Desenvolvimento de terapia genética multi alvo para tratamento do câncer cervical causado pelo HPV

Abstract

Cervical cancer is the second most common cancer among women worldwide and the fourth leading cause of death among women in Brazil. Human papillomavirus (HPV) plays a central role in this disease, as its infection is considered a necessary condition for the development of more than 90% of cervical cancer cases. The standard treatment for the disease consists of radiotherapy with cisplatin-based chemotherapy and, since 2014, the SUS has made available the vaccines Gardasil (against HPVs 6, 11, 16, and 18) and Cervarix (against HPVs 16 and 18). However, the efficacy of these vaccines for the treatment of patients with cervical cancer is not clear. HPV-associated carcinogenesis provides a classic model for cancer therapies based on RNA gene silencing (RNAi), as its progression depends significantly on the viral oncogenes E6 and E7. Therefore, most RNAi-based therapeutic strategies developed to date use RNAi molecules targeting the E6 and/or E7 genes, achieving 50 to 80% silencing. The aim of this research is to propose a therapy for the treatment of cervical lesions caused by HPV 16 and HPV 18 based on siRNA and circRNA, designed with the aid of bioinformatics tools and tested in silico and in vitro. The in silico tests were performed with bioinformatics tools that analyzed thermodynamic stability, possible off-target effects, and similarity with already patented RNAi molecules. The in vitro tests, in turn, consisted of transfection of the synthesized siRNA and circRNA molecules into commercial cell lines HeLa (HPV 18 positive), Caski (HPV 16 positive), and C-33a (HPV negative cervical cancer), followed by qPCR and statistical analyses. For siRNA silencing, a tool was developed to select siRNA sequences based on physicochemical parameters, which also allowed verifying the viability of the chosen siRNAs. Similarity of the siRNAs with some targets already described in patents registered with the National Institute of Industrial Property (INPI) was observed. In vitro tests performed to date have shown that the therapy was able to significantly reduce the expression of the E6 and E7 genes in HeLa cells. However, additional tests with circRNA and Caski (HPV 16 positive) and C-33a (HPV negative cervical cancer) cell lines are still being performed to verify the viability of the therapy proposed by this work.