p53 selective adenoviral vector for transgene expression and replication as a therapeutic tool for virotherapy of p53 altered cancers

Aims: A major obstacle of adenoviral cancer therapy is the tight restriction of therapeutic gene expression or viral replication to malignant cells. This study presents the bicistronic adenoviral vector Adp53dpR for gene therapy of tumor cells with altered p53-status.

Methods/Results: First, a luciferase version of Adp53dpR-(Luc) was constructed including one cistron containing luciferase controlled by an artificial GAL4-binding CMV-promoter which can be efficiently inhibited by GAL4-KRAB. A second cistron contained GAL4-KRAB under control of an artificial p53 selective promoter (prMinRGC). prMinRGC showed strong upregulation by active p53 but was almost inactive in p53 absence. The bicistronic vector and a comparable control virus AdR(Luc) lacking the p53 dependent promoter was constructed. Infection experiments in HepG2 (Huh7 as control) followed by luciferase measurements showed that the luciferase expression in Adp53dpR-(Luc)-infected HepG2 was repressed 65fold compared to AdR(luc)-infected cells. This repression was enhanced up to 200fold when p53 was preactivated by Doxorubicin. The results were confirmed in a subset of p53 altered vs. non-altered cell lines. For the construction of a replication competent form of the vector, luciferase was replaced by the adenoviral E1A/E1B unit. In Doxorubicin treated HepG2 cells infected by Adp53dpR viral replication was inhibited compared to the control whereas AdR was not inhibited. In Huh7 cells both vector types showed an equivalent replication rate. Inverse relation between GAL4-KRAB and E1A expressed in Adp53dpR infected HepG2 cells was shown by western blot. The vector showed slightly reduced oncolytic properties compared to the Ad5wt but was more efficient and p53-selective compared to ONYX–015 in a subset of cell lines. Most important p53 selectivity of Adp53dpR was additionally confirmed in Doxorubicin treated primary human hepatocytes.

Discussion: Together the data suggest that Adp53dpR provides an effective tool for specific transgene expression in p53-altered cancer cells and might help to reduce deleterious side effects on healthy tissue if used for the application of therapeutic genes. Furthermore, the replicative variant is capable to replicate selectively in p53 altered cancer cells.

Key words

p53 adenovirus virotherapy repressor