Optimization of antisense oligodeoxynucleotide structure for targeting bcr-abl mRNA

Abstract

Antisense oligodeoxynucleotides targeted to bcr-abl are potential ex vivo purging agents for use with autologous bone marrow transplantation in the treatment of chronic myeloid leukemia (CML). We investigated, in a cell-free system, the activity and nuclease resistance of phosphodiester, phosphorothioate, chimeric methylphosphonate/phosphodiester, and chimeric methylphosphonate/phosphorothioate antisense octadecamers directed against either b2a2 or b3a2 bcr-abl breakpoint RNAs. Certain chimeric compounds were shown to possess targeted activity broadly equal to the parent phosphodiester or phosphorothioate forms and greater resistance to the nucleases present in cell extracts. Selected chimeric structures were compared with phosphodiester and phosphorothioate analogues for antisense activity in human CML cells containing either b2a2 or b3a2 bcr-abl breakpoint mRNAs. We present results showing that all four structures can suppress bcr-abl mRNA level in vivo. The rank of in vivo activity is chimeric methylphosphonate/phosphodiester > or = phosphodiester > phosphorothioate > methylphosphonate/phosphorothioate. We show that b2a2 breakpoint RNAs can be more effectively targeted than b3a2 sequence RNAs both in vitro and in vivo and suggest that RNA secondary structure may be a possible explanation for this phenomenon.

True

“…376 Phosphorothioate ASODNs have a sulphur for oxygen substitution in the phosphate backbone of the DNA leading to higher nuclease resistance but also to a higher toxicity as well as aspecific interactions. 374,381 Methylphosphonate ASODNs are not charged thus generating less aspecific interactions. The poor solubility of these ASODNs, however, makes delivery via liposomes or phosphodiester tailing necessary.…”

A Suite of Therapeutically-Inspired Nucleic Acid Logic Systems for Conditional Generation of Single-Stranded and Double-Stranded Oligonucleotides

“…AONs were originally designed to inhibit gene expression through steric inhibition of the translation machinery but have evolved into several parallel regulatory approaches. These include targeted degradation of mRNA through RNaseH activity [5], as well as alteration of mRNA splicing patterns by limiting the accessibility of specific splice sites [6]. RNA interference (RNAi) methods then followed, again evolving from the general notion of double stranded RNA being able to silence a complementary target mRNA [7], to the design of synthetic short interfering RNA (siRNA) [8], Dicer substrate siRNA (DsiRNA) [9], and other related constructs [10,11].…”

Evaluation of folate‐PAMAM for the delivery of antisense oligonucleotides to rat C6 glioma cells in vitro and in vivo

“…Antisense strategies include the use of antisense oligodeoxynucleotides (ASODNs), antisense RNAs and ribozymes. ASODNs or expression vectors containing genes or gene fragments in the antisense orientation have been successfully developed for this purpose 6–8. Our previous study demonstrated that both antisense and siRNA approaches targeting the intracellular region of human EGFR could inhibit EGFR expression, thus exerting a suppressive effect on both U251 and C6 glioma cell growth in vitro and in vivo 5…”