ENHANCED ONCOLYTIC ACTIVITY OF BOHV-4-BASED VECTOR DELIVERING A MIRNA SEQUENCE AGAINST ENOLASE 2 TRANSCRIPT

MACCHI F; TEBALDI G; VERNA A; CAVIRANI S; DONOFRIO G

Conferencia; LXIX Convegno Nazionale SISVet; 2017

Cancer diseases are the second globally most frequent cause of mortality. To help to prevent and cure these diseases new innovative anticancer therapies and treatments are needed. Oncolytic viruses, because of their ability to infect and replicate selectively into tumor cells, could represent an interesting considerable anticancer strategy. [1] Experimental studies/data have shown how these viruses have marked tropism for brain tumor cells such as glioblastoma cells. Glioblastoma Multiforme (GBM) is a very aggressive brain tumor originating into the glia, characterized by high replication rate, which nowadays still has no cure. Genetic analysis revealed that progressive oncogenes and tumor suppressor genes mutations are involved in the development of GBM and one of the most frequent mutation observed in these tumor cells is enolase 1 (ENO1) homozygous deletion. ENO1 is an isoenzyme expressed in a variety of tissues, including brain, involved into glycolysis and gluconeogenesis cell energetic processes. The intense energetic demands of neuronal ENO1(-/-)tumor cells is modestly compensated by ENO2 gene expression, making ENO2 an ideal target for GBM anticancer therapy. [2] The aim of this work was to generate a recombinant Bovine Herpesvirus 4 (BoHV-4) based vector expressing a microRNA for ENO2 (miR-ENO2) to improve the BoHV-4 oncolytic activity in ENO1-deleted GBM cells. An expression cassette, caring miR-ENO2 downstream of Turbo RFP and under the transcriptional control of Cytomegalovirus Immediate Early promoter, was designed to subsequently generate the recombinant virus. [3] The oncolytic efficiency of recombinant BoHV-4-TurboRFP-ENO2 to selectively limit GMB cells growth and survival was tested on Gli56 and D423, ENO1(-/-)/ENO2(+/+),cell lines and evaluated by Cristal Violet, MTT assay and Western Immunoblotting. In vitro results showed how BoHV-4 is able to infect and replicate into ENO1-deleted GMB cells, inducing a diffuse cytopathic effect (CPE) on cells monolayer, and how ENO2 inhibition through miR-ENO2 expression has significantly increased its oncolytic activity. This increased activity could be, in fact, attributable to ENO2 post-transcriptional downregulation, thus causing the loss of energy source in GBM cells. These in vitro data could suggest a possible BoHV-4-TurboRFP-ENO2 use as a GBM therapeutic tool. However, in vivo studies on animal models will be necessary to consider BoHV-4 based vector as a valid alternative for GBM anticancer treatment. [1] Kelly E., Russell S.J. (2007) ?History of oncolytic viruses: genesis to genetic engineering.? Mol Ther. 15(4):651-9. [2] Muller F.L., Colla S. et al. (2012) ?Passenger deletions generate therapeutic vulnerabilities in cancer.? Nature. 488(7411):337-42. [3] Donofrio G., Franceschi V. et al. (2009) ?Cellular targeting of engineered heterologous antigens is a determinant factor for bovine herpesvirus 4-based vaccine vector development.? Clin Vaccine Immunol. 16(11):1675-86.