Fang Hongfei, Lai Ngai Chin, Gao Mei Hua, Miyanohara Atsushi, Roth David M, Tang Tong, Hammond H Kirk
Department of Medicine, University of California San Diego, San Diego, CA, USA.
Hum Gene Ther Methods. 2012 Aug;23(4):234-41. doi: 10.1089/hgtb.2012.105.
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.
心脏基因转移是一种对心血管疾病可能有用的策略。腺相关病毒（AAV）是在心脏中获得转基因表达的常用载体。最初在啮齿动物中进行的研究使用间接冠状动脉内给药进行心脏基因转移。最近，具有所谓心脏嗜性的AAV载体在静脉注射后能够实现显著的心脏转基因表达。然而，尚未对静脉内给药与冠状动脉内给药进行直接比较，并对心脏转基因表达进行严格定量。在本研究中，我们检验了冠状动脉内给予AAV在心脏转基因表达方面优于静脉内给药的假设。我们比较了AAV5、AAV6和AAV9（每只小鼠5×10¹¹个基因组拷贝）的静脉内给药和冠状动脉内给药。使用增强型绿色荧光蛋白作为报告基因，我们通过荧光强度和蛋白质印迹法定量转基因表达。还进行了定量聚合酶链反应（PCR）以评估载体DNA拷贝数，使用针对AAV5、AAV6和AAV9上共同序列的引物。与静脉内给药相比，根据AAV血清型，冠状动脉内给药在AAV注射后3周心脏中的转基因蛋白表达高2.6至28倍。冠状动脉内给予AAV9后实现了最高水平的心脏基因表达。冠状动脉内给予AAV9是心脏基因转移的首选方法。
