Coexpression of p21WAF1/CIP1 in adenovirus vector transfected human primary hepatocytes prevents apoptosis resulting in improved transgene expression

Abstract

Replication-deficient adenovirus (Ad vector) is one of the most effective gene transfer systems. However, its employment in human gene therapy trials is hampered by Ad vector associated cytotoxicity and induction of apoptosis of the infected cells. Here, we identify one underlying mechanism as uncoupling of S phase and mitosis of the cell cycle leading to apoptosis and decline of transgene expression. Moreover, we demonstrate a strategy to avoid Ad vector associated cytotoxicity and induction of apoptosis in human primary hepatocytes by coinfection of Ad vector carrying the cDNA of choice and the cell cycle regulator p21 WAF1/CIP1 (p21). In addition, animal experiments were performed using Ad vector directed coexpression of p21 and human a 1-antitrypsin. As serum analysis of a 1-antitrypsin after Ad vector mediated gene transfer to the liver of mice revealed, this strategy resulted also in the improvement of transgene expression by two orders of magnitude. These data suggest that coexpression of p21 and Ad vector carrying a therapeutic gene may be a promising strategy to avoid cytotoxicity and induction of apoptosis leading to improved safety in human gene therapy.

Co-expression of p21Waf1/Cip1 in adenovirus vectors improves expression of a second transgene

“…As NFkB is one of the transcription factors identified to be activated by p21 and as the RSV promoter driving a 1 -AT expression contains, besides C/ EBP and YY1 binding sites, RelA/NFkB responsive elements, it appears likely that the observed overexpression occurs through this mechanism. 23,24 This view is supported by the fact that the co-expression of p21 had an equally positive effect on RSV-and CMV-driven bgalactosidase expression (see Figure 5 in Wolff et al 5 ) and CMV has been proven to be activated by p21 18 . Further backing is provided by the varying efficiency in the a 1 -AT expression in HeLa and A549 despite similar p21 expressions (see Figure 1).…”

“…These data corroborate and extend earlier results, in which co-infection of an Ad vector expressing p21 with a second vector coding for a gene of interest augmented the expression from this second Ad vector. 5 In that study, the beneficial effect of p21 was ascribed to its function as a cell cycle regulator that counteracts Ad vector-mediated cell cycle dysregulation and cytotoxicity in infected cells. In this study, however, no cytotoxicity and vector-induced aberrant cell cycle profiles were observed.…”

“…[1][2][3] Approaches for reducing these detrimental effects include deletion of further or all coding sequences of the virus or incorporation of antiapoptotic genes like bcl-2 or p21 Waf1/Cip1 (p21). 4,5 Although bcl-2 is classified as an oncogene, p21 is not regarded as such and under certain circumstances even functions as a tumor suppressor. 6 p21 is a member of the Cip/Kip family of cyclin-dependent kinase inhibitors and, as such, acts as a cell cycle regulator at the G 1 /S and G 2 /M boundaries.…”

“…18 Co-infection with two Ad vectors expressing p21 together with a reporter gene has been shown recently to result in reduced cytotoxicity with a concomitant increased expression of the reporter gene. 5 This coinfection strategy also allowed improved and prolonged reporter gene expression in vivo. 5 However, co-infection results in a mosaic of infected cells and requires increased virus doses, which may compound immunologic consequences in in vivo applications.…”

“…5 This coinfection strategy also allowed improved and prolonged reporter gene expression in vivo. 5 However, co-infection results in a mosaic of infected cells and requires increased virus doses, which may compound immunologic consequences in in vivo applications.…”