Safety Evaluation of Ad5CMY- p53 In Vitro and In Vivo

In preparation for a clinical trial of the recombinant p53 adenovirus Ad5CMV-p53 for the treatment of lung cancer, the potential adverse effects of Ad5CMV-p53 were assessed in vitro and in vivo. No infectious replication of Ad5CMV-p53 was detectable in HeLa cells infected with extracts from HeLa cells previously infected with Ad5CMV-p53. No Ad5CMV-p53 DNA replication was detected by ³²Pi labeling in lung cancer cells infected with Ad5CMV-p53 at multiplicities of infection (moi) up to 1,000 pfu/cell (total of 5 × 10⁹ pfu viruses). The infectivity and cytotoxicity of Ad5CMV-p53 were examined in vitro in normal human bronchial epithelial (NHBE) cells. At a moi of 50 pfu/cell, Ad5CMV-p53 infection and expression were detectable in 80% of the treated cells. The exogenous p53 protein was first detected by western blotting at 8 hr and peaked at 48 hr after infection. Growth of NHBE cells was not affected by Ad5CMV-p53 infection at a moi of 100 pfu/cell. The pathogenicity of Ad5CMV-p53 was assessed in BALB/c mice. The virus was given to four groups of mice by intratracheal injection at dosages from 10⁷ to 10¹⁰ pfu; a fifth group received phosphate-buffered saline alone. None of the viral injections proved to be lethal. Mild to moderate peribronchiolar and perivascular infiltration by mononuclear cells and lymphocytes, with patches of pneumonitis, was the most acute toxic effect detected by histologic analysis in the two high-dose groups. Immunohistochemical analysis of the same paraffin-embedded sections showed that infectivity and level of expression of p53 in lung tissue were dose-dependent. Our results demonstrate that Ad5CMV-p53 is a replication-defective virus that yields a relatively low degree of acute toxicity in mice; these data document a safety profile encouraging for clinical trials of Ad5CMV-p53 in the therapy of lung cancer.

Overview summary

Documentation of the safety of a recombinant virus is essential for approval of clinical trials of gene therapy. In preparation for clinical trials of adenovirus Ad5CMV-p53 in gene therapy of lung cancer, a set of in vitro and in vivo experiments was designed to assess the potential adverse effects of the virus on normal cells and lung tissue. These experiments described in this paper focused on clarifying (i) whether Ad5CMV-p53 will replicate in target cells; (ii) the degrees of infectivity and cytotoxicity Ad5CMV-p53 has in normal human bronchial epithelial cells; and (iii) whether Ad5CMV-p53 is pathogenic when intratracheally injected in mouse models. The results reported here demonstrate that Ad5CMV-p53 has an encouraging safety profile for clinical trials.