Strategie di Costruzione di Vettori Erpetici Oncolitici per la Cura del Tumore Epatico

Background: Several studies show that Hepatocellular carcinoma (HCC) represents the 80% of primary hepatic tumours and it is one of the most common malignancies in the world. Because of the large numbers of cases and the lack of effective therapy, new therapeutic procedures are urgently needed. In this contest oncolytic viruses are emerging as a safe and effective promising therapeutic approach for cancer therapy in association with traditional therapies. Aims: In the contest of an European project (THOVLEN), our research group is involved in the development and construction of Herpes Simplex (HSV-1) based oncolytic vectors against HCC. To this purpose, the main goal is to construct replication-competent HSV-1 vectors strictly targeted to hepatocellular carcinomas (HCC), such that they will infect, replicate and spread efficiently only in the tumour mass, while displaying no virulence or toxicity in normal tissues, by means of receptorial and transcriptional targeting Methods: Design and construction of fusion molecules using cloning and PCR techniques, construction and purification of recombinant viruses using standard methods of: marker transfer/marker rescue, limiting dilution and Southern Blot; analysis in vitro and in vivo of growth and oncolytic ability of oncolytic viruses. Results: To target viral replication to hepatocytes and/or tumor cell lines, the first step was the construction of recombinant viruses H6HRE-gD e H6Ealb-gD in which expression of glycoprotein D, essential for HSV-1 replication, is under the transcriptional control of a hypoxia-induced tumour specific promoter, (HRE) or a tissue-specific promoter of albumin (EalbP), protein over expressed in liver. Successively, either an EGFP or a lucyferase expression cassette has been inserted in an intergenic HSV-1 region, not encoding viral genes, in order to facilitate the screening of recombinant viruses in vitro and to allow the in vivo tracking of such viruses in the murine model. In order to modify the reptional targeting, we used as a starting point recombinant virus previously constructed in our laboratory in which glycoproteins B and C, responsible of viral attachment and entry after interaction with Heparansulfate cellular receptor, have been engineered with insertion of a fusion molecule between gC and PreSap (gCPreSap), an active peptide of HBV involved in the attachment process of the virus to Hepatocytes. This recombinant virus shown a preferential binding to hepatocytes, giving a productive infection, will be further modified in order to delete the gD binding domain to its cellular receptor HVEM. To this purpose the binding domain of gD have been deleted and substitute by PreSap, creating a gD-PreSap fusion molecule. This elimination will eliminate viral wild type tropism to obtain a recombinant virus selective for hepatocytes. Conclusions: the growth ability of recombinant viruses has been evaluated in several cells lines and the in vivo viral biodistribution and oncolytic activity are ongoing. If the results will be promising, we plain to incorporate all the modifications into a single herpetic oncolytic vector in order to obtain a viral recombinant strictly targeted to hepatocellular carcinomas.