Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution

Parts used

Show 3 moreparts→

Key findings

• A truncated cardiac troponin T (cTnT) promoter spanning −375 to +43 relative to the transcriptional start site restricts AAV-delivered transgene expression to the heart even after systemic intravenous administration. Packaged in an AAV-6 capsid and injected into one-week-old mice, a cTnT-luciferase vector gives luciferase activity nearly 100-fold higher in heart than in any other tissue assayed, whereas the same vector driven by the CMV promoter expresses body-wide, with intercostal muscle 9-fold higher than heart.
• Capsid serotype, not promoter, sets the efficiency of cardiac AAV gene delivery after intravenous injection: relative to AAV-2, cardiac luciferase expression from the cTnT promoter is 26-fold higher with AAV-1, 28-fold with AAV-6, 271-fold with AAV-8 and 358-fold with AAV-9. Cardiac restriction is preserved with every serotype tested, and the AAV-9 capsid combined with the cTnT promoter gives luciferase expression more than 640-fold higher in heart than in the next highest tissues, liver and intercostal and gastrocnemius muscle.
• The AAV-9 capsid transduces cardiomyocytes more efficiently than AAV-1, AAV-2, AAV-6 or AAV-8 after intravenous injection into one-week-old mice, reaching 96 ± 2% eGFP-positive cardiomyocytes at only 3.15 × 10^10 vector genomes per mouse. AAV-8 needs 1 × 10^11 vg to reach a comparable 95 ± 3%, AAV-1 and AAV-6 plateau near 75% at that same dose, and AAV-2 transduces cardiomyocytes poorly even at 1 × 10^11 vg. The AAV-9 advantage widens at low dose: at 3.15 × 10^9 vg per mouse it transduces cardiomyocytes 5.8-fold more efficiently than AAV-8 and 22-fold more efficiently than AAV-1 or AAV-6.
• Mean vector genome copies per cardiomyocyte rise linearly with dose for AAV-1, AAV-6, AAV-8 and AAV-9 across 3.15 × 10^9 to 1 × 10^11 vg per mouse, while AAV-2 copy number increases by only 60% over the same range, marking AAV-2 as poorly suited to cardiac gene delivery. Counting transgene-positive cells is a saturating measure of gene transfer: the percentage of transduced cells plateaus at 100% while mean vector copy number per cell continues to climb with dose.
• Cardiomyocyte restriction by the cTnT promoter holds at single-cell resolution within the heart itself. In hearts transduced with AAV-9-packaged cTnT-eGFP, vascular smooth muscle and endothelial cells of intramyocardial arterioles are entirely devoid of eGFP while the cardiomyocytes immediately surrounding those arterioles are intensely positive, confirmed by immunohistochemistry on serial sections, and expression is distributed homogeneously throughout both right and left ventricles.
• AAV transduction of cardiomyocytes in vivo is quantal at the single-cell level rather than continuously variable. Per-cardiomyocyte eGFP fluorescence intensity clusters around integer values by K-means analysis, with every cluster significantly distinct from the others (one-way ANOVA with Dunnett's T3, p < 0.05 for all pairwise comparisons), and the measured proportions of cells carrying n = 0, 1, 2, 3, 4 or ≥ 5 expressed genomes match the theoretical Poisson distribution generated from the observed mean, correlating across 24 measured values with R² = 0.85. Fluorescence intensity per cell therefore reads out vector genome copy number per cell.
• Because AAV gene delivery to cardiomyocytes follows Poisson statistics, the dose needed for near-complete transduction is set by the mean genomes per cell rather than by any per-cell threshold: an average of approximately two expressed vector genomes per cardiomyocyte is required for 85% of cardiomyocytes to express at least one genome.

Abstract

Newly-isolated serotypes of AAV readily cross the endothelial barrier to provide efficient transgene delivery throughout the body. However, tissue-specific expression is preferred in most experimental studies and gene therapy protocols. Previous efforts to restrict gene expression to the myocardium often relied on direct injection into heart muscle or intracoronary perfusion. Here, we report an AAV vector system employing the cardiac troponin T promoter (cTnT). Using luciferase and eGFP, the efficiency and specificity of cardiac reporter gene expression using AAV serotype capsids: AAV-1, 2, 6, 8 or 9 were tested after systemic administration to 1 week old mice. Luciferase assays showed that the cTnT promoter worked in combination with each of the AAV serotype capsids to provide cardiomyocyte-specific gene expression, but AAV-9 followed closely by AAV-8 was the most efficient. AAV9-mediated gene expression from the cTnT promoter was 640-fold greater in the heart compared to the next highest tissue (liver). eGFP fluorescence indicated a transduction efficiency of 96% using AAV-9 at a dose of only 3.15×10^10 viral particles per mouse. Moreover, the intensity of cardiomyocyte eGFP fluorescence measured on a cell-by-cell basis revealed that AAV-mediated gene expression in the heart can be modeled as a Poisson distribution; requiring an average of nearly two vector genomes per cell to attain an 85% transduction efficiency.