Canine and human oral melanomas: identification of the mutation profile by sequencing exomas and evaluation of innovative therapies using genomic editing and recombinant BCG techniques

• Grant number: 19/15261-0
• Support Opportunities: Regular Research Grants
• Start date: January 01, 2020
• End date: December 31, 2022
• Field of knowledge: Agronomical Sciences - Veterinary Medicine - Animal Pathology
• Agreement: CONFAP - National Council of State Research Support Foundations
• Principal Investigator: Maria Lucia Zaidan Dagli
• Grantee: Maria Lucia Zaidan Dagli
• Host Institution: Faculdade de Medicina Veterinária e Zootecnia (FMVZ). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated researchers: Silvia Vanessa Lourenço

Abstract

Oral melanomas are very common neoplasms in dogs, while in humans they are considered rare but extremely aggressive. Treatment options for oral melanoma include surgery, chemotherapy, radiotherapy, and immunotherapy, however relapses and metastases are common. In addition, few studies related to mutations present in canine and human oral melanomas are available. Currently, exome sequencing techniques have been used in Oncology to detect mutations. In addition, modern genome-editing techniques have been intensively employed for the correction of mutations in neoplasms, with the CRISPR / Cas9 technique being the most promising for therapeutic purposes. New therapies involving recombinant BCG are being tested in UFPel, which are based on strengthening the immunomodulatory properties of BCG by entering neoplastic cells and expressing in them proteins of interest. The present research project aims to identify the mutation profile in canine and human oral melanomas by means of sequencing of exome, and in this way characterize therapeutic targets to perform the genetic editing of oral canine and human melanoma cell lines through of the CRISPR / Cas9 system and to apply recombinant BCG strains to produce innovative therapies for this neoplasm, using modern biotechnology. Thus, samples of oral melanomas from dogs (from the Laboratory of Experimental and Comparative Oncology of FMVZ-USP) will be used for the sequencing of exome. Also included in the sequencing of exome are 5 cell lines of canine oral melanomas and 2 cell lines of human melanomas. These samples will be submitted to extraction of genomic DNA, capture and enrichment of the exomas (preparation of the libraries), which will be sequenced by new generation sequencing (NGS) at CEFAP (Center for Research Support Facilities of USP). Afterwards, the data will be analyzed by bioinformatics and compared with the genome of non-neoplastic cells. Based on sequencing data and existing mutations, the UFPel Cancer Biotechnology Laboratory used these targets in the editing of melanoma tumor lines through CRISPR / Cas9, in order to evaluate the response of these edited cells to conventional therapies as well as Immunotherapy with recombinant BCG in vitro. It is hoped, therefore, to better understand the mutations in oral melanomas of dogs and humans, and to create therapeutic innovations for this neoplasia. (AU)