Comparison of adeno-associated virus serotypes and delivery methods for cardiac gene transfer

Abstract

Cardiac gene transfer is a potentially useful strategy for cardiovascular diseases. The adeno-associated virus (AAV) is a common vector to obtain transgene expression in the heart. Initial studies conducted in rodents used indirect intracoronary delivery for cardiac gene transfer. More recently AAV vectors with so-called cardiac tropism have enabled significant cardiac transgene expression following intravenous injection. However, a direct comparison of intravenous versus intracoronary delivery with rigorous quantification of cardiac transgene expression has not been conducted. In the present study we tested the hypothesis that intracoronary AAV delivery would be superior to intravenous delivery vis-à-vis cardiac transgene expression. We compared intravenous and intracoronary delivery of AAV5, AAV6, and AAV9 (5×10(11) genome copies per mouse). Using enhanced green fluorescent protein as a reporter, we quantified transgene expression by fluorescence intensity and Western blotting. Quantitative polymerase chain reaction (PCR) was also performed to assess vector DNA copies, employing primers against common sequences on AAV5, AAV6, and AAV9. Intracoronary delivery resulted in 2.6- to 28-fold higher transgene protein expression in the heart 3 weeks after AAV injection compared to intravenous delivery depending on AAV serotype. The highest level of cardiac gene expression was achieved following intracoronary delivery of AAV9. Intracoronary delivery of AAV9 is a preferred method for cardiac gene transfer.

FIG. 1.

EGFP fluorescence intensity-area product as a better measurement for cardiac gene transfer efficiency. (A) Schematic presentation of AAV vectors encoding EGFP. (B) Representative panoramic photomicrograph showing EGFP fluorescence in epicardium after indirect intracoronary injection of AAV9.EGFP. (C) Three-dimensional plot of EGFP fluorescence intensity for the representative photomicrography shown in (B). (D) Product of EGFP fluorescence intensity-area (EGFP intensity-area) is highly correlated to EGFP protein content (r²=0.93), while EGFP percentage area is less correlated to EGFP protein content (r²=0.58) (p<0.0001). AAV, adeno-associated virus; CMV, cytomegalovirus; EGFP, enhanced green fluorescent protein; ITR, inverted terminal repeat; poly A, poly A from SV40 virus genome. Color images available online at www.liebertpub.com/hgtb

FIG. 2.

Representative photomicrographs showing cardiac EGFP expression after AAV5-, AAV6-, and AAV9-mediated gene transfer. EGFP fluorescence was evaluated 3 weeks after indirect intracoronary (IC) or intravenous (IV) delivery for each of the three vectors. Color images available online at www.liebertpub.com/hgtb

FIG. 3.

Higher transgene protein expression in left ventricular (LV) samples after indirect IC injection than IV injection. (A) Representative Western blots showing LV expression of EGFP after indirect IC and IV deliveries of AAV5.EGFP, AAV6.EGFP, and AAV9.EGFP. (B) The bar graph summarizes EGFP protein content in LV samples measured by Western blotting (n=6 for each group). Probability values are from Student's t-test (unpaired, two-tailed). Error bars denote 1 SE. **p<0.0001; *p<0.002.

FIG. 4.

Similar transgene protein expression in liver samples after indirect IC and IV injections. (A) Indirect IC injection and IV injection did not affect EGFP protein content in LV samples after gene transfer of AAV5.EGFP (p=0.66), AAV6.EGFP (p=0.76), or AAV9.EGFP (p=0.95). (B) Indirect IC injection and IV injection did not affect AAV copy numbers in LV samples after gene transfer of AAV5.EGFP (p=0.33), AAV6.EGFP (p=0.07), or AAV9.EGFP (p=0.85). Probability values are from Student's t-test (two-tailed, unpaired). Numbers in bars indicate group size; error bars denote 1 SE.