Terada Yoshio, Tanaka Hiroyuki, Okado Tomokazu, Shimamura Haruko, Inoshita Seiji, Kuwahara Michio, Akiba Takashi, Sasaki Sei
Homeostasis Medicine and Nephrology, Tokyo Medical and Dental University, Tokyo, Japan.
Kidney Int. 2002 Dec;62(6):1966-76. doi: 10.1046/j.1523-1755.2002.t01-1-00650.x.
The development of an in vivo gene transfer approach to deliver physiologic levels of recombinant proteins to the systemic circulation would represent a significant advance in the treatment of protein deficiency disorders. However, the ability to regulate transgene expression is of paramount importance for safe and effective gene transfer therapy.
We developed two plasmids, one encoder of chimeric GeneSwitch protein, and the other an inducible transgene for human erythropoietin (Epo). The level of secretion of Epo into the serum was modulated by intraperitoneal administration of mifepristone (MFP). Rats were divided into four groups: one group administered Epo plasmid with MFP for 50 days, a second group administered Epo plasmid with MFP for 15 days and then again from day 30 to day 50, a third group administered Epo plasmid without MFP, and a fourth group administered control plasmid. A pair of electrodes was inserted into the muscle of the right thigh, 100 mg of each plasmid was injected, and in vivo electroporation (8 pulses at 100 V for 50 msec) was performed.
The presence of vector-derived Epo mRNA was detected by RT-PCR only in the Epo plasmid and MFP(+) groups. The hematocrit levels increased continuously, from the pre-injection level of 41.2% to 55.0% on day 30 and 53.8% on day 50 in the Epo plasmid and MFP(+) groups. In the MFP re-challenged group, the hematocrit levels rose up to day 15, fell after 20 to 30 days, and then rose again after MFP re-administration. The serum Epo levels increased only in the Epo plasmid and MFP(+) groups. There were no significant changes in hematocrit levels and Epo levels in the Epo plasmid and MFP(-) group.
Epo gene transfer with the GeneSwitch system by in vivo electroporation is a useful procedure for efficient drug-regulated delivery of Epo.
开发一种体内基因转移方法，将生理水平的重组蛋白输送到体循环中，这将代表在蛋白质缺乏症治疗方面取得重大进展。然而，调节转基因表达的能力对于安全有效的基因转移治疗至关重要。
我们构建了两种质粒，一种编码嵌合基因开关蛋白，另一种是人促红细胞生成素（Epo）的诱导型转基因。通过腹腔注射米非司酮（MFP）来调节Epo分泌到血清中的水平。将大鼠分为四组：一组给予Epo质粒和MFP，持续50天；第二组给予Epo质粒和MFP，持续15天，然后从第30天到第50天再次给药；第三组给予Epo质粒但不给予MFP；第四组给予对照质粒。将一对电极插入右大腿肌肉，注射每种质粒100毫克，并进行体内电穿孔（100伏，8个脉冲，持续50毫秒）。
仅在Epo质粒和MFP（+）组中通过RT-PCR检测到载体衍生的Epo mRNA的存在。在Epo质粒和MFP（+）组中，血细胞比容水平持续升高，从注射前的41.2%升至第30天的55.0%和第50天的53.8%。在MFP再次激发组中，血细胞比容水平在第15天之前上升，在20至30天后下降，然后在再次给予MFP后再次上升。血清Epo水平仅在Epo质粒和MFP（+）组中升高。Epo质粒和MFP（-）组的血细胞比容水平和Epo水平没有显著变化。
通过体内电穿孔利用基因开关系统进行Epo基因转移是一种有效的药物调节Epo递送的有用方法。
