The correction of ETV6/RUNX1 translocation in acute lymphocytic leukemia cells: a new gene targeting system by homologous recombination mechanism.

Regarding the uncertainty of the exact cause of the acute lymphocytic leukemia (ALL) caused by ETV6-RUNX1t(12;21) translocation, correcting genes of the ETV6 and RUNX1 in ETV6/RUNX1 fusion gene simultaneously on chromosome 12 may be effective in reducing leukemia malignancy. Thus, we designed an homologous recombination (HR) plasmid to target of the ETV6/RUNX1 fusion gene in the REH cell line containing the ETV6-RUNX1t(12;21) translocation. Cells were cultured and transfected by HR plasmid. The presence of the replacement cassette at specific location in the ETV6/RUNX1 fusion gene was verified by PCR and sequencing method. A quantitative gene expression assay was performed to evaluate changes in expression of ETV6, RUNX1, and ETV6/RUNX1 genes following editing. The cell viability was measured by trypan blue staining. The expression of the ETV6 gene was significantly increased in modified cells than unmodified cells by 10.9-fold. In contrast, the expression of the ETV6-RUNX1 fusion gene was significantly decreased in the modified cells compared with unmodified cells by 0.26-fold. The expression of the RUNX1 gene had no significant difference between modified and unmodified cells. The survival rate of edited cells was significantly decreased than unedited cells (p = 013). We designed a gene targeting system based on HR method to correct genes of ETV6 and RUNX1 simultaneously in ETV6/RUNX1 fusion gene on chromosome 12 containing ETV6-RUNX1t(12;21) translocation. The modification of this translocation may lead to reducing effects of the fusion gene's damaging and the dosage compensation related to ETV6 and RUNX1 genes and subsequently reduce the effects of leukemia. This targeting system may open a window for treating leukemia as ex vivo.