TP53 is the most commonly altered tumor-suppressor gene in cancer and is currently being tested in Phase II/III gene replacement trials. Many tumors contain wild-type TP53 sequence with elevated MDM2 protein levels, targeting p53 for degradation. These tumors are more refracto-ry to treatment with exogenous wild-type p53. Here we generate a recombinant adenovirus expressing a p53 variant, rAd-p53 (d 13–19), that is deleted for the amino acid sequence neces-sary for MDM2 binding (amino acids 13–19). We compared the apoptotic activity of rAd-p53 (d 13–19) with that of a recombinant adenovirus expressing wild-type p53 (rAd-p53) in cell lines that differ in endogenous p53 status. rAd-p53 (d 13–19) caused higher levels of apoptosis in p53 wild-type tumor lines compared with wild-type p53 treatment, as measured by annexin V-FITC staining. In p53-altered tumor lines, rAd-p53 (d 13–19) showed apoptotic activity similar to that seen with wild-type p53 treatment. In normal cells, no increase in cytopathicity was detected with rAd-p53 (d 13ndash;19) compared with wild-type p53 treatment. This variant protein displayed synergy with chemotherapeutic agents to inhibit proliferation of ovarian and breast cell lines. The p53 variant showed greater antitumor activity in an established p53 wild-type tumor com-pared with treatment with wild-type p53. The p53 variant represents a means of expanding TP53 gene therapy to tumors that are resistant to p53 treatment due to the cellular responses to wild-type p53.
Understanding where genes are being expressed in normal, wounded and wound impaired skin, along with the duration of expression after transduction by recombinant adenovirus (rAd) is an important factor relevant to gene therapy strategies. Recombinant adenoviral vector encoding green fluorescent protein (rAd-GFP), or platelet-derived growth factor-B (rAd-PDGF-B), was intradermally or topically administrated into either mouse or rabbit non-ischemic or ischemic ear skin. Quantitative characterization of GFP expression with or without wounds was performed in these animal models. Mice and rabbits received 3x1010 and 3|[times]|109 viral particles of rAd-GFP per injection site or wound, respectively. We observed reproducible differences in gene expression profiles in wounded and wound impaired skin when compared to normal skin. GFP expression was visible from 1-7 days post intradermal injection, with a decrease in visual expression after day 5 in normal skin of mice and rabbits. Gene expression was extended by 1 day in the mouse skin punch model. In the rabbit ischemic ear model, the onset of rAd-GFP expression was delayed 1-3 days as compared with non-ischemic conditions. Interestingly, GFP expression was visibly brighter, covered a larger area and lasted approximately 1.4-fold (no wounds) or 4-fold longer (with wounds) in the ischemic ear when compared with non-ischemic conditions after intradermal or topical delivery, respectively. Quantitative analyses of normal mouse skin tissue and rabbit ischemic ear wound bed tissue by PCR and RT-PCR confirmed the presence of rAd-PDGF-B RNA out to 14 and 21 days, respectively. In summary, increased gene expression, larger visual area and longer duration of gene expression under hypoxic wound environments may be beneficial for gene therapy in chronic wound healing.
