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Chronic myeloid leukaemia is one of the few cases of cancer driven by a single genetic event. The constitutive kinase activity of BCR/ABL is necessary and enough for the development of leukaemia. The suppression of this kinase activity is the basis of the main current treatment of the disease: tyrosine kinase inhibitors. However, these treatments continue to have a serial of problems, such as the need for chronic administration, the appearance of resistance or the risk of relapse..

For this reason, CML becomes a perfect model to test the possibilities of gene therapy. In our lab, we are using the novel CRISPR/Cas9 technology to Knock out BCR-ABL at the genomic level in order to revert the disease. For that purpose, we have designed two sgRNA directed against the tyrosine kinase domain of the gene, because this domain is the one that confers oncoprotein activity.

Moreover, our guides are close enough to produce a specific deletion rather than random Indels. This kind of edition allows us, on one side, ensure that we knocked out the gene, and on the other side, detect the edition by a simple PCR.

This would bring us closer to the ideal possibility of being able to extract the leukemic stem cells from a CML patient, edit them in vitro eliminating the expression of BCR/ABL and finally reinfuse these corrected stem cells into the patient himself, avoiding with that the possibility of rejection.

Once our editing system had been successfully developed, we proceeded to evaluate its therapeutic potential in two models of bone marrow transplantation, using for that immunodeficient mice (NSG) as a transplant recipient ¹.

On one side, we used as a donor cells, the leukemic stem cells of a murine CML model, which expresses human BCR/ABL oncogene and partially mimics the disease. On the other side, we used the human leukemic stem cells from CML patients as donor cells for transplantation.

In this way, and by analysing the derived blood populations, at 2 and 4 months post transplantation, we will be able to answer our main questions. First, if the edited leukemic stem cells can repopulate the bone marrow of a host mouse, and secondly, if these edited leukemic stem cells are capable of giving rise to physiological haematopoiesis.

Finally, 4 months after transplantation, we observed significant differences between transplants made with control cells (only electroporated with Cas9) and those made with corrected cells. 

In both murine and human strategy, control transplantations of unedited cells show levels similar to those found in a pathological haematopoiesis, characterized by abnormally high levels of myeloid populations and low levels of lymphoid populations, exhibiting the characteristic myeloid bias of CML. However, the mice transplanted with edited cells present significantly lower levels of myeloid cells and higher levels of lymphoid cells, reaching physiological values and restoring normal haematopoiesis.

Our study ¹ demonstrates that our CRISPR/Cas9 deletion system is able to efficiently eliminate the BCR/ABL fusion oncogene in leukemic stem cells (LSCs), maintaining its stem characteristics and developing a phisiological haematopoyesis into inmunodeficent mice. These constitutes the proof of concept that the CRISPR / Cas9 system is a potential therapeutic tool to destroy cancer gene drivers.

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