Towards fibroid gene therapy: adenovirus-mediated delivery of herpes simplex virus 1 thymidine kinase gene/ganciclovir shrinks uterine leiomyoma in the Eker rat model.

BACKGROUND/AIMS: The objective of this study was to assess in vivo gene therapy of uterine leiomyomas in the Eker rat model using adenovirus (Ad)-mediated delivery of herpes simplex virus 1 thymidine kinase gene (HSV1TK) followed by ganciclovir (GCV) treatment. METHODS: We randomized 27 female Eker rats with MRI-confirmed uterine leiomyomas to a single treatment with direct intra-tumor injection of Ad-HSV1TK/GCV, Ad-LacZ/GCV, or medium alone. Samples were collected from tumors, other body organs, and blood at 10, 20, and 30 days after treatment to assess the safety and efficacy of the treatment. RESULTS: Ad-HSV1TK/GCV treatment significantly decreased uterine fibroid volume by 75 +/- 16, 58.7 +/- 6.3, and 67.5 +/- 27.5%, of the pretreatment volume at days 10, 20, and 30, respectively. Ad-HSV1TK/GCV increased caspase-3 activity, Bax expression, and TUNEL apoptosis marker, and it decreased cyclin D1, PCNA, Bcl2, and PARP protein expressions. Ad transfection induced local CD4+ and CD8+ infiltration and serum anti-Ad antibodies. Additionally, Ad transfection was tumor-localized and safe to non-target tissues. CONCLUSION: These studies demonstrate a marked efficiency and high safety for the Ad-HSV1TK/GCV therapeutic approach in the context of Eker rat uterine leiomyomas and provide essential preclinical data for the development of Ad-HSV1TK/GCV gene therapy for uterine fibroids.

Gene therapy of uterine leiomyoma: adenovirus-mediated herpes simplex virus thymidine kinase/ganciclovir treatment inhibits growth of human and rat leiomyoma cells in vitro and in a nude mouse model.

Uterine leiomyomas (LM) affect a high percentage of reproductive-age women. They develop as discrete, well-defined tumors that are easily accessible with imaging techniques--making this disease ideal for localized gene therapy approaches. In this study, we determined the efficacy of adenovirus-mediated herpes simplex virus thymidine kinase gene transfer in combination with ganciclovir (Ad-TK/GCV) as a potential therapy for LM. Rat ELT-3 LM cells and human LM cells were transfected with different multiplicity of infections (10-100 plaque forming units [PFU]/cell) of Ad-TK and treated with GCV (5, 10, or 20 microg/ml) for 5 days. To test the bystander effect, Ad-TK-transfected ELT-3 cells (100 PFU/cell) or LM cells (10 PFU/cell) were cocultured with corresponding nontransfected cells at increasing percentages and treated with GCV followed by cell counting. In ELT-3 cells transfected with Ad-TK/GCV (10, 20, 50, or 100 PFU/cell), the cell count was reduced by 24, 42, 77, and 87%, respectively, compared with the control cells (transfected with Ad-Lac Z/GCV). Similarly, in LM cells transfected with Ad-TK/GCV (10, 50, or 100 PFU/cell), the cell count was reduced by 31, 62, and 82%, respectively, compared with the control. A strong bystander effect was noted in both ELT-3 and LM cells with significant killing (p = 0.001) at a ratio of infected:uninfected cells of only 1:99 and maximal killing at 1:4. This study demonstrates the potential efficacy of the Ad-TK/GCV gene therapy approach as a viable nonsurgical alternative treatment for uterine LM.

Photochemically enhanced gene transfection increases the cytotoxicity of the herpes simplex virus thymidine kinase gene combined with ganciclovir.

Tumor targeting is an important issue in cancer gene therapy. We have developed a gene transfection method, based on light-inducible photochemical internalization (PCI) of a transgene, to improve gene delivery and expression selectively in illuminated areas, for example, in tumors. In the present work, we demonstrate that PCI improved the nonviral vector polyethylenimine (PEI)-mediated transfection of a therapeutic gene, the 'suicide' gene encoding herpes simplex virus thymidine kinase (HSVtk). In U87MG glioblastoma cells in vitro, the photochemical treatment stimulated expression of the HSVtk transgene, and, consequently, enhanced cell killing by the subsequent treatment with the prodrug ganciclovir (GCV). When relatively low doses of DNA (1 microg/ml) and the PEI vector (N/P 4) were used, HSVtk gene transfection followed by the GCV treatment did not have an effect on cell survival unless the photochemical treatment was performed, which potentiated the cytotoxicity to 90%. These findings indicate that photochemical transfection allows: (i) selective enhancement in gene expression and gene-mediated biological effects (cell killing by the Hsvtk/GCV approach) in response to illumination; (ii) the use of low, suboptimal for the nonviral transfection methods without PCI, doses of both DNA and the vector, which may be relevant and advantageous for therapeutic gene transfer in vivo.

Adenovirus-mediated tumor-specific combined gene therapy using Herpes simplex virus thymidine/ganciclovir system and murine interleukin-12 induces effective antitumor activity against medullary thyroid carcinoma.

The present treatment of advanced and metastatic medullary thyroid carcinoma (MTC) is unsatisfactory. Tissue-specific cancer gene therapy is a novel alternative approach. We developed a recombinant adenovirus expressing Herpes simplex type 1 thymidine kinase (HSVtk) driven by a modified CALC-I promoter TCP (AdTCPtk). Infection with this virus showed efficient cytotoxic effect on MTC cell lines (rMTC and TT cells) after treatment with ganciclovir (GCV) in vitro. In a syngenic WAG/Rij rat model, the combination of AdTCPtk/GCV treatment with administration of murine interleukin-12 (mIL-12) expressing adenovirus under control of TCP (AdTCPmIL-12) resulted in effective growth suppression of tumor at the treated site and also at a distant untreated site, in comparison to treatment with AdTCPtk/GCV or AdTCPmIL-12 alone. Moreover, intravenous injection of AdTCPtk, or AdTCPtk+AdTCPmIL-12, followed by administration of GCV, did not cause evident toxicity after administration of GCV. These results indicate that this combined system can provide an effective therapy for metastatic MTC with minimal toxicity.

Use of H19 regulatory sequences for targeted gene therapy in cancer.

We present a tumor gene therapy approach based on the use of regulatory sequences of the H19 gene that are differentially expressed between normal and cancer cells. We constructed expression vectors carrying the gene for the A fragment of diphtheria toxin (DT-A) or herpes simplex virus thymidine kinase (HSV-tk), under the control of a 814 bp 5'-flanking region of the H19 gene. The cell killing activity of these constructs was in accordance with the relative activity of the H19 regulatory sequences in the transfected cells. We evaluated the therapeutic potential of the gene expression constructs driven by H19 regulatory sequences in an animal model of bladder cancer induced by subcutaneous injection of syngeneic bladder tumor cell lines. Intratumoral injection of these constructs caused a significant suppression of subcutaneous tumor growth, with no obvious toxicity toward the host.

In situ gene transfer and suicide gene therapy of gastric cancer induced by N-ethyl-N'-nitro-N-nitrosoguanidine in dogs.

Gene therapy could potentially revolutionize the treatment of gastrointestinal (GI) tract cancer. The aim of this study was to establish a practical method of gene transfer which would be applicable to human gastric cancer. Retrovirus or/and adenovirus vectors carrying the lacZ marker gene were transferred in situ by needle through an endoscopic biopsy channel into primary gastric cancer in six male beagle dogs that had been treated with N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG). In addition, an adenovirus vector carrying the herpes simplex virus thymidine kinase (Ad.CAGHSV-TK) gene was introduced in situ into cancer tissues in the stomach of three dogs, and the animals were treated with intravenous ganciclovir (GCV). Retrovirus-producing cells which expressed the lacZ gene were specifically localized to the injection site in the stomach. The lacZ gene was more widely transferred into the tumor by the adenovirus vector than by retrovirus-producing cells. Improvement of the needle used for gene transfer and the use of multiple injections per tumor led to more diffuse transfer of the vector into the tumor. The Ad.CAGlacZ gene was also transferred into regional lymph nodes of the stomach. Moderate to diffuse degeneration of the primary cancer tissues of the stomach was found after Ad.CAGHSV-TK/GCV gene therapy. Moreover, almost complete tissue degeneration was observed in the regional lymph nodes of the stomach. An adverse effect of HSV-TK/GCV gene therapy was acute hepatotoxicity, which was not found after Ad.CAGlacZ gene transfer, but was found after high-titer Ad.CAGHSV-TK gene transfer followed by GCV. These findings suggest that in situ gene transfer of a suicide gene followed by prodrug treatment may be applicable not only to primary tumors, but also to lymph node metastases of gastric cancer, though further study of both beneficial and adverse effects is required before clinical usage.