David M. Tidd

Clinical use of streptolysin-O to facilitate antisense oligodeoxyribonucleotide delivery for purging autografts in chronic myeloid leukaemia

Antisense oligodeoxyribonucleotides (ODN) targeted against the breakpoint in BCR-ABLmRNA will specifically decrease BCR-ABL mRNA, provided cells are first permeabilised with Streptolysin-O (SL-O). We used 18-mer chimeric methylphosphonodiester: phosphodiester linked (4–9-4) ODN complementary to 9 bases either side of the BCR-ABL junction to purge harvests ex vivo in three CML patients who remained completely Ph positive after multiple chemotherapy courses. After CD34+ cell selection and SL-O permeabilisation, harvests were purged with 20 μM ODN. After purging, all individual CFU-GM colonies grown from the two b3a2 breakpoint cases remained positive for BCR-ABL mRNA. In contrast, all 24 colonies grown from the b2a2 breakpoint case were BCR-ABL mRNA negative. Patients were conditioned with busulphan 16 mg/kg. The initial post-transplant course was uneventful, although the time to return to 0.5 × 109/l neutrophils was slow at 25–51 days. Both chronic phase patients remain in haematological remission at +724 and +610 days, although each has cytogenetic evidence of relapse. The b2a2 accelerated phase patient died of myeloid blast transformation at day +91. The present SL-O-facilitated ODN purging strategy appears to be without significant toxicity, and offers considerable improvements in ODN delivery to the cytosol.

ENHANCED ANTISENSE EFFECTS RESULTING FROM AN IMPROVED STREPTOLYSIN-O PROTOCOL FOR OLIGODEOXYNUCLEOTIDE DELIVERY INTO HUMAN LEUKAEMIA CELLS

An improved protocol for delivering oligodeoxynucleotides into the cytoplasm and nucleus of human cells in culture using streptolysin O is described. The new procedure permitted reduced target protein expression to result from antisense suppression of target mRNA levels.

Preclinical studies of streptolysin-O in enhancing antisense oligonucleotide uptake in harvests from chronic myeloid leukaemia patients

Antisense oligodeoxyribonucleotides (ODN) have been shown to produce a sequence-specific cleavage of BCR-ABL mRNA. They may therefore have clinical potential for purging harvests from chronic myeloid leukaemia (CML) patients, prior to autografting. Whilst ODN are highly effective in cell-free systems, their uptake into intact cells is very poor. We have previously reported that reversible permeabilisation of CML cell lines with Streptolysin-O (SL-O) can dramatically increase intracytoplasmic and nuclear ODN uptake. In this study, we examined whether SL-O permeabilisation could be used to enhance ODN uptake into bone marrow (BM) and peripheral blood stem cell (PBSC) harvests from CML patients, without undue toxicity. All 19 harvests studied were from patients in stable chronic phase of CML. Samples studied were either fresh BM harvests following leucoconcentration, fresh PBSC collections, or from previously cryopreserved harvests. Cells were permeabilised by SL-O to load them with fluorescein-labelled ODN. The proportion of permeabilised and viable cells was assessed by fluorescein uptake and propidium iodide exclusion, respectively, by flow cytometry. The effect of SL-O on ODN uptake and cell toxicity was unpredictable on simple mononuclear fractions of harvests. In contrast, SL-O consistently significantly enhanced ODN uptake in samples which were first selected for CD34-positive cells, and this was achieved without either direct toxicity or inhibition of CFU-GM growth. The SL-O concentration required for optimal permeabilisation varied considerably from case to case, in line with previous data on cell lines. PBSC harvests positively selected for CD34-positive cells tended to achieve superior permeabilisation to CD34 positively selected BM harvests. SL-O can be used to safely enhance the intracellular uptake of antisense ODN. This is best achieved on harvests which are first selected for CD34-positive cells.

Specificity of antisense oligonucleotides

Antisense oligodeoxynucleotides that are sufficiently long to specify unique species of mRNA may direct ribonuclease H (RNase H) to cleave nontargeted mRNAs at sites of partial complementarity, both in cell-free systems and in living cells. Specificity of antisense action against selected gene expression may be achieved by increasing the stringency of hybridization under constant physiological conditions through incorporation of a limited number of helix-destabilizing methylphosphonate analogue backbone modifications in the molecules. Chimeric antisense oligodeoxynucleotides with terminal, nonionic methylphosphonodiester linkages were protected from exonuclease degradation, and exhibited reduced sensitivity to endonuclease attack relative to unmodified oligodeoxynucleotides. The reduced hybridization potential of the chimeric oligomers correlated with increased RNase H-mediated antisense activity at the target site in cell-free systems, and reduced rates of cleavage at sites of partial complementarity. Using this strategy, single base discrimination was achieved for destruction of p53 mRNA in living human leukemia cells.

Expression of c- myc is not critical for cell proliferation in established human leukemia lines

BackgroundA study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides.ResultsRNase H-active, chimeric methylphosphonodiester / phosphodiester antisense oligodeoxynucleotides targeting bases 1147–1166 of c-myc mRNA downregulated c-Myc protein and induced apoptosis and cell cycle arrest respectively in cultures of MOLT-4 and KYO1 human leukemia cells. In contrast, an RNase H-inactive, morpholino antisense oligonucleotide analogue 28-mer, simultaneously targeting the exon 2 splice acceptor site and initiation codon, reduced c-Myc protein to barely detectable levels but did not affect cell proliferation in these or other leukemia lines. The RNase H-active oligodeoxynucleotide 20-mers contained the phosphodiester linked motif CGTTG, which as an apoptosis inducing CpG oligodeoxynucleotide 5-mer of sequence type CGNNN (N = A, G, C, or T) had potent activity against MOLT-4 cells. The 5-mer mimicked the antiproliferative effects of the 20-mer in the absence of any antisense activity against c-myc mRNA, while the latter still reduced expression of c-myc in a subline of MOLT-4 cells that had been selected for resistance to CGTTA, but in this case the oligodeoxynucleotide failed to induce apoptosis or cell cycle arrest.ConclusionsWe conclude that the biological activity of the chimeric c-myc antisense 20-mers resulted from a non-antisense mechanism related to the CGTTG motif contained within the sequence, and not through downregulation of c-myc. Although the oncogene may have been implicated in the etiology of the original leukemias, expression of c-myc is apparently no longer required to sustain continuous cell proliferation in these culture lines.

Chimeric oligodeoxynucleotide analogs: chemical synthesis, purification, and molecular and cellular biology protocols.

Publisher Summary The consequences of forming an intracellular antisense oligodeoxynucleotide (ON)-RNA heteroduplex may include inhibition of splicing, or other RNA-processing events, and inhibition of translation. Perhaps the most widely anticipated result is cleavage of the RNA, within the heteroduplex region, resulting from the action of the apparently ubiquitous endogenous enzyme ribonuclease H (RNase H). RNases H have been convincingly demonstrated to mediate many of the antisense effects observed in in vitro systems, following ON microinjection into Xenopus oocytes, eggs, and embryos, and following delivery of antisense effectors into human cells in culture. Clearly, if the transcript becomes degraded through the action of RNase H, then translation of the cognate protein is impossible. This chapter focuses on chimeric ON constructed with terminal methylphosphonodiester sections and a central phosphodiester region. Procedures for the synthesis, fluorescent labeling, purification, and use of chimeric ON are provided. All of the procedures described have been found to be robust and most are generally applicable to a wide range of antisense ON structures.

Identification of a Good C-MYC Antisense Oligodeoxynucleotide Target Site and the Inactivity at This Site of Novel NCH Triplet-Targeting Ribozymes

A region of c-myc mRNA was identified which permitted very efficient antisense effects to be achieved in living cells using chimeric methylphosphonate--phosphodiester antisense effectors. Novel inosine--containing ribozymes (which cleave after NCH triplets) were directed to an ACA triplet within this region and delivered into living cells. No ribozyme intracellular activity could be identified. Very low ribozyme function was also observed in in vitro assays using a 1700nt substrate RNA.

Abrogation of c-MYC protein degradation in human lymphocyte lysates by prior precipitation with perchloric acid

Conventional lysis buffers, though containing cocktails of protease inhibitors, did not prevent the degradation of c-MYC recombinant protein added immediately prior to lysis to cell pellets from human mixed lymphocyte cultures. Treatment of the cells with 4.2% perchloric acid, however, prevented protein degradation and facilitated the detection of c-MYC protein by Western blotting even in unstimulated lymphocytes, where previously it had been reported to be undetectable or barely detectable using this technique. PHA stimulation of lymphocytes induced an approximately six fold increase in measured c-MYC protein within 5 h if cell extracts were prepared using perchloric acid precipitation. However, using conventional lysis buffer the proto-oncogene protein was undetectable until 48-72 h after mitogen addition. Pretreatment with perchloric acid may be useful for Western blotting analysis of protein in other systems where it may be desirable to dispense with the use of toxic protease inhibitors or where these may be incompletely effective.