MODELS OF IN VIVO MYOMETRIAL GENE TRANSFER

研究已经证明了将基因转移到 肌肉以治疗遗传异常或作为体内抗原的方法 用于疫苗生产的表达。最近，基因转移已经被 在血管平滑肌中使用疱疹病毒 胸苷激酶/更昔洛韦（HSV-tk/GCV）细胞毒性基因治疗方法 消融动脉壁损伤后的平滑肌增殖。的 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，或者作为一种新颖的 治疗异常子宫平滑肌增生（平滑肌瘤）。 子宫肌瘤是子宫肌层的良性肿瘤，发生在30%的女性中 在他们的生育年龄，并代表最常见的迹象， 子宫切除术初步研究表明，在体外HSV-tk/GCV细胞毒性， 基因治疗在人和ELT-3大鼠平滑肌瘤细胞中非常有效， 由于极高的旁观者效应而诱导细胞死亡。我们还 在体内模型中显示，当稳定ELT-3 HSV-tk表达平滑肌瘤 细胞系用于产生平滑肌瘤， 观察PI假设通过注射直接质粒载体转移 将HSV-tk基因导入平滑肌瘤，再加上GCV治疗， 体内平滑肌瘤消退。具体目标是：一。以确定子宫是否 平滑肌细胞对质粒基因治疗载体的转移敏感 并确定用于基因转移的最佳载体。平滑肌瘤将是 通过将ELT-3细胞注射到肩胛上间隙中实验诱导 裸小鼠然后将质粒基因治疗载体注射到 平滑肌瘤结节。编码氯霉素的质粒载体 乙酰转移酶和β-半乳糖苷酶基因将用于监测 转染效率并比较裸质粒DNA的转移与 脂质体复合DNA。二.为了确定是否直接质粒载体注射 HSV-tk基因后用GCV治疗可诱导小鼠肿瘤消退。 体内平滑肌瘤模型没有证据表明远端药代动力学分布 质粒载体超出注射部位。PI将注射HSV-tk 或对照质粒导入实验性平滑肌瘤中， GCV治疗后的肿瘤大小。将研究远距离器官，以确定 肿瘤注射导致质粒DNA的播散。三.确定 HSV-tk/GCV介导的基因治疗的体内“旁观者效应”的程度 平滑肌瘤，并确定这种影响是否增加肿瘤发展 在雌二醇环境中。一种稳定表达HSV-tk的细胞系（ELT-3-tk） 将用于评估体内旁观者效应。使用混合种群 稳定的HSV-tk表达细胞和野生型ELT-3细胞 GCV治疗后，旁观者效应和连接蛋白-43水平将 在存在或不存在雌二醇的情况下发展的肿瘤中评估。在一起， 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.