title: The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses.
abstract: During the course of a study aimed at improving antisense oligodeoxynucleotide-mediated ex vivo bone marrow purging of patients suffering from chronic myeloid leukemia (CML), the properties of a number of antisense structures intended to reduce the expression of c-myc, mutant p53, and bcr-abl mRNAs and proteins were examined. The majority of the antisense oligodeoxynucleotides were designed to be capable of directing ribonuclease H (RNase H) cleavage of their target mRNAs. Streptolysin O (SLO) reversible permeabilization was used to deliver the oligodeoxynucleotides into the CML line KYO-1. We found that the efficiency and specificity of antisense oligonucleotide-induced reductions of target protein expression depended on target protein half-life, the oligonucleotide structure, and the specific sequence within the target mRNA. Transient reductions of c-myc mRNA and protein were achieved with a chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to the initiation codon, but cell proliferation was unaffected. In contrast, a chimeric oligodeoxynucleotide of similar structure targeted to an alternative site in the coding region of c-myc mRNA reduced target mRNA and protein levels for over 24 hours and halted cell proliferation. Chimeric methylphosphonate-phosphodiester oligodeoxynucleotide antisense to a point mutation in KYO-1 p53 mRNA efficiently reduced target mRNA expression, but only small, transient reductions in p53 protein expression were observed. However, a chimeric methylphosphonate-phosphorothioate oligodeoxynucleotide targeted to the same site reduced p53 protein to 30% of control levels over a 48-hour period. BCR-ABL protein expression was unaffected by chimeric oligodeoxynucleotides targeted to the breakpoint in bcr-abl mRNA, even when mRNA levels at early times were substantially reduced.
authors.affiliation: Department of Haematology, University of Liverpool, UK.
authors.forename: D G
authors.initials: DG
authors.lastname: Spiller
authors.forename: R V
authors.initials: RV
authors.lastname: Giles
authors.forename: C M
authors.initials: CM
authors.lastname: Broughton
authors.forename: J
authors.initials: J
authors.lastname: Grzybowski
authors.forename: C J
authors.initials: CJ
authors.lastname: Ruddell
authors.forename: D M
authors.initials: DM
authors.lastname: Tidd
authors.forename: R E
authors.initials: RE
authors.lastname: Clark
mesh_terms: D001483:Base Sequence; D016044:Fusion Proteins, bcr-abl / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D006207:Half-Life; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D016271:Proto-Oncogene Proteins c-myc / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism
chemical_list: D016376:Oligonucleotides, Antisense; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D016044:Fusion Proteins, bcr-abl