MODELS OF IN VIVO MYOMETRIAL GENE TRANSFER

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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.