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Use of Hypoxia- and Radiation-activated Cre/loxP Switch Vectors for Gene Therapy of Breast Cancer
Tumor hypoxia has long been recognized as a critical issue in the treatment of breast cancer. Resistance of hypoxic areas affects the outcome after radiotherapy, chemotherapy and surgery. However, despite being an adverse prognostic factor, hypoxia represents a physiological difference that can be exploited for selective cancer therapy. In particular, hypoxia-targeted gene therapy exploits the cellular response to low oxygen concentration to activate therapeutic gene expression within the tumor mass. In the proposed project, hypoxia-regulated gene therapy will be combined with radiation therapy. Ionizing radiation (IR) will be exploited not only to directly kill tumor cells, but also to further activate selective transgene expression via novel IR-sensitive promoters that we have included in our gene therapy constructs.
The central hypothesis of this study is that hypoxia- and IR-activated ‘molecular switch’ vectors expressing the herpes simplex virus thymidine kinase (HSVtk) suicide gene can be used to improve radiation treatment of breast cancer. DNA constructs containing gene promoters inducible by hypoxia and/or IR will be delivered to the tumor. In the presence of the stimuli of either hypoxia (within the tumor mass), or IR (externally targeted to the tumor), or both, these promoters activate the expression of the therapeutic gene HSVtk. HSVtk is non-toxic per se, but is able to convert the prodrug ganciclovir (GCV) into a cytotoxin (suicide gene therapy). HSVtk/GCV gene therapy has been shown to kill transfected as well as neighboring non-transfected tumor cells (bystander effect), resulting in tumor eradication even if only a fraction of the target cells are genetically modified. To achieve tight and specific gene regulation and produce high levels of therapeutic gene product, a transgene expression amplification system based on Cre/loxP recombination will be employed. This ‘molecular switch’ permits gene expression from a strong secondary promoter following activation of a primary hypoxia- and IR-activated promoter. To deliver the switch vectors, modified adenoviral vehicles, well characterized and extensively used in pre-clinical and clinical studies, will be used.
The specific aims of the project are to:
1) Investigate whether the growth of suicide switch vector-modified breast cancer cells can be selectively and efficiently controlled in vivo after radiation, using human tumor xenograft models;
2) Construct adenoviral vectors for the delivery of the hypoxia- and IR-activated ‘molecular switch’ vectors and test them in vitro;
3) Test the efficacy of adenoviral delivery in vivo to wild-type human tumor xenografts, and measure the efficiency of radiation- and gene therapy-induced tumor growth delay.
The efficacy of the hypoxia- and IR-activated ‘molecular switch’ vectors for cancer gene therapy will be tested in human breast adenocarcinoma MCF-7 cells. For Specific Aim 1, vector-modified cell lines will be established and grown as xenografts in nude mice. Tumor growth delay will be measured after delivery of the prodrug GCV and radiation. Tumor biopsies will be analyzed for transgene expression and hypoxia. This study will demonstrate the efficacy of our gene therapy system in an animal model. For efficient gene delivery in a clinical setting, adenoviral vectors will be constructed and tested (Specific Aims 2 and 3). Experiments in vitro and in vivo in wild-type MCF-7 cells will determine the applicability of such a gene therapy system and assess dosing and timing of the combinational therapy in a pre-clinical model.
Addressing the problem of tumor hypoxia may significantly improve the radiation treatment of breast cancer. In particular, a combinational approach, exploiting hypoxia- and IR-responsive promoters within a ‘molecular switch’ vector could overcome some of the limitations associated with radiotherapy regimes and specifically target the tumor areas that are refractive to treatment. In particular, the efficacy of each radiation dose fraction will be increased, allowing a reduction of the total dose to be delivered to the patient. Since gene activation would be controlled and restricted to hypoxic and/or irradiated cells, damage to surrounding normal tissue would be limited, thereby improving the therapeutic ratio. This should result in increased local control and survival, with reduced normal tissue damage and improved quality of life.
Radiation therapy is one of the most common treatment modalities for breast cancer. Unfortunately, some tumors do not fully respond to therapy, resulting in patient relapse and reduced survival. One of the reasons of tumor resistance to treatment is the presence of areas in the tumor at low oxygen concentration. Oxygen concentration below normal physiological levels, commonly referred to as hypoxia, has been shown to be one of the most significant adverse factors affecting the response to radiotherapy, chemotherapy and surgery. On the other hand, severe hypoxia does not occur in healthy, non-cancerous tissues and so this differential can be utilized to selectively kill cancer cells. A novel approach to target hypoxic tumor cells is hypoxia-activated ‘suicide’ gene therapy. Foreign DNA is delivered to the tumor, but only hypoxic cells will process it and produce a protein that will cause their death. This selectivity can be achieved by using gene regulators or ‘promoters’ (DNA sequences that control gene expression) activated only under hypoxic conditions. To optimize tumor response, we intend here to use such a gene therapy protocol in conjunction with standard radiation treatment. Furthermore, to take maximum advantage of the selectivity provided by both stimuli, we will employ novel gene promoters that respond to hypoxia and are also stimulated by radiation. In the presence of either hypoxia (within the tumor mass), or radiation (externally targeted to the tumor during treatment), or both, these promoters will activate the expression of a therapeutic ‘suicide’ gene (which kills the cells that express it). Normal tissues, which are neither hypoxic, nor irradiated, will remain unaffected. Moreover, gene therapy vectors able to further amplify the expression of the therapeutic gene and increase tumor cell kill will be tested.
In the proposed study, breast cancer cells containing the gene therapy vectors will be grown as tumors in an animal model and pre-clinical studies will be conducted. Subsequently, for future clinical applications, safe, non-pathogenic viruses containing the gene therapy vectors will be constructed and tested. Modified adenoviruses have been extensively used in clinical trials for gene therapy and are the most efficient delivery system available.
By combining radiotherapy with such a gene therapy protocol, tumor cells that are resistant to radiotherapy will be eradicated. This will result in increased local tumor control and therefore survival. Furthermore, by increasing the efficacy of each dose of radiation, it will be possible to actually reduce the total amount of radiation needed to cure the cancer. Consequently, long-term side effects of breast radiotherapy such as fibrosis, lymphedema, telangiectasia and impaired shoulder movement would be alleviated, with a significant improvement of quality of life for the patients.