Hedi Haddada

Highly Efficient Adenovirus-Mediated Gene Transfer to Cardiac Myocytes after Single-Pass Coronary Delivery

Efficient and homogeneous gene transfer to cardiac myocytes is a major target in myocardial gene therapy. The aim of this study was to determine the conditions permitting efficient, homogeneous, adenovirus-mediated gene transfer to cardiac myocytes, with a view to application during coronary artery catheterization. Gene transfer to adult rat ventricular myocytes was conducted using type 5 adenoviruses carrying the lacZ reporter gene. Adenovirus delivery via coronary arteries was performed on isolated perfused rat hearts, and gene transfer efficiency was analyzed on whole ventricles, freshly isolated myocytes, and cultured myocytes. Single-pass delivery of 1 X 10(9) PFU associated with 1 min of no-flow yielded only 1 +/- 0.5% of positive myocytes. Pretreatment by histamine perfusion (10(-5) M final concentration) increased this value to 30 +/- 9% (p < 0.001), and pretreatment by Ca2+-free buffer perfusion increased it to 67 +/- 8% (p < 0.001). Combination of the two pretreatments had no additional effect. Increasing the viral dose to 3 X 10(9) PFU increased transfection efficiency only in permeabilized vessels. The 1-min no-flow period after adenovirus delivery was crucial for efficient gene transfer: despite histamine pretreatment, only 2 +/- 1% positive myocytes were observed without flow interruption (p < 0.05 versus 1 min of no-flow). Gene transfer was shown to occur in situ during cardiac perfusion, rather than during heart digestion or myocyte isolation. This study shows that highly efficient adenovirus-mediated gene transfer to cardiac myocytes in situ can be achieved by single-pass intracoronary vector delivery, provided that vascular permeability is first increased and coronary flow is briefly interrupted.

Differential response of p53 target genes to p73 overexpression in SH-SY5Y neuroblastoma cell line

p73, the first p53 gene homologue, encodes an array of p73 proteins including p73α full-length (TAp73α) and amino-truncated isoforms (ΔNp73α), two proteins with opposite biological functions. TAp73α can induce tumor suppressive properties, while ΔNp73α antagonizes p53 as well as TAp73 in a dominant-negative manner. In human malignant neuroblasts, p53 protein is wild-type but known to be excluded from the nucleus, therefore disabling its function as a tumor suppressor. The present study investigates whether there is a functional link between p73 isoforms and p53 in neuroblastoma. Experiments were performed on two neuroblastoma cell lines differing in their p53 status, e.g. wild-type p53 SH-5Y5Y cells and mutated p53 IGR-N-91 cells. Data indicate that (i) both TA- and ΔN-p73α enhance p53 protein level in SH-SY5Y cells, whereas level remains unchanged in IGR-N-91 cells; (ii) only in SH-SY5Y cells does forced TAp73α overexpression markedly induce nuclear accumulation of p53 protein; (iii) p21 protein expression is increased in both cell lines infected with TAp73, suggesting that, in IGR-N-91 cells, p21 is induced by p73 through a p53-independent pathway; (iv) in the SHSY5Y cell line, Btg2 expression is strongly enhanced in cells overexpressing TA, and to a lesser extent in cells overexpressing ΔN. Taken together our results suggest that TAp73 may restore p53 function in NB with wild-type nonfunctional p53, but not in NB with mutated p53.

Regression of AK7 malignant mesothelioma established in immunocompetent mice following intratumoral gene transfer of interferon gamma

Malignant mesothelioma (MM) is a lethal tumor linked with a prior exposure to asbestos in which limited progress has been made so far using conventional therapies. MM is an example of a “nonimmunogenic” tumor characterized by a fibrous stroma and an absence of infiltrating T lymphocytes. High levels of transforming growth factor-beta (TGF-β) produced by mesothelioma cells have been related to the immune tolerance towards the tumor. In order to evaluate the effect of local delivery of cytokines such as interferon gamma (IFN-γ) by gene transfer, we characterized and used a murine model, AK7, which appeared very similar to human mesothelioma. AK7 cells expressed low levels of major histocompatibility class I and class II antigens and secreted high levels of latent TGF-β. The TGF-β pathway in AK7 cells is operative but inefficient because endogenous TGF-β is predominantly inactive. Treatment of pre-established AK7 tumors by direct intratumoral injection of an adenovirus vector expressing murine IFN-γ, Ad.mIFN-γ, led to significant tumor regression. Peripheral tumor infiltration by CD4+ and CD8+ T lymphocytes in the treated tumors appeared to be because of the induction of an immune response. Tumor relapse was observed, which could be due to local TGF-β secretion by remaining tumor cells.