Hassan M H, Khatoon N, Curiel D T, Hamada F M, Arafa H M, Al-Hendy A
Department of Obstetrics and Gynecology, University of Texas Medical Branch, Galveston, TX 77555-0587, USA.
Hum Reprod. 2008 Mar;23(3):514-24. doi: 10.1093/humrep/dem410. Epub 2008 Jan 8.
To circumvent the paucity of the primary adenovirus (Ad5) receptor and the non-specific Ad5 tropism in the context of uterine leiomyoma cells, Ad5 modification strategies would be beneficial.
We screened several modified adenoviruses to identify the most efficient and selective virus toward human leiomyoma cells to be used as candidate for delivering therapeutic genes. We propagated: wild-type Ad5-luc, fiber-modified viruses: ad5 RGD-luc, Ad5-Sigma-luc, Ad5/3-luc and Ad5-CAV2-luc, as well as transcriptional targeted viruses: ad5 survivin-luc, Ad5-heparanase-luc, Ad5-MSLN-CRAD-luc and Ad5-SLPI-luc, on 293 cells and purified them by double CsCL density centrifugation. Then we transfected primary cultures of human leiomyoma cells derived from fibroids of four different patients, telomerase-immortalized human leiomyoma cell line (huLM), telomerase-immortalized normal human myometrial cell line (HM9) and immortalized normal human liver cells (THLE3) with the viruses at 5, 10 and 50 plaque-forming units (PFU)/cell. After 48 h, luciferase activities were measured and normalized to the total cellular protein content.
Ad5-RGD-luc and Ad5-CAV2-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc at 5, 10 and 50 pfu/cell showed significantly higher expression levels of luciferase activity in both primary and immortalized human leiomyoma cells when compared with Ad5-Luc. Additionally, these modified viruses demonstrated selectivity toward leiomyoma cells, compared with myometrial cells and exhibited lower liver cell transduction, compared with Ad5-luc, at the same dose levels.
Ad5-CAV2-luc, Ad5-RGD-luc, Ad5-SLPI-luc and Ad5-MSLN-CRAD-luc are promising delivery vehicles in the context of leiomyoma gene therapy.
鉴于子宫平滑肌瘤细胞中主要腺病毒（Ad5）受体数量稀少以及Ad5存在非特异性嗜性，对Ad5进行修饰的策略将大有裨益。
我们筛选了几种修饰腺病毒，以确定对人平滑肌瘤细胞最有效且最具选择性的病毒，用作递送治疗性基因的候选病毒。我们在293细胞上繁殖了野生型Ad5 - luc、纤维修饰病毒：ad5 RGD - luc、Ad5 - Sigma - luc、Ad5/3 - luc和Ad5 - CAV2 - luc，以及转录靶向病毒：ad5 survivin - luc、Ad5 - 乙酰肝素酶 - luc、Ad5 - MSLN - CRAD - luc和Ad5 - SLPI - luc，并通过双重CsCL密度离心法对其进行纯化。然后，我们用病毒以5、10和50个噬斑形成单位（PFU）/细胞的剂量转染来自四名不同患者肌瘤的人平滑肌瘤细胞原代培养物、端粒酶永生化人平滑肌瘤细胞系（huLM）、端粒酶永生化正常人子宫肌层细胞系（HM9）和永生化正常人肝细胞（THLE3）。48小时后，测量荧光素酶活性并将其标准化为总细胞蛋白含量。
与Ad（5） - Luc相比，ad5 RGD - luc和Ad5 - CAV2 - luc、Ad5 - SLPI - luc和Ad5 - MSLN - CRAD - luc在5、10和50 pfu/细胞时，在原代和永生化人平滑肌瘤细胞中均显示出显著更高的荧光素酶活性表达水平。此外，与子宫肌层细胞相比，这些修饰病毒对平滑肌瘤细胞具有选择性，并且在相同剂量水平下，与Ad5 - luc相比，对肝细胞的转导作用更低。
在平滑肌瘤基因治疗中，Ad5 - CAV2 - luc、Ad5 - RGD - luc、Ad5 - SLPI - luc和Ad5 - MSLN - CRAD - luc是很有前景的递送载体。
