HOST EPIGENETIC REGULATOR BMI1 AND VIRAL FIBER PROTEIN FOR EQUILIBRIUM BETWEEN ADENOVIRUS AND HOST

Sammanfattning: Human adenoviruses (Ads) are broadly used in cancer gene therapy, vaccine development and gene delivery. The modified Ad5 or Ad2 viruses have been successfully used as oncolytic agents in pre-clinical studies. However they could not be translated into clinic utility due to a number of limitations including inefficient Ad spread among tumor cells. The cancer cell killing capacity of oncolytic Ad is dependent not only on the efficiency of Ad replication, but also on the efficiency of progeny dispersal and propagation of infection within cancer tissue. In this thesis, we believe that both viral and host cell factors regulate Ad propagation and spread process and consequently affect the co-existence interplay between Ad and host cells. Our projects aim to identify such factors from Ad as well as host cell side, and to clarify their role in regulating Ad propagation and spread. To identity viral factors, we investigated the kinetics of cell killing and Ad propagation following Ad infection at low multiplicity. Our results showed that prior to the release of Ad progenies, Ad infected cells secrete free fiber molecules in an excess, which mask Ad receptor molecules on non-infected bystander host cells, thus preventing these cells from efficient Ad infection and thereby promoting Ad and host cell co-existence. This is advantageous to Ad propagation and persistency of infection compared to the killing of all host cells with rapid kinetics. However, this is disadvantageous if Ad is used as oncolytic agents for therapeutic purposes. To identity host factors, we investigated the effect of polycomb gene BMI1 on Ad propagation. BMI1 is broadly overexpressed in various cancers. By retroviral vector mediated enforced BMI1 overexpression or siRNA mediated down-regulation of endogenous BMI1 expression, we demonstrated an inverse correlation between the Ad progeny production and the levels of BMI1 expression. This effect was not related to the cell cycle status and the receptor dependent Ad infectivity in host cells; nor to the replication of Ad genome and the production of Ad structural proteins. Instead, BMI1 overexpression impaired the morphogenesis of Ad particles, which could be reversed by TSA mediated inhibition of HDAC activity. Our findings indicate overexpression of BMI1 as a limiting factor in cancer therapy based on oncolytic Ad. So inhibition of BMI1 expression or BMI1-related HDAC activity may improve the functionality of oncolytic Ads in cancer therapy. To explore new approaches in Ad vector mediated dual gene expression in human hematopoietic stem cells (HSC), we generated an Ad vector, Ad5F35- ΔLNGFR-BiDp-GFP, encoding kinase domain deleted low-affinity NGF receptor (ΔLNGFR) and green fluorescent protein (GFP) expression cassette controlled by a synthetic bi-directional promoter. Our data showed that Ad5F35-ΔLNGFR-BiDp vector is highly active in directing dual gene expression in HSCs and most leukemic cell types tested, but the relative levels of dual gene expression by this vector is strongly cell-type dependent.