heart and B liver tissues were collected for DNA/RNA extraction followed by NGS analysis. For each capsid and tissue combination, results on the left are displayed as boxplots comparing performance of ubiquitous promoter CMV, liver-specific promoter (LSP) and cardiac-specific promoters (CSP), and on the right as boxplots comparing performance of cardiac-specific promoters only. Statistical analyses were performed using the Kruskal-Wallis test followed by Dunn’s post-hoc test with Bonferroni correction (*p < 0.05).

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  Fig. 4. Competitive transduction assay to compare efficiency of barcoded rAAV promoters in sheep and pigs. Adult sheep and pig (n = 1 per capsid) were intramyocardially injected with a rAAV promoter kit packaged in AAV6 at 5 × 1011 vg/animal and AAV9 at 1 × 1012 vg/animal. A Endocardial gene delivery using the injection catheter with a retractable 27 gauge needle under B fluoroscopic and C intracardiac echocardiography guidance. D GFP fluorescence signal at RV was visualised two weeks post transduction. GFP-expressing heart tissues were collected for DNA and RNA extraction for NGS. Heat maps are expressed as expression index for E AAV Capsid 6 and F AAV Capsid 9.

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  Fig. 5. Summary of promoter efficiencies across all models tested. The size and colour of the dots indicate the mean expression intensity (EI_mean), with larger dots and darker colours corresponding to higher proportion.