Suicidal gene therapy in an NF-κB-controlled tumor environment as monitored by a secreted blood reporter

Abstract

The nuclear factor-κB (NF-κB) is known to be activated in many cancer types including lung, ovarian, astrocytomas, melanoma, prostate as well as glioblastoma, and has been shown to correlate with disease progression. We have cloned a novel NF-κB-based reporter system (five tandem repeats of NF-κB responsive genomic element (NF; 14bp each)) to drive the expression cassette for both a fusion between the yeast cytosine deaminase and uracil phosphoribosyltransferase (CU) as a therapeutic gene and the secreted Gaussia luciferase (Gluc) as a blood reporter, separated by an internal ribosomal entry site (NF-CU-IGluc). We showed that malignant tumor cells have high expression of Gluc, which correlates to high activation of NF-κB. When NF-κB was further activated by tumor necrosis factor-α in these cells, we observed up to 10-fold increase in Gluc levels and therefore transgene expression in human glioma cells served to greatly enhance the sensitization of these cells to the prodrug, 5-fluorocytosine both in cultured cells and in vivo subcutaneous tumor xenograft model. This inducible system provides a tool to enhance the expression of imaging and therapeutic genes for cancer therapy.

Secreted blood reporters: Insights and applications

“…A reporter system was generated by cloning Gluc under the control of tandem repeats of NFκB responsive elements. This reporter demonstrated to be a highly sensitive for non-invasive continuous monitoring of the kinetics of NFκB activation and inhibition over time using blood or urine samples in mice (Badr et al, 2010a; Badr et al, 2009; Yang and Richmond, 2009). Recently, An apoptosis blood assay was generated by fusing GFP to the N-terminus of Gluc (including its signal sequence) separated by a short peptide consisting of aspartic acid, glutamic acid, valine, and aspartic acid (DEVD).…”

“…Gluc blood assay revealed virus reactivation 3 days after latent infection, preceding the detection of infectious virus by approximately 4 days (Marquardt et al, 2011). In another study, the efficiency of lentivirus infection and replication in tumors was monitored using the Gluc blood assay (Badr et al, 2010a; Wurdinger et al, 2008). …”

Bioluminescence imaging: progress and applications

“…In addition to the spatio-temporal evaluation of gene therapy, BLI allows the evaluation of viral tropism and transduction as well as viral replication [76, 77]. To evaluate the therapeutic response to suicidal gene therapy in a subcutaneous tumor model expressing Fluc, the therapeutic gene cytosine deaminase fused to uracil phosphoribosyltransferase [converts the nontoxic compound 5-fluorocytosine (5FC) into the drug 5-fluorouracil] was linked with Gluc through an internal ribosomal entry site, all under the control of the tumor-specific NFkB promoter[78]. Upon gene transfer into tumors, Gluc allows monitoring of the duration and magnitude of transgene expression.…”

Suicidal gene therapy in the effective control of primary human hepatocellular carcinoma as monitored by noninvasive bioimaging

“…All of these studies require co-expression of reporter gene with gene of interest. This can be achieved by two coordinately acting promoters, [10][11][12] gene fusion 13 and by including proteolytic cleavable sites such as fusagene or translational linkers such as the internal ribosome entry site (IRES) or self-processing peptides. 14 Over the last 10 years, the IRES element has been the most common way to express multiple genes in a single vector.…”