MODELS OF IN VIVO MYOMETRIAL GENE TRANSFER

研究已经证明了将基因转移到 肌肉治疗遗传异常或作为体内抗原的一种方法 用于生产疫苗的表达。最近，基因转移已经被 使用疱疹病毒显示对血管平滑肌有效 胸苷激酶/更昔洛韦(HSV-tk/GCV)细胞毒基因治疗的研究进展 消融动脉壁损伤后的平滑肌增殖。这个 子宫平滑肌基因转移的潜在临床应用 包括改变子宫收缩行为的能力或作为一种新的 子宫平滑肌异常增生(平滑肌瘤)的治疗。 子宫肌瘤是发生在30%的女性子宫肌层的良性肿瘤 在它们的生殖年限内，这是最常见的迹象 子宫切除术。初步研究表明HSV-tk/GCV在体外具有细胞毒作用 基因治疗对人和ELT-3大鼠子宫肌瘤细胞非常有效 由于极高的旁观者效应而导致细胞死亡。我们还有 体内模型显示，当ELT-3HSV-tk稳定表达时，肌瘤 细胞系被用来制造肌瘤，旁观者被高度杀死 观察到的。PI假设通过注射直接转移质粒载体 HSV-tk基因导入肌瘤联合GCV治疗将诱导 子宫肌瘤在体内消退。具体目标是：一、确定子宫是否 血管平滑肌细胞对质粒基因治疗载体的转移敏感 并确定最佳的基因转移载体。肌瘤将会是 肩关节上腔内注射ELT-3细胞的实验研究 裸鼠。然后将质粒基因治疗载体注射到 子宫肌瘤结节。氯霉素编码基因的载体构建 乙酰转移酶和β-半乳糖苷酶基因将用于监测 并比较裸质粒DNA的转染率和转染率。 脂质体-复杂的DNA。II.确定直接注射质粒载体是否 HSV-tk基因联合GCV治疗诱导裸鼠肿瘤消退 无药动学分布证据的活体子宫肌瘤模型 注射部位以外的质粒载体。PI将注射HSV-TK 或对照质粒导入实验性子宫肌瘤并测定其对子宫肌瘤的影响 GCV治疗后肿瘤大小。将对远处的器官进行研究，以确定 肿瘤注射会导致质粒DNA的扩散。三、确定 HSV-tk/GCV基因治疗对小鼠体内“旁观者效应”的影响 并确定这一效应是否在发生的肿瘤中增加 在雌二醇环境中。稳定表达HSV-tk的细胞株(ELT-3-tk) 将被用来评估体内的旁观者效应。使用混合种群 不同比例稳定表达HSV-tk和野生型ELT-3细胞 在GCV治疗后，旁观者效应和连接蛋白-43水平将 在缺乏雌二醇的情况下发生的肿瘤中进行评估。一起， 这些研究将为未来提供体内疗效和安全性数据。 子宫肌层基因治疗的临床应用。

Studies have demonstrated the feasibility of transferring genes to muscle to treat genetic abnormalities or as a method of in vivo antigen expression for the production of vaccines. Recently, gene transfer has been shown to be effective in vascular smooth muscle using the herpes virus thymidine kinase/ganciclovir (HSV-tk/GCV) cytotoxic gene therapy approach to ablate smooth muscle proliferation following arterial wall injury. The potential clinical applications for gene transfer to uterine smooth muscle include the ability to alter contractile behavior of the uterus or as a novel treatment for abnormal uterine smooth muscle proliferation (leiomyomas). Leomyomas are benign tumors of the myometrium which occur in 30% of women during their reproductive years and represent the most common indication for hysterectomy. Preliminary studies have shown in vitro that HSV-tk/GCV cytotoxic gene therapy is very effective in human and ELT-3 rat leiomyoma cells in inducing cell death due to an extremely high bystander effect. We have also shown in an in vivo model that when a stable ELT-3 HSV-tk expression leiomyoma cell line was used to create leiomyomas, a high degree of bystander killing was observed. The PI hypothesize that direct plasmid vector transfer by injection of the HSV-tk gene into leiomyomas coupled with GCV treatment will induce leiomyoma regression in vivo. Specific aims are: I. To determine if uterine smooth muscle cells are susceptible to transfer of plasmid gene therapy vectors in vivo and determine the optimum vehicle for gene transfer. Leiomyomas will be experimentally induced by injection of ELT-3 cells into the suprascapular space of nude mice. Plasmid gene therapy vectors will then be injected into the leiomyoma nodules. Plasmid vectors encoding for chloramphenicol acetyltransferase and beta-galactosidase genes will be used to monitor transfection efficiency and to compare transfer of naked plasmid DNA vs. liposome-complexed DNA. II. To determine if direct plasmid vector injection of the HSV-tk gene followed with GCV treatment induces tumor regression in the in vivo leiomyoma model without evidence of distal pharmacokinetic distribution of the plasmid vector beyond the site of injection. The PI will inject the HSV-tk or control plasmid into experimental leiomyomas and determine the effect on tumor size after GCV treatment. Distant organs will be studied to determine if tumor injection results in dissemination of plasmid DNA. III. To determine the extent of the in vivo "bystander effect" of HSV-tk/GCV mediated gene therapy in leiomyomas and determine whether this effect is increased in tumors developed in an estradiol environment. A stable HSV-tk expressing cell line (ELT-3-tk) will be used to assess the bystander effect in vivo. Using mixed populations of stable HSV-tk expressing and wild type ELT-3 cells in various percentages followed by GCV treatment, the bystander effect and connexin-43 levels will be assessed in tumors developed in the presence of absence of estradiol. Together, these studies will provide in vivo efficacy and safety data for the future clinical application of gene therapy in uterine myometrium.