Circular Antisense Oligonucleotides Inhibit Growth of Chronic Myeloid Leukemia Cells

Abstract

Background: Antisense represents a conceptually powerful method for regulating gene expression. However, antisense oligonucleotides developed to date manifest two serious limitations-nuclease susceptibility and nonspecific hybridization. Circular oligonucleotides may be superior to conventional linear oligonucleotides in both respects. First, circular agents, having no ends, are exonuclease-resistant. Second, they bind to complementary strands of RNA and DNA with a higher affinity than corresponding linear agents. Methods and Results: We assessed the activity of circular phosphodiester deoxynucleotides using chronic myeloid cell lines by targeting polypurine sequences. To represent cells having a bcr3/abl2-type junction, we used K(562 cells. A circle targeting a bcr polypurine sequence 385 nucleotides 5' to the junction decreased the cell number by day 5 with an IC 50 of 9 ,uM. To represent cells having a bcr2/ab/2-type junction, we used BV173 cells. A circle targeting the bcr-abl junction itself decreased the cell number by day 7 with an IC50 of 8 ,uM. Control oligonucleotides, whether the same sequence uncircularized or circles with the same nucleotide composition but in scrambled sequence, had little effect. Unlike linear agents, circles were stable when incubated in 10% serum. The amount of bcr-abl protein detected by Western blotting using a specific anti-bcr-abl antibody at 24 hr in antisense-treated BV173 cells was only 10% of that of cells treated with control circles, which demonstrates an antisense mechanism of action.Conclusions: Circular oligodeoxyribonucleotides (1) inhibit the accumulation of CML cells, (2) decrease the amount of bcr-abl protein per cell, (3) have sequenceselective activity, and (4) are more active than linear oligonucleotides containing only the base-pairing region.

Synthesis and “DNA Interlocks” Formation of Small Circular Oligodeoxynucleotides

“…Blumenfeld et al managed to control gene expression using a circular dumbbell as decoy DNA which contains a binding site for hepatocyte nuclear factor I while Worm et al constructed circular dumbbell CpG oligodeoxynucleotides (ODNs) as immunoadjuvants . Moreover, circular antisense ODNs targeting bcr polypurine sequence were utilized to inhibit the growth of chronic myeloid leukemia cells as well as the mRNA imaging and gene therapy in living cells . Through in situ intercross-linked “click” cyclization, Raj et al successfully established the ClampFISH (click-amplifying FISH) method to detect an individual nucleic acid molecule .…”

Antigene Effect in K562 Cells of a PEG-Conjugated Triplex-Forming Oligonucleotide Targeted to the bcr/abl Oncogene

“…Maxam and Gilbert G reactions were performed according to the standard procedure (35). In brief, a 50-mer target duplex (20 nM), end-labeled with [γ- 32 P]ATP at the purine strand, was dissolved in a final volume of 20 µL containing 50 mM Tris-acetate, 10 mM MgCl 2 , and 0.05 mg/mL salmon sperm DNA. Two microliters of 5% DMS in ethanol was added to this reaction mixture.…”

BCR/ABL: from molecular mechanisms of leukemia induction to treatment of chronic myelogenous leukemia

“…However, ribozymes result in only imperfect cleavage of target mRNAs (James and Gibson, 1998). New modifications to the antisense system, such as DNAzymes (Hamada et al, 1999;Kuwabara et al, 1998Kuwabara et al, , 2001aTanabe et al, 2000;Warashina et al, 1999), BCR/ABL junction-specific catalytic subunits of RNase P (Cobaleda and Sanchez-Garcia, 2000) or maxizymes; novel allosterically controllable ribozymes (Hamada et al, 1999;Kuwabara et al, 1998Kuwabara et al, , 2001aTanabe et al, 2000); may increase specificity and increase cleavage of BCR/ABL mRNA (Maran et al, 1998;Mendoza-Maldonado et al, 2002;Rowley et al, 1999).…”