Dataset Information

Efficient disruption of bcr-abl gene by CRISPR RNA-guided FokI nucleases depresses the oncogenesis of chronic myeloid leukemia cells.

ABSTRACT:

Background

The bcr-abl fusion gene encodes BCR-ABL oncoprotein and plays a crucial role in the leukemogenesis of chronic myeloid leukemia (CML). Current therapeutic methods have limited treatment effect on CML patients with drug resistance or disease relapse. Therefore, novel therapeutic strategy for CML is essential to be explored and the CRISPR RNA-guided FokI nucleases (RFNs) meet the merits of variable target sites and specificity of cleavage enabled its suitability for gene editing of CML. The RFNs provide us a new therapeutic direction to obliterate this disease.

Methods

Guide RNA (gRNA) expression plasmids were constructed by molecular cloning technique. The modification rate of RFNs on bcr-abl was detected via NotI restriction enzyme digestion and T7 endonuclease 1 (T7E1) assay. The expression of BCR-ABL and its downstream signaling molecules were determined by western blotting. The effects of RFNs on cell proliferation and apoptosis of CML cell lines and CML stem/progenitor cells were evaluated by CCK-8 assay and flow cytometry. In addition, murine xenograft model was adopted to evaluate the capacity of RFNs in attenuating the tumorigenic ability of bcr-abl.

Results

The RFNs efficiently disrupted bcr-abl and prematurely terminated its translation. The destruction of bcr-abl gene suppressed cell proliferation and induced cell apoptosis in CML lines and in CML stem/progenitor cells. Moreover, the RFNs significantly impaired the leukemogenic capacity of CML cells in xenograft model.

Conclusion

These results illustrate that the RFNs can target to disrupt bcr-abl gene and may provide a new therapeutic option for CML patients affiliated by drug resistance or disease relapse.

SUBMITTER: Luo Z

PROVIDER: S-EPMC6537404 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe pathogenesis of chronic myeloid leukemia (CML) is the formation of the BCR/ABL protein, which is encoded by the bcr/abl fusion gene, possessing abnormal tyrosine kinase activity. Despite the wide application of tyrosine kinase inhibitors (TKIs) in CML treatment, TKIs drug resistance or intolerance limits their further usage in a subset of patients. Furthermore, TKIs inhibit the tyrosine kinase activity of the BCR/ABL oncoprotein while failing to eliminate the pathologenic oncoprotein. To develop alternative strategies for CML treatment using therapeutic antibodies, and to address the issue that antibodies cannot pass through cell membranes, we have established a novel intracellular delivery of anti-BCR/ABL antibodies, which serves as a prerequisite for CML therapy.MethodsAnti-BCR/ABL antibodies were encapsulated in poly(D, L-lactide-co-glycolide) nanoparticles (PLGA NPs) by a double emulsion method, and transferrin was labeled on the surface of the nanoparticles (Ab@Tf-Cou6-PLGA NPs). The characteristics of nanoparticles were measured by dynamic light scattering (DLS) and transmission electron microscopy (TEM). Cellular uptake of nanoparticles was measured by flow cytometry (FCM). The effect of nanoparticles on the apoptosis and proliferation of CML cells was testified by FCM and CCK-8 assay. In addition, the anti-cancer impact of nanoparticles was evaluated in mouse models of CML.ResultsThe results demonstrated that the Ab@Tf-Cou6-PLGA NPs functioned as an intracellular deliverer of antibodies, and exhibited an excellent effect on degrading BCR/ABL oncoprotein in CML cells via the Trim-Away pathway. Treatment with Ab@Tf-Cou6-PLGA NPs inhibited the proliferation and induced the apoptosis of CML cells in vitro as well as impaired the oncogenesis ability of CML cells in vivo.ConclusionsIn conclusion, our study indicated that this approach achieved safe and efficient intracellular delivery of antibodies and degraded BCR/ABL oncoprotein via the Trim-Away pathway, which provides a promising therapeutic strategy for CML patients, particularly those with TKI resistance.