A novel gene transfer method for prostate-specific and potent expression and its utilization in gene therapy

我们发现一个大约5.3 kb的前列腺特异性抗原（PSA）基因启动子区域可以在雄激素敏感的人前列腺癌LNCaP细胞中复制内源性表达模式。然后，我们开发了一种新的两步转录激活系统（TSTA系统），其中PSA启动子驱动人工转录激活子GAL4-VP16融合蛋白，然后在gal4响应元件的控制下激活转基因表达。通过使用该系统，可以在保持前列腺特异性表达的同时大大增加转基因表达。我们应用该系统驱动扩展的聚谷氨酰胺（前聚q），一种有效的促凋亡分子，选择性地诱导psa阳性前列腺癌细胞凋亡。为了在体内证实这一系统，我们试图构建两种携带psa -启动子加GAL4-VP16或gal4 -响应元件加前聚q的腺病毒载体。然而，尚未分离到转染psa -启动子加GAL4-VP16的腺病毒。为了建立评估基因治疗效果的动物模型，我们分离了绿色荧光蛋白（GFP）基因稳定表达的LNCaP细胞。当我们将这种LNCaP-GFP细胞原位接种到SCID小鼠的前列腺中时，可以比以往报道的常规方法更早地检测到肺转移灶（4周比12周）。因此，该系统被认为是用于评估抗转移治疗的触觉体内模型。

We showed that an approximately 5.3-kb promoter region of the prostate-specific antigen (PSA) gene can replicate the endogenous expression pattern in an androgen-sensitive human prostate cancer LNCaP cells. We then developed a novel two-step transcriptional activation system (TSTA system) in which the PSA promoter drives an artificial transcriptional activator, GAL4-VP16 fusion protein, and it in turn activates transgene expressions under the control of GAL4-responsive element. By using this system, transgene expressions can be greatly augmented while maintaining prostate-specific expression. We applied this system to drive an expanded polyglutamine (ex-poly-Q), a potent proapoptotic molecule, to induce apoptosis selectively in PSA-positive prostate cancer cells. In order to confirm this system in vivo, we tried to construct two adenovirus vectors carrying PSA-promoter plus GAL4-VP16 or GAL4-resposive element plus ex-poly Q. We have, however, not yet isolated the adenovirus in which PSA-promoter plus GAL4-VP16 is transfected.To-establish an animal model for the assessment of gene therapy, we isolated LNCaP cells that showed stable expression of green fluorescence protein(GFP) gene. When we inoculated this LNCaP-GFP cells orthotopically into the prostate of SCID mice, pulmonary metastatic foci could be detected much earlier than the conventional method that ever reported (4 weeks vs. 12 weeks). This system thus is considered to be utilized as a tactile in vivo model for the assessment of anti-metastatic therapy.