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Evaluation of the effect of MMP9 silencing by CRISPR-Cas9 system concomitant to the use of BCG and anti-PDL1 in the treatment of Bladder Cancer: in vivo study


Bladder Cancer (BC) is the tenth most common type of cancer, responsible for about 5% of all new cancer cases. It is a disease with important morbidity and mortality, and its prognostic is directly related to tumor staging. When the disease is restricted to the superficial urothelial layer of the bladder wall (pTa and pT1), the selected treatment is based on the preservation of the bladder, thus reducing its impact on the patient´s quality of life. In high-risk superficial tumors, the local recurrence and progression rates to muscle-invasive (pT2) disease are important, corresponding to almost 50% of the cases. Many factors are related to the tumor´s invasion capacity, including the high expression of the matrix metalloproteinases, such as the MMP-9. OBJECTIVE: Analyze the effect of intra-vesical treatment using CRISPR-Cas9 gene editing technique of localized urothelial bladder tumor, and the synergistic effect of this blockade with the use of BCG and anti-PDL1. METHODS: Initially, the insertion of guide RNA sequences (sgRNA) for target sequences in the MMP-9 gene will be performed in the plasmid PX-330. Then, the already modified plasmids will be transfected into MB49 cell line, then gene and protein expression analysis will be performed by qRT-PCR and Western-blotting, respectively, as well as cell migration assay by wound closure test. Finally, the orthotopic animal model in mice (C57-black) for CaB using cells of the MB49-luc lineage will be established. After establishing the animal model, these will be divided into seven groups: control group, CRISPR-MMP9 group, BCG group, anti-PDL1 group, CRISPR-MMP9 + BCG group, BCG + anti-PDL1 group and CRISPR-MMP9 + BCG + anti-PDL1 group. (AU)

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