Development and Characterization of Recombinant Adenoviruses Encoding Human p53 for Gene Therapy of Cancer

Abstract

We have constructed recombinant human adenoviruses that express wild-type human p53 under the control of either the Ad 2 major late promoter (MLP) or the human cytomegalovirus (CMV) immediate early gene promoter. Each construct replaces the Ad 5 E1a and E1b coding sequences necessary for viral replication with the p53 cDNA and MLP or CMV promoter. These p53/Ad recombinants are able to express p53 protein in a dose-dependent manner in infected human cancer cells. Tumor suppressor activity of the expressed p53 protein was assayed by several methods. [3H]Thymidine incorporation assays showed that the recombinant adenoviruses were capable of inhibiting DNA synthesis in a p53-specific, dose-dependent fashion. Ex vivo treatment of Saos-2 tumor cells, followed by injection of the treated cells into nude mice, led to complete tumor suppression using the MLP/p53 recombinant. Following a single injection of CMV/p53 recombinant adenovirus into the peritumoral space surrounding an in vivo established tumor derived from a human small cell lung carcinoma cell line (NIH-H69), we were able to detect p53 mRNA in the tumors at 2 and 7 days post-injection. Continued treatment of established H69 tumors with MLP/p53 recombinant led to reduced tumor growth and increased survival time compared to control treated animals. These results indicate that recombinant adenoviruses expressing wild-type p53 may be useful vectors for gene therapy of human cancer.

Comparison of high-capacity and first-generation adenoviral vector gene delivery to murine muscle in utero

“…18 The FG-Ad vector is a first-generation Ad vector with deletions of the E1 and E3 viral genes. 19 Both recombinant Ad vectors encode the Escherichia coli lacZ gene driven by the human CMV promoter. Each virus was titered by counting BFU after infection of 293 cells to limiting dilution as previously described.…”

CAR is a cell–cell adhesion protein in human cancer cells and is expressionally modulated by dexamethasone, TNFα, and TGFβ

“…For monitoring adenoviral transduction efficiency, a p53-(ACNp53) 30 or b-galactosidase-expressing adenovirus of serotype 5 (AdGal) 30 was employed. Cells were seeded at a cell density of 1 x 10 5 cells in 24-wells (1.9 cm 2 ) and incubated the following day for 2 h with different titers of AdGal or ACNp53 in serum-free DMEM at 371C.…”

Biological activity and safety of adenoviral vector-expressed wild-type p53 after intratumoral injection in melanoma and breast cancer patients with p53-overexpressing tumors

“…30 An adenoviral expression vector system was chosen because of its established safety in clinical trials and its organotropism, 34 -40 and because of its capability to effect wt p53 gene transfer in vitro and in preclinical animal models. 27,32 Unlike retroviruses, integration into the cell genome is not an obligate part of the adenoviral life cycle; rather, adenovirus DNA functions in the extrachromosomal portion of the cell's nucleus. 41 Thus, in comparison with retroviral vectors for gene delivery, this process has the therapeutic advantage of reducing the risk of insertional mutagenesis and reducing the chance that viral DNA will be active in cell progeny after cell division.…”

“…27,32,33 SCH 58500 was manufactured under current good manufacturing practice compliance in validated plant facilities under strict environmental monitoring and control conditions by Schering-Plow Werthenstein Chemie AG (Schachen, Switzerland) 17 and was supplied in vial strengths of 1 ϫ 10 8 plaque-forming units (PFU)/mL and 1 ϫ 10 9 PFU/mL (7.5 ϫ 10 10 particles/mL and 7.5 ϫ 10 11 particles/mL). All patients were treated once with a calculated total dose of SCH 58500 (based on tumor size) divided and administered as four injections of equal volume, with one injection given per tumor quadrant within the tumor at its perimeter.…”

“…32 In brief, total cellular RNA was isolated from homogenized tumor biopsies and reverse transcribed into cDNA. PCR was performed for 28 cycles on all samples.…”