Transcriptional targeting of gene expression in breast cancer by the promoters of protein regulator of cytokinesis 1 and ribonuclease reductase 2.

For cancer gene therapy, cancer-specific over- expression of a therapeutic gene is required to reduce side effects derived from expression of the gene in normal cells. To develop such an expression vector, we searched for genes over-expressed and/or specifically expressed in cancer cells using bioinformatics and have selected genes coding for protein regulator of cytokinesis 1 (PRC1) and ribonuclease reductase 2 (RRM2) as candidates. Their cancer-specific expressions were confirmed in both breast cancer cell lines and patient tissues. We compared each promoter's cancer-specific activity in the breast normal and cancer cell lines using the luciferase gene as a reporter and confirmed cancer-specific expression of both PRC1 and RRM2 promoters. To test activities of these promoters in viral vectors, the promoters were also cloned into an adeno-associated viral (AAV) vector containing green fluorescence protein (GFP) as the reporter. The GFP expression levels by these promoters were various depending on cell lines tested and, in MDA-MB-231 cells, GFP activities derived from the PRC1 and RRM2 promoters were as strong as that from the cytomegalovirus (CMV) promoter. Our result showed that a vector containing the PRC1 or RRM2 promoter could be used for breast cancer specific overexpression in gene therapy.

Cancer gene therapy using adeno-associated virus vectors.

Gene therapy has offered highly possible promises for treatment of cancers, as many potential therapeutic genes involved in regulation of molecular processes may be introduced by gene transfer, which can arrest angiogenesis, tumor growth, invasion, metastasis, and/or can stimulate the immune response against tumors. Therefore, viral and non-viral gene delivery systems have been developed to establish an ideal delivery vector for cancer gene therapy over the past several years. Among the currently developed virus vectors, the adeno-associated virus (AAV) vector is considered as one of those that are closest to the ideal vector mainly for genetic diseases due to the following prominent features; the lack of pathogenicity and toxicity, ability to infect dividing and non-dividing cells of various tissue origins, a very low host immune response and long-term expression. Particularly, the most important attribute of AAV vectors is their safety profile in clinical trials ranging from CF to Parkinson's disease. Although adenovirus and several other oncolytic viruses have been more frequently used to develop cancer gene therapy, AAV also has many critical properties to be exploited for a cancer gene delivery vector. In this review, we will briefly summarize the basic biology of AAV and then mainly focus on recent progresses on AAV vector development and AAV-mediated therapeutic vectors for cancer gene therapy.

A novel way of therapeutic angiogenesis using an adeno-associated virus-mediated angiogenin gene transfer.

To develop a novel therapeutic angiogenesis for the treatment of cardiovascular diseases, angiogenin (ANG1) was examined as a potential therapeutic gene. An adeno-associated virus (AAV)-mediated gene delivery system was used to measure the therapeutic efficacy of ANG1. Using a triple co-transfection technique, rAAV-ANG1-GFP, rAAV- VEGF-GFP and rAAV-GFP vectors were produced, which were then used to infect human umbilical vein endothelial cells (HUVECs) in order to evaluate in vitro angiogenic activities. Their protein expressions, tagged with green fluorescent protein (GFP), were monitored by confocal microscopy. The functional activities were measured using wound- healing HUVEC migration assays. The number of migrated cells stimulated by both the expressed ANG1 and the VEGF in rAAV-infected HUVECs increased almost twice the number observed in the expressed GFP control. In vivo angiogenic activities of the expressed ANG1 or VEGF were determined using mouse angiogenesis assays. The angiogenic activities of ANG1 or VEGF expressed in the injected mice were increased by 1.36 and 2.16 times, respectively, compared to those of the expressed GFP control. These results demonstrate that the expressed ANG1 derived from rAAV infection has in vitro and in vivo angiogenic activities and suggest that the rAAV-ANG1 vector is a potential strategy for therapeutic angiogenesis.