An innovative epigenetic strategy for retinoblastoma treatment

Retinoblastoma (Rb) is a rare cancer that nonetheless represents the most common ocular malignancy in children. The management of retinoblastoma often requires intensive chemotherapy and sometimes even surgery to preserve useful vision and quality of patient's life. Between the two retinoblastoma forms (familiar and sporadic), the incidence of the sporadic one is increasing and its pathogenesis is mostly unknown. Recently, the discovery of the epi/genetic nature of nonhereditable/ sporadic Rb led to the development of high-throughput precision investigation strategies for the study of its epigenetic landscape. Epigenetic alteration in gene silencing in Rb can become target for the reprogramming of cancer cells leading to apoptosis. There is therefore the need to expand the field of investigation to identify new more effective molecules and/or reliable minimally invasive treatments using drug delivery systems for more targeted therapy. This thesis evaluated the effect of the demethylating agent 5-Aza-2'-deoxycytidine (DAC) and of a new DAC-formulation based on erythrocyte drug delivery system (EMHVs DDS) for the treatment of Rb. The results showed that DAC induced an anti-proliferative effect in Rb cells by activating genes involved on apoptotic response. Using cDNA microarray analysis, a gene-gene network interaction study in cells treated with DAC identified numerous differential expressed genes (DEG). Among those, fifteen genes involved in initiation and progression of Rb, were chosen as candidates for targeted therapy. qRT-PCR validation confirmed the time-dependent variation of genes expression. Methylation analysis indicated the direct effect of DAC on both CASP8 and BIK while showing an indirect regulation on the other genes, such as FAS. Preclinical investigation of DAC therapy and epigenetic combinatory treatments (DAC and Trichostatin A) was carried out in xenograft and orthotopic models, indicating that DAC alone induced the most significant anti-cancer effect. EMHVs DDS was used to improve efficacy of DAC therapy as well as to deliver a new biodrug, Survivin Molecular Beacon (SURV-MB) with theranostic activity, targeting mRNA of BIRC5 oncogene overexpressed in Rb. The beneficial effect of using EMHVs DDS was lowering the dose of DAC and improving the bioavailability of both the active molecules. These results will open new perspective for localized, safe and target specific therapies in Rb. This work is seminal for further investigation on the possibility of using both the carrier and the formulations for the development of new therapeutic intervention in precision medicine.