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

我们发现，前列腺特异性抗原（PSA）基因的大约5.3 kb的启动子区域可以复制内源性表达模式在雄激素敏感的人前列腺癌LNCaP细胞。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因子GAL 4-VP 16融合蛋白，并反过来激活GAL 4-响应元件控制下的转基因表达。通过使用该系统，转基因表达可以大大增强，同时保持前列腺特异性表达。我们应用这个系统来驱动一个扩展的聚谷氨酰胺（前聚Q），一个强大的促凋亡分子，诱导PSA阳性前列腺癌细胞的凋亡选择性。为了在体内验证该系统，我们尝试构建了两个携带PSA启动子+GAL 4-VP 16或GAL 4-应答元件+ex-poly Q的腺病毒载体。为了建立基因治疗的动物模型，我们分离了稳定表达绿色荧光蛋白（GFP）基因的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，肺转移灶可以比以往报道的常规方法早得多地检测到（4周对12周）。因此，认为该系统可用作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.