Specific Inhibition of BCR-ABL Gene Expression in Acute Lymphoblastic Leukemia by Catalytic DNAzymes.

The BCR-ABL fusion protein p190 resulting from the translocation t(9;22) exhibits dysregulated tyrosine kinase activity and was shown to cause acute lymphoblastic leukemia (ALL). Detection of the BCR-ABL fusion gene in childhood ALL is associated with an adverse prognosis and defines a group of high risk patients. Because the BCR-ABL gene fusion is specific for leukemic cells it represents an ideal target for leukemia specific treatment approaches.

Catalytic DNAzymes are able to cleave mRNA in a sequence specific manner, causing inhibition of protein translation from the DNAzyme targeted mRNA both in vitro and in vivo. In order to cut off the BCR-ABL driven malignant proliferation, we designed DNAzymes to impede the expression of p190 BCR-ABL by cleaving the BCR-ABL mRNA adjacent to the fusion site. One construct was found that cleaved the target mRNA efficiently and specifically leaving BCR and ABL, relevant for normal cell survival and proliferation, unaffected. Activity and specificity of the BCR-ABL DNAzyme was investigated in cleavage assays with in vitro transcribed BCR-ABL, BCR and ABL mRNA. DNAzymes were delivered to cultured BCR-ABL+ ALL cells by lipid transfection. The efficiency of cellular delivery reached 90% as studied by flow cytometry, fluorescence microscopy and confocal microscopy after transfection of FITC labeled DNAzymes. To control for unspecific effects of DNAzyme delivery as well as for antisense effects, a catalytically inactive DNAzyme still exhibiting BCR-ABL antisense activity was designed. Fourty-eight hours after a single treatment of BCR-ABL+ ALL-cells with DNAyzmes the BCR-ABL mRNA concentration, as measured by quantitative real-time RT-PCR, was significantly reduced by 56% and 66% compared to controls treated with the inactivated DNAzyme and to untreated cells, respectively. Western blot analysis showed a decrease in p190 protein levels after DNAzyme treatment in comparison to the control treated with inactive DNAzyme as well as to the untreated cells. Most noteworthy, four days after a single DNAzyme treatment the net growth of BCR-ABL+ ALL cells treated with the active DNAzyme was inhibited by 68% compared to the untreated control.

From these data we conclude, firstly, DNAzymes targeting mRNA coding for the minor BCR-ABL variant are able to significantly reduce the amount of fusion mRNA in the cells, leading to a reduction in protein expression, followed by the inhibition of BCR-ABL driven proliferation of ALL cells. Secondly, this exemplified setting gives a hint that DNAzymes might be of therapeutic use in hematopoietic malignancies associated with specific mutations, expressing oncogenic fusion genes or overexpressing oncogenic genes.