Bularreko minbiziaren prebentziorako BRCA1 gene mutatuaren edizioa CRISPR/Cas9 teknikaren bidez

Abstract

[EUS] Gaur egungo gizartean gero eta minbizi kasu gehiago diagnostikatzen dira, eta horien artean % 23a bular-minbiziari dagokio. Gainera, nagusienetarikoa da emakumeen heriotza kausen artean, % 14a alegia. Hori dela eta, azterketa asko egin izan dira bular minbiziaren inguruan eta bi gene izan dira, nagusiki, bular minbiziarekin lotu direnak, BRCA1 eta BRCA2, hain zuzen. Lan honetan bi hauetako bat aztertuko da, BRCA1 genea, gizakiotan 17. kromosoman kokatzen dena (17q21 ), gene honen zenbait mutazio komunak direlako bular-minbizia duten gaixoen artean eta baita familia berekoen artean. Gene honek tumore-supresorea den p220 proteina kondetzen du. Proteina horren ziklo zelularraren kontrolean funtsezko papera betetzen du, izan ere, zelulen zikloko zenbait bidezidor konplexutan parte hartzen du, beste proteina batzuekin elkar eraginez, p53 proteinarekin adibidez. BRCA1 genean mutazioak gertatzean, baina, ziklo zelularraren kontrola inhibitu egiten da eta zelulen gehiegizko proliferazioa gertatzen da, bular zein obario minbizia dakarrena. Gene honetan deskribatutako mutazioen artean bi dira nagusitzen direnak: 2. exonean ematen den 2 nukleotidotako delezioa, 187delAG, eta 19. exonean ematen den nukleotido baten insertzioa, 5382insC. Lan honetan azken mutazio hau landu da, izan ere, handia da mutazio honengatik bular-minbizia garatzeko arriskua (% 67). Lan honetan beraz, mutazioa honen edizioak bular minbizia garatzeko arrisku murrizten inpaktua izango duela proposatzen dugu. Hortaz, kontzeptuen proba moduan hurrengo proiektu pilotua proposatzen da. Mutatutako HCC 1937 lerro zelularra erabiliko dugu, BRCA1 5382insC mutaziorako homozigotoa dena, in vitro modelo esperimental moduan. Edizioa aurrera eramateko CRISPR/Cas9 edizio sistema erabiltzea proposatzen da mutatutako zelulak basati bihurtzeko helburuarekin. Edizioa zuzentzeko, HCC 1937 zelulak 2 plasmido erabilita transfektatuko dira: bat RNA gudaren eta Cas9 nukleasaren ko-espreziorako, eta bestea DNA emailea eramango duena, basatia BRCA1 generako, DNAren konponketa faboratzeko. Edizioaren ondorioak aztertzeko, proliferazio indizea eta kimioterapiarekiko sentikortasuna analizatuko dira HCC 1937 zelula editatuetan.

[EN] Nowadays, a large amount of cancer cases are diagnosed every year, of which 23% correspond to breast cancer. In addition, breast cancer is the main cause (14%) of death among women and therefore, many studies have been accomplished in it. Those studies have proven that two genes are mainly related to breast cancer, namely BRCA1 and BRCA2. In this project we are going to focus on one of these two genes will be analysed, the BRCA1 gene, which in humans is found in chromosome 17 (17q21), because some of the mutations of this gene are common among breast cancer patients and among the people from the same family. This gene encodes for a tumor-suppressor protein, known as p220. This protein has a key role in the control of cell cycle, as it interacts with other proteins, like p53. Moreover, when mutations in the BRCA1 gene appears cell cycle control is inhibited, and an over-proliferation of cells occurs. This can lead to the developing of a breast or ovarian cancer. Among the mutations described in this gene, two are those that prevail: the 2-nucleotide deletion in exon 2, 187delAG, and the insertion of a nucleotide in exon 19, 5382insC. Because of the high risk of developing cancer in carriers of this latest mutation (67%), BRCA1 5382insC has been addressed in the present work. Here we propose that editing this mutation would have an impact in reducing the risk of developing cancer. Thus, as a proof of principle the following pilot project is proposed. We will use HCC 1937 mutated cell line is proposed, which is a homozygous for BRCA1 5382insC mutation, as an experimental in vitro model. It is proposed to perform the edition using the CISPR/Cas9 edition-system in order to make the mutated cell line become wild-type. To conduct the edition, we will transfect HCC 1937 cells with 2 plasmids: one coexpressing a guide-RNA to the mutant sequence and Cas9 nuclease, and another one providing the donor DNA wild-type for BRCA1 to facilitate the repair of the cleaved DNA by the cells. In order to test the outcome of the edition, we will analyzed the proliferative index and the sensitivity to chemotherapy in edited HCC 1937 cells.