In vivo CRISPR/Cas9 targeting of fusion oncogenes for selective elimination of cancer cells

Fusions oncogenes (FOs) are chimeric genes that drive the development of many human cancers and are only expressed in cancer cells. Some of them are the only genetic drivers for cancer, which makes them attractive therapeutic targets. The present study describes a strategy using CRISPR/Cas9 technology for cancer treatment by targeting an FO formed by two consecutive sequences from the EWSR1 and FLI1 genes. The expression of this FO leads to a dominant transcription factor in Ewing sarcoma cancer.

CRISPR/Cas9 (more specifically, LVCas9_EF) was used for the silencing of this FO, namely the deletion of specific intronic sequences within the FO in question that are essential for its viability. The same two guide RNAs allowed targeting of a large variety of the breakpoints specific to the intron sequences, making the method non-patient-specific.

To test their method in vitro, they exposed LVCas9_EF to the A673 Ewing sarcoma cell line and used LVCas9_NT, which is non-specific to A673 cells genome, as control. The gene-edited cells demonstrated a significant decrease in cell proliferation and increase in apoptosis levels in comparison to the control (Fig. 1b). When tested on heathy cells, they did not observe a significant difference in EWSR1 and FLI1 gene expression, which demonstrates a high specificity of LVCas9_EF for the FO. They tested their approach in vivo by injecting a delivery vectors containing LVCas9_EF in tumours resulting from A673 cell proliferation in mice and compared to a standard chemotherapeutic option for Ewing sarcoma (DOX) and the LVCas9_NT control. A reduction in tumour size at sacrifice (Fig. 1b) and an increase in survival (Fig. 1c) was observed with LVCas9_EF treatment, which were even greater when LVCas9_EF was combined with DOX.

In conclusion, FO silencing has been shown to be a simple, robust, efficient and cancer cell-specific strategy for cancer treatment. The method reduced tumour size, reduced FO-driven cancer cell proliferation and did not damage tissues as chemotherapy does. However, targeting efficiencies, off-target effects and the delivery systems should be further investigated to use the approach clinically.

Fig. 1 – Results Obtained from the LVCas9_EF Method

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