Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has synergistic inhibitory effect on gastric cancer

Gastric cancer (GC) is one of the major causes of cancer related mortality. The use of oncolytic virus for cancer gene-virotherapy is a new approach for the treatment of human cancers. In this study, a novel Survivin promoter driven recombinant oncolytic adenovirus carrying mK5 or MnSOD gene was constructed, which was modified after deletion of E1B gene. Human plasminogen Kringle 5 mutant ( mK5 ) and manganese superoxide dismutase ( MnSOD ) are both potential tumor suppressor genes. To construct Ad-Surp- mK5 and Ad-Surp- MnSOD oncolytic adenovirus, we hypothesized that the combination of the two viruses would enhance the therapeutic efficacy of GC as compared to the virus alone. The results of the in vitro experiments revealed that the combination of adenovirus carrying mK5 and MnSOD gene exhibited stronger cytotoxicity to GC cell lines as compared to the virus alone, Additionally, the virus could selectively kill cancer cells and human somatic cells. Cell staining, flow cytometry and western blot analysis showed that the combination of two adenovirus containing therapeutic genes could promote the apoptosis of cancer cells. In vivo experiments further verified that Ad-Surp- mK5 in combination with Ad-Surp- MnSOD exhibited significant inhibitory effect on the growth of GC tumor xenograft as compared to the virus alone, and no significant difference was observed in the body weight of treatment and the normal mice. In conclusion, the combination of our two newly constructed recombinant oncolytic adenovirus containing mK5 or MnSOD therapeutic genes could significantly inhibit gastric cancer growth by inducing apoptosis, suggestive of its potential for GC therapy.

A Novel Branched DNA-Based Flowcytometric Method for Single-Cell Characterization of Gene Therapy Products and Expression of Therapeutic Genes

Many preclinical and clinical studies of hematopoietic stem cell-based gene therapy (GT) are based on the use of lentiviruses as the vector of choice. Assessment of the vector titer and transduction efficiency of the cell product is critical for these studies. Efficacy and safety of the modified cell product are commonly determined by assessing the vector copy number (VCN) using qPCR. However, this optimized and well-established method in the GT field is based on bulk population averages, which can lead to misinterpretation of the actual VCN per transduced cell. Therefore, we introduce here a single cell-based method that allows to unmask cellular heterogeneity in the GT product, even when antibodies are not available. We use Invitrogen’s flow cytometry-based PrimeFlow™ RNA Assay with customized probes to determine transduction efficiency of transgenes of interest, promoter strength, and the cellular heterogeneity of murine and human stem cells. The assay has good specificity and sensitivity to detect the transgenes, as shown by the high correlations between PrimeFlow™-positive cells and the VCN. Differences in promoter strengths can readily be detected by differences in percentages and fluorescence intensity. Hence, we show a customizable method that allows to determine the number of transduced cells and the actual VCN per transduced cell in a GT product. The assay is suitable for all therapeutic genes for which antibodies are not available or too cumbersome for routine flow cytometry. The method also allows co-staining of surface markers to analyze differential transduction efficiencies in subpopulations of target cells.

Strategies for Targeting Gene Therapy in Cancer Cells With Tumor-Specific Promoters

Cancer is the second cause of death worldwide, surpassed only by cardiovascular diseases, due to the lack of early diagnosis, and high relapse rate after conventional therapies. Chemotherapy inhibits the rapid growth of cancer cells, but it also affects normal cells with fast proliferation rate. Therefore, it is imperative to develop other safe and more effective treatment strategies, such as gene therapy, in order to significantly improve the survival rate and life expectancy of patients with cancer. The aim of gene therapy is to transfect a therapeutic gene into the host cells to express itself and cause a beneficial biological effect. However, the efficacy of the proposed strategies has been insufficient for delivering the full potential of gene therapy in the clinic. The type of delivery vehicle (viral or non viral) chosen depends on the desired specificity of the gene therapy. The first gene therapy trials were performed with therapeutic genes driven by viral promoters such as the CMV promoter, which induces non-specific toxicity in normal cells and tissues, in addition to cancer cells. The use of tumor-specific promoters over-expressed in the tumor, induces specific expression of therapeutic genes in a given tumor, increasing their localized activity. Several cancer- and/or tumor-specific promoters systems have been developed to target cancer cells. This review aims to provide up-to-date information concerning targeting gene therapy with cancer- and/or tumor-specific promoters including cancer suppressor genes, suicide genes, anti-tumor angiogenesis, gene silencing, and gene-editing technology, as well as the type of delivery vehicle employed. Gene therapy can be used to complement traditional therapies to provide more effective treatments.