Linking epigenetics and gene therapy: enhanced replication and therapeutic effect of a CRAd by placing a nucleosome positioning sequence upstream of a tumor specific promoter

NUÑEZ FELIPE; CAFFERATA EDUARDO; DE FERRARI GIANCARLO; BRAVO SORAYA; MONTECINO MARTIN; PODHAJCER OSVALDO

Philadelphia

Congreso; American Society of Gene and Cell Therapy 15th Annual Meeting; 2012

Linking epigenetics and gene therapy: enhanced replication and therapeutic effect of a CRAd by placing a nucleosome positioning sequence upstream of a tumor specific promoter Felipe Núñez1,3, Soraya Bravo2,3, Eduardo Cafferata1, Giancarlo De Ferrari2, Martín Montecino2,4 and Osvaldo Podhajcer1,4. Fundación Instituto Leloir, Buenos Aires, Argentina (1); Centro de Investigaciones Biomédicas, Universidad Andrés Bello, Santiago de Chile, Chile (2). Both contributed equally (3). Both should be considered corresponding authors (4). Cancer development is the result of global changes driven by the epigenetic machinery such as DNA methylation, histone modification and nucleosome positioning. During the last years the concept that adenoviral replication can be driven by tumor specific promoters (TSP) gained adherence and several Conditionally Replicative Adenoviruses (CRAd) have demonstrated enhanced therapeutic effect in preclinical trials and few of them entered clinical trials as well. Few of the promoter sequences that drive viral replication were modified by introducing DNA motives that respond to tumor microenvironment features such as hypoxia, reactive oxygen species and Wnt/b-catenin-responsive motives among others. Here, we show for the first time that placing a nucleosome positioning sequence (NUC) upstream of a TSP combined with wnt-responsive motives enhanced TSP transcriptional activity and hence viral lytic capacity. The synthetic enhancer sequence (named pART) involved 4 TCF/LEF-responsive element (TBE sites) obtained from the COX-2 gene promoter (i.e. WWCAAAGS; S=C/G; W=A/T), placed upstream of the NUC obtained from the osteocalcin gene promoter extending from ?287bp to ?106bp. In order to assess the role of pART on TSP activity we placed downstream a variant of the gastrointestinal cancer-associated-REG1A promoter (REG1A-Pr) sequence extending from ?571 to +75. Through gene reporter assays with plasmid constructs, we observed more than 2-fold enhanced REG1A-Pr activity in gastric cancer cells when pART was placed upstream of the TSP. Constructs containing pART (∆NUC) upstream of REG1A-Pr showed luciferase activity similar to that of REG1A-Pr alone. In terms of viral oncolysis, placing pART upstream of REG1A-Pr reduced by one order of magnitude the MOI necessary for a CRAd to achieve an in vitro lytic effect on different gastric cancer, colon cancer and pancreatic cancer cells. This CRAd (named Ad-pART(REG1A) showed no activity on WI38 fibroblasts and A549 lung cancer cells that do not express REG1A. This enhanced in vitro CPE was highly dependent on the presence of NUC as removal of this nucleosome positioning sequence from pART reduced Ad-pART(REG1A)  activity to levels similar to those observed with the CRAd whose replication is driven by REG1A-Pr alone. We observed a strong increase in E1A expression immediately after 2 hr of infection with Ad-pART(REG1A) compared to a low E1A expression observed only after 8 hr infection when a CRAd whose replication was driven by REG1A(Pr) alone. pART also enhanced the transcriptional activity of other heterologous promoters such as cdc25B(Pr). Ad-pART(REG1A) was also therapeutically effective on s.c. growing pancreatic tumors as it induced the remission of 4/5 tumors compared to none in control nude mice. It can be concluded that nucleosome positioning sequences could be very useful in gene therapy approaches in cancer as in other diseases as well.