Late Expression of p53 from a Replicating Adenovirus Improves Tumor Cell Killing and Is More Tumor Cell Specific than Expression of the Adenoviral Death Protein

Abstract

Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.

Modification of the p53 transgene of a replication-competent adenovirus prevents mdm2- and E1b-55kD-mediated degradation of p53

“…An adenovirus expressing the modified p53 cDNA (Adp53W23S) was constructed using identical methods as described for Adp53. 21 Thus, Adp53 and Adp53W23S are identical except for amino acid 23 of the p53 transgene.…”

“…21 Adp53 is based on an E1-wild-type, E3-deleted, Ad5 viral backbone and has the p53 cDNA inserted into the fiber transcription unit, utilizing an internal ribosomal entry site. Ad-control has the same viral backbone without the p53 transgene.…”

“…21,35 Antibodies against p53 (Bp53-12 or DO-1) and actin (I-19) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and used at a dilution of 1:500 and 1:200, respectively. Antibodies against adenoviral E1a (M73) and p21 (6B6) were purchased from NeoMarker (Fremont, CA) and BD Biosciences (San Jose, CA), and used at 1:500 and 1:200 dilution, respectively.…”

“…Immunoblotting was then performed using antibodies against E1b-55kD and p53. 21 For the detection of p53-mdm2 complexes, 1 Â 10 7 cells were seeded in 15-cm plates. Lysate preparation and immunoprecipitation were carried out as above.…”

“…[18][19][20] To improve the efficacy of oncolytic adenoviruses, we have previously constructed a replication-competent adenoviral vector that expresses p53. 21 To prevent impairment of viral propagation, the virus was engineered to express p53 late in the viral life cycle. Improved oncolytic activity and viral spread were demonstrated with this construct without impairment of viral replication.…”