Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat

Abstract

Heart disease is the leading cause of morbidity and mortality. Cardiac gene transfer may serve as a novel therapeutic approach. This investigation was undertaken to compare cardiac tropisms of adeno-associated virus (AAV) serotypes 1, 6, 7, 8, and 9. Neonatal mice were injected with 2.5 x 10(11) genome copies (GC) of AAV serotype 1, 6, 7, 8, or 9 expressing LacZ under the control of the constitutive chicken beta-actin promoter with cytomegalovirus enhancer promoter via intrapericardial injection and monitored for up to 1 year. Adult rats were injected with 5 x 10(11) GC of the AAV vectors via direct cardiac injection and monitored for 1 month. Cardiac distribution of LacZ expression was assessed by X-Gal histochemistry, and beta-galactosidase activity was quantified in a chemiluminescence assay. Cardiac functional data and biodistribution data were also collected in the rat. AAV9 provided global cardiac gene transfer stable for up to 1 year that was superior to other serotypes. LacZ expression was relatively cardiac specific, and cardiac function was unaffected by gene transfer. AAV9 provides high-level, stable expression in the mouse and rat heart and may provide a simple alternative to the creation of cardiac-specific transgenic mice. AAV9 should be used in rodent cardiac studies and may be the vector of choice for clinical trials of cardiac gene transfer.

FIG. 1.

Representative photomicrographs of sections from mouse heart, diaphragm, and liver 6 weeks after intrapericardial injection of 50 μl containing 2.5 × 10¹¹ GC of AAV-CB-LacZ of the indicated serotype. Sections have been stained with X-Gal and counterstained with eosin. Scale bars: 1 mm for heart, 200 μm for liver and diaphragm.

FIG. 2.

Dose response of LacZ expression in the mouse heart 6 weeks after intrapericardial injection of 50 μl containing the indicated dose of AAV-CB-LacZ. (a) Representative photomicrographs of mouse heart stained with X-Gal and counterstained with eosin. Scale bars: 200 μm. (b) Graph displaying β-galactosidase activity as determined by quantitative chemiluminescence assay of samples from mice treated with the intermediate dose. Columns and error bars represent means and SD. Note that AAV9 is superior to the other serotypes evaluated at the intermediate dose (*p < 0.05 vs. other serotypes).

FIG. 3.

Time course of LacZ expression after intrapericardial injection of 50 μl containing 2.5 × 10¹¹ GC of AAV9-CB-LacZ. Shown are representative photomicrographs of sections stained with X-Gal and counterstained with eosin. Scale bars: 1 mm for low magnification (top row), 200 μm for high magnification (bottom row).

FIG. 4.

Representative photomicrographs of sections from rat heart 4 weeks after direct myocardial injection into the left ventricular free wall of 250 μl containing 5 × 10¹¹ GC of AAV-CB-LacZ of the indicated serotype in five equal aliquots. Sections have been stained with X-Gal and counterstained with eosin. Scale bars: 2.4 mm.

FIG. 5.

Dose response of LacZ expression in the rat heart 4 weeks after direct myocardial injection of 250 μl of the indicated dose of AAV-CB-LacZ in five equal aliquots. (a) Representative photomicrographs of rat heart stained with X-Gal and counterstained with eosin. Scale bars: 200 μm. (b) Graph displaying β-galactosidase activity as determined by quantitative chemiluminescence assay of samples from rats treated with the high dose. Columns and error bars represent means and SD. Note that AAV9 is superior to the other serotypes evaluated (*p < 0.05 vs. other serotypes).

FIG. 6.

Cardiac hemodynamic measurements in rats 4 weeks after direct myocardial injection of 5 × 10¹¹ GC of AAV8-and AAV9-CB-LacZ compared with uninjected control. Representative pressure–volume loops were recorded via a Millar conductance catheter. No significant differences in pressure or volume were observed. RVU, relative volume units.

FIG. 7.

Biodistribution of LacZ expression and vector genomes 4 weeks after direct injection of 5 × 10¹¹ GC of AAV8- and AAV9-CB-LacZ into the rat heart. (a) Representative photomicrographs of LacZ expression in several organs examined. Sections were stained with X-Gal and counterstained with eosin. Scale bars: 200 μm. (b) Graph displaying vector genome distribution in several organs examined via TaqMan PCR. He, heart; Li, liver; Lu, lung; Br, brain; Te, testis; Ki, kidney; Sp, spleen; St, stomach; Ga, gastrocnemius. Columns and error bars represent means and SD.