Huang Jianhua, Ito Yoshinori, Kobune Masayoshi, Sasaki Katsunori, Nakamura Kiminori, Dehari Hironari, Takahashi Kazuhiro, Ikeda Katsuya, Uchida Hiroaki, Kato Kazunori, Hamada Hirofumi
Department of Molecular Medicine, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 0608556, Japan.
Division of Gene Therapy, Sapporo Medical University, South-1, West-17, Chuo-ku, Sapporo 0608556, Japan.
J Gene Med. 2003 Oct;5(10):900-908. doi: 10.1002/jgm.429.
Although naked plasmid injection is the safest and most convenient method for gene delivery, a major limitation of this approach is currently poor transgene expression. The CA promoter (chicken beta-actin promoter with cytomegalovirus, CMV, enhancer) is one of the strongest transcriptional control modules found; however, it is uncertain whether a CA promoter-based vector is efficient enough for naked gene therapy in a cardiovascular context.
The beta-galactosidase (LacZ) expression provided by CA promoter plasmid (pCAZ2) injection into the skeletal muscle or the heart of Lewis rats was compared with CMV promoter plasmid or adenoviral vector (AxCAZ3). The effect of Simian virus 40 of the replication origin (SV40ori) deletion from pCAZ2 on transgene expression was also evaluated.
pCAZ2 showed the highest LacZ expression in both skeletal muscle and heart in comparison with the CMV promoter-based vector 5 days after naked plasmid injection. LacZ expression in the heart obtained using 20 micro g of pCAZ2 was almost equivalent to that shown with AxCAZ3 at 6.0 x 10(9) optical particle units. The time course of transgene expression driven by CMV and CA promoters in the heart were similar, with the CA promoter providing significantly higher gene expression than the CMV promoter across all time points examined. SV40ori deletion from pCAZ2 did not affect transgene expression in either skeletal muscle or heart.
Transgene expression mediated by naked CA promoter-based plasmid injection was shown to be quite efficient in the heart. We propose that the CA promoter vector is suitable for myocardial gene therapy.
尽管裸质粒注射是基因递送最安全、最便捷的方法，但目前这种方法的一个主要局限性是转基因表达不佳。CA启动子（带有巨细胞病毒增强子的鸡β-肌动蛋白启动子）是已发现的最强转录控制模块之一；然而，基于CA启动子的载体在心血管领域进行裸基因治疗时是否足够有效尚不确定。
将注射CA启动子质粒（pCAZ2）到Lewis大鼠骨骼肌或心脏所提供的β-半乳糖苷酶（LacZ）表达与巨细胞病毒启动子质粒或腺病毒载体（AxCAZ3）进行比较。还评估了从pCAZ2中缺失猿猴病毒40复制起点（SV40ori）对转基因表达的影响。
与基于巨细胞病毒启动子的载体相比，裸质粒注射5天后，pCAZ2在骨骼肌和心脏中均显示出最高的LacZ表达。使用20μg pCAZ2在心脏中获得的LacZ表达几乎与在6.0×10⁹光学粒子单位下AxCAZ3所显示的表达相当。巨细胞病毒和CA启动子在心脏中驱动的转基因表达时间进程相似，在所有检测的时间点，CA启动子提供的基因表达均显著高于巨细胞病毒启动子。从pCAZ2中缺失SV40ori对骨骼肌或心脏中的转基因表达均无影响。
基于裸CA启动子的质粒注射介导的转基因表达在心脏中显示出相当高效。我们认为CA启动子载体适用于心肌基因治疗。
