Tumor-specific adenoviral gene therapy: transcriptional repression of gene expression by utilizing p53-signal transduction pathways

Abstract

Adenoviral gene expression that is repressed by p53 in nontransformed cells could provide a tumor-specific gene therapy approach for a large subset of tumors. Adenoviral infection in vivo induces stabilization of p53, which can be utilized for a strategy that includes p53-dependent expression of a transcriptional repressor and a target promoter ,which is highly susceptible for transcriptional repression. Therefore, we constructed different versions of CMV-promoters (CMV gal ) with binding sites for GAL4-DBD and investigated 11 GAL4-DBD fusion proteins to elucidate the most effective repressor domain to silence CMV gal activity.The transcriptional repressor GAL4-KRAB-A under control of a p53-dependent promoter facilitates strong CMV gal -mediated gene expression specifically in p53 mutant cells by a double-recombinant adenoviral vector (Ad-RGCdR). GAL4-KRAB-A mediates strong transcriptional repression of Ad-RGCdR in p53 wild-type cells, which could be further enhanced by preactivation of p53-signalling following low-dose chemotherapy prior to adenoviral infection. By utilizing p53 signalling involved in chemotherapy and adenoviral infection, more than 99% of Ad-RGCdR gene expression could be repressed in p53 wild-type cells. Controlled gene expression from CMV gal promoters by transcriptional repression utilizing functional p53 signalling thus provides a very effective tool for tumor-specific adenoviral gene therapy.

p53-dependent antiviral RNA-interference facilitates tumor-selective viral replication

“…To allow for p53-selective expression of shRNA we chose the artificial, p53-responsive promoter prMinRGC that we have described previously (18). As shown in Figure 1C, this promoter is highly active in p53-normal cells but silent in p53-dysfunctional cells.…”

“…However, fibroblasts are highly resistant against adenoviral transduction. For efficient transduction at low MOI, viral particles were pretreated with the bispecific CAR ex -Tat as described before (18). This allows for efficient cell entry and subsequent monitoring of replication for at least one cycle.…”

“…Expression vectors for miR30-shRNA-motifs were constructed using pBluescript provided with synthetic polylinker, a SV40-polyadenylation signal derived from AdApt (Introgene), and a promoter, such as the wildtype CMV-promoter (from pBK-CMV, Stratagene), U6-promoter (pSuperRetro, Addgene), or the p53-responsive promotor prMinRGC from prMinRGC-Luc (18). For the generation of miR30-shRNA-motifs, two partially overlapping oligonucleotides were annealed and overhangs were then filled-in using Herculase (Stratagene).…”

Targeting of P53-transcriptional dysfunction by conditionally replicating adenovirus is not limited by P53-homologues

“…CMVgal consists of a 474 base-pair long CMV-promoter sequence equipped with internal and flanking clusters of Gal4-binding sites to attract Gal4-KRAB as described previously. 12 This genetic setup allows for inhibition of E1A expression under nonpermissive conditions and for high E1A levels under permissive conditions (Figure 1e). The CMVgal-promoter shows a higher transcriptional activity compared to the wild-type E1A-promoter, but is susceptible for efficient transcriptional repression by Gal4-KRAB (Figure 1f).…”

“…12 For construction of the plasmid prE1A-Luc, the Ad5-E1A-promoter (pos. 2-558) was PCR-amplified from adenoviral DNA (primers: 5′-CC GAGCTCATCAATAATATACCTTATTTTGGATTGAAGCC-3′ and 5′-GTAAGCTTCAGTCCCGGTGTCGGAGCGG-3′) and cloned into pGL2-Basic (Promega, Mannheim, Germany).…”