Safety Evaluation of Ad5CMY-p53 In VitroandIn Vivo

Abstract

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 x 10(9) 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 10(7) to 10(10) 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.

Superinduction of wild-type p53 protein after 2-methoxyestradiol treatment of Ad5p53-transduced cells induces tumor cell apoptosis

“…The MOI was de®ned as the ratio of the total number of plaque-forming units used in a particular infection to the total number of cells to be infected. Adenovirus preparations were free of replication-competent adenovirus as determined by previously described techniques (Zhang et al, 1995). Cell growth was measured in untreated controls, in cells treated with 5 mM 2-MeOE 2 , and in cells infected with Ad5p53.…”

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

“…2 In preclinical studies, many neoplastic cell types have been shown to be sensitive to p53 gene transfer, whereas normal cells appear largely unaffected. [3][4][5][6][7][8][9] In addition, several clinical trials with replication-deficient adenoviruses that express p53 have demonstrated an excellent safety profile. [10][11][12][13][14][15] Recently, such a vector has been approved for clinical application in China and several phase 3 trials are ongoing in the United States.…”

Development and application of adenoviral vectors for gene therapy of cancer

“…60 Biological assays for the detection of RCA were designed to detect the replication capability of rAd at the viral DNA level as well as virus. 59 The labeling of viral DNA with 32 P or consecutive infections with cell extracts in HeLa cells was used to determine RCA potential or the contamination of a given preparation of Ad-p53 vector. An additional development involved the application of A549 cells to determine RCA through a consecutive infection process using cell extracts that were initially infected with samples of the Ad vectors being tested.…”