Development of new adeno-associated virus capsid variants for targeted gene delivery to human cardiomyocytes

Figures

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  Figure 1. Cardiotropic rAAV variants obtained by directed evolution were closely related to WT AAV6 and AAV3 Directed evolution was performed using the hiPSC-CM line WTCWT. After six rounds of selection, the enriched novel AAVs were sequenced and phylogenetic analysis performed to compare with parental AAVs. (A) Clustering of amino acid sequences from novel variants (red) to WT AAV parental serotypes (black), with relationships depicted as a phylogenetic tree. (B) Heatmap illustrating the percentage identity among select parental AAVs and enriched novel variants. (C) Parental sequence contribution analysis of variants isolated after directed evolution. Black line represents the most probable composition of individual shuffled clones based on the longest sequence of identity to parental variants in a 5′ to 3′ direction. (D) Surface representations of the new capsid variants KK01, KK02, KK03, and KK05. The colored regions (yellow, orange, purple) denote surface residues that differ from the parental AAV6 capsid. Amino acid changes not located on the capsid surface are listed below. (D) Depiction as in (E) for the capsid variant KK04. The colored regions (red and purple) denote surface…

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  Figure 2. rAAV.KK04 was most efficient at gene delivery to hiPSC-CMs (A) hiPSC-CMs competitively transduced with a barcoded library of rAAV.CMV.GFP vectors, then harvested at D5 post transduction. Extraction of DNA/RNA was performed, followed by analysis using next-generation sequencing (n = 3). The relative proportions of barcode reads post NGS analysis are given at the level of cell entry (gDNA) and gene expression (mRNA) for MOT 100, 1,000, and 10,000. Results are expressed as percentage of total reads for each cell line. (B) hiPSC-CMs were transduced with unbarcoded rAAV.CBA.GFP vectors at MOT 1,000, followed by analysis using microscopy and flow cytometry to quantify GFP (n = 3 per group for SCVI 8 and 100, n = 4 per group for SCVI 480) on day 5 post transduction. Fluorescence images showing GFP auto-fluorescence in live cells. Scale bars, 100 μM. (C) Flow cytometry dot plots quantifying proportions of GFP-positive cardiomyocytes (cTnT+ cells) in three hiPSC lines. Data are mean ± SEM. Statistical significance of differences was calculated using an ordinary one-way ANOVA, and the difference between the mean of the variants and AAV6 was calculated with Dunnett’s multiple comparison…

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  Figure 3. Competitive transduction assay and functional validation of novel AAV variants in hCO (A) Human cardiac organoids were transduced with a barcoded library of rAAV.CMV.GFP vectors at MOT 100, 1,000, and 10,000. This was followed by analysis using next-generation sequencing (n = 8). The relative proportions of barcode reads post NGS analysis were given for all cell lines at the level of cell entry (gDNA) and gene expression (cDNA). Results are expressed as percentage of total reads for each cell line. (B) Human cardiac organoids were then transduced with unbarcoded rAAV vectors at MOT 10,000, followed by analysis by microscopy to quantify GFP (n = 6 or 7 per group). Fluorescence images showing GFP auto-fluorescence in live organoids. (C) GFP fluorescence intensity was quantified from microscopy acquired imaging by MATLAB. Data are mean ± SEM. Statistical significance of differences was calculated using an ordinary one-way ANOVA, and the difference between the mean of the variants and AAV6 was calculated with Dunnett’s multiple comparison test (∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

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  Figure 4. Competitive transduction assay to compare efficiency of barcoded AAV variants in human myocardial slices Cardiac slices were generated from the left ventricular myocardium of fresh human heart tissue. Slices were transduced with a barcoded library of rAAV.CMV.GFP vectors at MOT 10,000 (n = 6 from two human hearts). Two days post transduction, DNA and RNA were extracted from the slices and analyzed by next-generation sequencing. The relative proportions of barcode reads post NGS analysis are given at the level of cell entry and gene expression. Results are expressed as percentage of total reads for each condition. Only five data points are shown for PCM1+ gene expression as the AAV amplicon could not be amplified from one replicate of heart 2. Data are mean ± SEM. Statistical significance of differences was calculated using an ordinary one-way ANOVA, and the difference between the mean of the variants and AAV6 was calculated with Dunnett’s multiple comparison test (∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

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  Figure 5. Competitive transduction assay to compare efficiency of barcoded AAV variants in pig myocardial slices Cardiac slices were generated from the left ventricular myocardium of fresh pig heart tissue. Slices were transduced with a barcoded library of rAAV.CMV.GFP vectors at MOT 10,000 (n = 4). Two days post transduction, DNA and RNA were extracted from the slices and analyzed by next-generation sequencing. The relative proportions of barcode reads post NGS analysis are given at the level of cell entry and gene expression. Results are expressed as percentage of total reads for each condition. Data are mean ± SEM. Statistical significance of differences was calculated using an ordinary one-way ANOVA, and the difference between the mean of the variants and AAV6 was calculated with Dunnett’s multiple comparison test (∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

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  Figure 6. AAV variants exhibiting human cardiotropism did not show increased risk of liver transduction in vivo, although efficient cardiac expression was also not seen in the mouse heart C57Bl6 mice (6–8 week males, n = 6) were injected with a barcoded library of rAAV.CMV.GFP vectors at 1011 vg per animal via the tail vein. Four weeks post transduction, mice were sacrificed. Heart and liver tissues were collected for DNA and RNA extraction for next-generation sequencing. The relative proportions of barcode reads post NGS analysis are given at the level of cell entry and gene expression. Results are expressed as percentage of total reads for each condition. Data are mean ± SEM. Statistical significance of differences was calculated using an ordinary one-way ANOVA, and the difference between the mean of the variants and AAV9 (for heart) or AAV8 (for liver) was calculated with Dunnett’s multiple comparison test (∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, ∗∗∗∗ p ≤ 0.0001).

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  Figure 7. Summary of rAAV transduction efficiencies across all cardiac models tested Relative proportion of barcode reads showing rAAV performance at cell entry (gDNA) and gene expression (mRNA). With the exception of the hiPSC-CM, hCO and mouse heart, all other models show the barcode reads from the myocyte fraction (identified by PCM1+ staining for myocyte nuclei in the human, NHP, and pig cardiac slices). Higher proportions are indicated by both larger dot size and darker color. Lower proportions are indicated by smaller dot size and lighter color. NA shows variants which were not tested in that particular model.