Iwatate M, Gu Y, Dieterle T, Iwanaga Y, Peterson K L, Hoshijima M, Chien K R, Ross J
Institute of Molecular Medicine and Department of Medicine, University of California San Diego, La Jolla, CA 92093, USA.
Gene Ther. 2003 Oct;10(21):1814-20. doi: 10.1038/sj.gt.3302077.
High-efficiency somatic gene transfer in adult mouse heart has not yet been achieved in vivo. Here, we demonstrate high-efficiency in vivo transcoronary gene delivery to the adult murine myocardium using a catheter-based technique with recombinant adenovirus (AdV) and adeno-associated virus (AAV) vectors in normal and genetically engineered mice. The method involves immersion hypothermia followed by transient aortic and pulmonary artery occlusion with proximal intra-aortic segmental injection of cardioplegic solution containing substance P and viral vectors. Gene expression measured using a LacZ marker gene was observed throughout both ventricles. The expression efficiency of a cytoplasmic LacZ marker gene in the left ventricular myocardium was 56.4+/-14.5% (mean+/-s.d.) at 4 days with an AdV vector, and with an AAV vector it was 81.0+/-5.9% at 4 weeks. Following AAV gene transfer, no gene expression was found in kidney, brain, lung, and spleen, but there was slight expression in liver. In addition, we demonstrate temporally controlled genetic manipulation in the heart with an efficiency of 54.6+/-5.2%, by transferring an AdV vector carrying Cre recombinase in ROSA26 flox-LacZ reporter mice. Procedure-related mortality was 16% for AdV and zero for AAV transfer. Thus, this method provides efficient, relatively homogeneous gene expression in both ventricles of the adult mouse heart, and offers a novel approach for conditional gene rescue or ablation in genetically engineered mouse models.
在成年小鼠心脏中尚未实现高效的体内体细胞基因转移。在此，我们展示了一种基于导管技术，利用重组腺病毒（AdV）和腺相关病毒（AAV）载体，在正常和基因工程小鼠中向成年鼠心肌进行高效体内冠状动脉基因递送。该方法包括低温浸泡，随后短暂阻断主动脉和肺动脉，并在主动脉近端节段注射含P物质和病毒载体的心脏停搏液。使用LacZ标记基因测量发现，两个心室均有基因表达。AdV载体在4天时，左心室心肌细胞质LacZ标记基因的表达效率为56.4±14.5%（平均值±标准差），AAV载体在4周时为81.0±5.9%。AAV基因转移后，在肾脏、大脑、肺和脾脏中未发现基因表达，但在肝脏中有轻微表达。此外，我们通过在ROSA26 flox-LacZ报告基因小鼠中转移携带Cre重组酶的AdV载体，证明了心脏中可进行时间控制的基因操作，效率为54.6±5.2%。AdV转移的操作相关死亡率为16%，AAV转移为零。因此，该方法在成年小鼠心脏的两个心室中提供了高效、相对均匀的基因表达，并为基因工程小鼠模型中的条件性基因拯救或消融提供了一种新方法。
