Determination of optimal sites of antisense oligonucleotide cleavage within TNFalpha mRNA.

Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFalpha mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity.

Oligodeoxynucleotide 5mers containing a 5'-CpG induce apoptosis through a mitochondrial mechanism in T lymphocytic leukaemia cells.

A chimeric methylphosphonodiester/phosphodiester 15mer oligodeoxynucleotide of randomly selected sequence was observed to rapidly induce apoptosis in MOLT-4 and Jurkat E6 T lymphocytic leukaemia cells following intracytoplasmic delivery. A series of further methylphosphonate substitutions and mutations and truncations of the oligodeoxynucleotide served to establish that the phosphodiester-linked sequence CGGTA present in the 15mer was responsible for this biological activity. End-protected CpG oligodeoxy-nucleotide 5mers of sequence type CGNNN exhibited a range of apoptosis-inducing potencies, with CGTTA being the most active. The latter was shown to significantly reduce the rate of RNA synthesis in MOLT-4 cells within 1 h; DNA laddering and redistribution of phosphatidylserine to the outer surface of the plasma membrane were marked by 160 min and mitochondrial transmembrane potential collapsed over roughly the same time scale. Pro-caspase 8 was reduced within 130 min and the proteolytically activated caspase 8 substrate Bid was also down by this time, implicating release of cytochrome c from mitochondria by the active 15 kDa fragment of Bid. Substantial proteolytic activation of pro-caspase 3 was relatively delayed. These findings support a mitochondrial amplification mechanism for apoptosis triggered by CpG 5mers.

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.

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

BACKGROUND: A study was undertaken to resolve preliminary conflicting results on the proliferation of leukemia cells observed with different c-myc antisense oligonucleotides. RESULTS: RNase 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. CONCLUSIONS: We 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.

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.

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-ABL mRNA 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 microM 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 x 10(9)/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.

A potential role for antisense oligonucleotide analogues in the development of oncogene targeted cancer chemotherapy.

The identification of activated oncogenes as the basic biochemical difference between tumour cells and normal cells has opened up the possibility for development of truly tumour specific chemotherapy. It may be hypothesized that malignant cells would revert to a more normal phenotype and might even be triggered into terminal differentiation if expression of the appropriate oncogenes were inhibited. Although, at present, it is not possible to anticipate what form future therapy based upon this approach would take, it is clear that the immediate priority must be to establish the general validity of the hypothesis with a variety of tumour cell types in vitro. For this purpose antisense oligonucleotide analogues appear to offer considerable promise as sequence specific inhibitors of oncogene expression. However, no analogue structure yet devised fulfils all the requirements of an ideal antisense effector in terms of biological stability, cell uptake, non-toxicity, hybridization efficiency and mechanism of action on target nucleic acids. Inhibition of oncogene expression in certain cell types has been reported using antisense oligonucleotides but the technique is not universally applicable and more detailed biochemical investigations of the interactions of oligonucleotides with intact cells are required before improved structures may be developed.

Specific and sensitive combined high-performance liquid chromatographic-flow fluorometric assay for intracellular 6-thioguanine nucleotides metabolites of 6-mercaptopurine and 6-thioguanine.

A new non-radioisotopic technique is described for measuring rates of intracellular formation by human leukemic blasts of 6-thioguanine nucleotide metabolites, obligatory intermediates in the antineoplastic action of both 6-mercaptopurine and 6-thioguanine itself. The method is both specific and sensitive, and involves combined high-performance liquid chromatography and flow fluormetric detection of oxidized 6-thioquanine nucleotides in alkaline permanganate-treated cell extracts. Non-metabolized 6-thioguanine and 6-thioxanthine are also separated and quantitated in this system, permitting complementary in vivo pharmacokinetic analysis. The assay may be applied to detect resistant disease at an early stage in therapy, and thereby provides the opportunity for alternative treatments to be instituted.

Partial protection of oncogene, anti-sense oligodeoxynucleotides against serum nuclease degradation using terminal methylphosphonate groups.

Under certain circumstances sequence-specific inhibition of gene expression may be achieved in intact cells using exogenous anti-sense oligodeoxynucleotides. The efficacy of this approach to investigating gene function is limited in part by the rapid serum nuclease mediated degradation of oligodeoxynucleotides in culture media. In order to determine the relative contributions of 3'-exonuclease, 5'-exonuclease and endonuclease activity in fetal calf serum to oligodeoxynucleotide destruction, we have tested chimeric N-ras anti-sense sequence molecules protected against exonuclease attack with terminal methylphosphonate diester linkages. An 18-mer with two methylphosphonate diester linkages at the 3'-terminus, a 20-mer with two methylphosphonate diester groups at both ends, and the 16-mer 3'-methylphosphonate monoester components of their respective piperidine hydrolysates were totally resistant to venom phosphodiesterase, whereas the 16-mer 3'-hydroxyl components of the hydrolysates were rapidly degraded. Both the chimeric oligodeoxynucleotides and 3'-methylphosphonate monoesters were considerably more stable than normal 3'-hydroxyl oligodeoxynucleotides at 37 degrees C in McCoy's 5A medium containing 15% heat inactivated fetal calf serum. Typically 20-30% of the former (initial concentration 10-100 microM) remained intact at 20 h as compared to the latter which were 88-100% degraded in 4 h and undetectable at 20 h. We conclude that a 3'-phosphodiesterase activity is a predominant nuclease responsible for oligodeoxynucleotide degradation by fetal calf serum, and that for cell culture studies, significant protection of oligodeoxynucleotides may be achieved by incorporating 3'-terminal methylphosphonate diester or even monoester end groups.

Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/phosphodiester structures.

One of the inherent problems in the use of antisense oligodeoxynucleotides to ablate gene expression in cell cultures is that the stringency of hybridization in vivo is not subject to control and may be sub-optimal. Consequently, phosphodiester or phosphorothioate antisense effectors and non-targeted cellular RNA may form partial hybrids which are substrates for RNase H. Such processes could promote the sequence dependent inappropriate effects recently reported in the literature. We have attempted to resolve this problem by using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides. In contrast to the extensive RNA degradation observed with all-phosphodiester oligodeoxynucleotides, highly modified chimeric antisense effectors displayed negligible, or undetectable, cleavage at non-target sites without significantly impaired activity at the target site. We also note that all of the all-phosphodiester oligodeoxynucleotides tested demonstrated inappropriate effects, and that such undesirable activity could vary widely between different sequences.

Enhanced RNase H activity with methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides.

Chimeric oligodeoxynucleotides, comprised of internal phosphodiester and terminal methylphosphonodiester sections, possess many beneficial characteristics as antisense effectors. We have investigated the effects of progressive replacement of phosphodiester by methylphosphonodiester linkages on hybrid stability with complementary RNA and DNA. The melting temperatures (Tms) of oligodeoxynucleotide/RNA heteroduplexes were found to decrease dramatically with increasing methylphosphonate substitution. In contrast, a smaller reduction in Tm was observed for comparable DNA heteroduplexes. This disparate reduction in hybrid stability was found with both the G + C-rich human c-myc and A + T-rich human c-Ha-ras sequences used, suggesting that methylphosphonate oligodeoxynucleotide analogues generally hybridize with less affinity to RNA than DNA. RNase H assays were employed to determine if the noted decreases in Tm impaired the ability of chimeric oligodeoxynucleotides to direct the degradation of RNA. Contrary to expectation, increasing methylphosphonate substitution gave rise to increasing rates of RNA degradation for both the c-myc and c-Ha-ras series. The present results suggest that chimeric oligodeoxynucleotide analogues may be of considerable utility as antisense agents in systems where RNase H is thought to make a major contribution to inhibition of gene expression.

The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells.

Chimeric oligodeoxynucleotides with terminal nonionic methylphosphonate analogue sections and internal phosphodiester regions offer several advantages as antisense effectors over either structure alone. These include enhanced biological stability relative to all-phosphodiester molecules, increased activity in directing ribonuclease H mediated destruction of target RNA, increased specificity and reduced non-specific toxicity. However, another important parameter, the ability of these molecules to enter intact mammalian cells, has not previously been investigated. Therefore, oligodeoxynucleotides were tagged at their 5'-termini with fluorescein reporter groups and a detailed study of uptake kinetics in human leukaemia MOLT-4 cells undertaken by calibrated flow cytometry. Baseline measurements with all-phosphodiester and all-methylphosphonate molecules confirmed that uptake of oligodeoxynucleotides by intact cells is a highly inefficient process. The kinetic data were in agreement with previous reports of mechanisms of cell uptake involving receptor mediated endocytosis in the case of phosphodiester molecules and simple diffusion for methylphosphonates. Chimeric oligodeoxynucleotides exhibited saturable cell uptake kinetics similar to all-phosphodiester oligodeoxynucleotides, suggesting that uptake was receptor-mediated and distinct from concentration-dependent uptake of all-methylphosphonate molecules. Similarly, chimeric molecules were apparently confined to the endosomal compartment within cells. These results imply that reversible masking of the negative charges of the phosphodiester sections of chimeric oligodeoxynucleotides may be required to change the uptake mechanism back to simple diffusion and allow intracellular delivery to the site of the target RNA.

Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with streptolysin O.

Most mammalian cell types appear to take up antisense oligonucleotides and oligonucleotide analogs from the bathing medium by highly inefficient endocytic mechanisms, and most if not all intracellular oligomer is sequestered in vesicles, still separated by a membrane from the target mRNA. On the other hand, oligonucleotides introduced directly into the cytoplasm by microinjection rapidly accumulate in the cell nucleus. Poor delivery to the designated site of action of antisense oligonucleotides is a major problem limiting their routine use in genetic research and their development as potential therapeutic agents. In view of this difficulty, various means of membrane permeabilization were applied to cultured human leukemia cells in an attempt to enhance intracytoplasmic delivery of fluorescein-tagged oligodeoxynucleotides. The outcome of the manipulations was monitored by flow cytometry and fluorescence microscopy. This work has directly confirmed the conclusion suggested by reported antisense effects, that streptolysin O reversibly permeabilizes the plasma membrane toward oligonucleotides and may be utilized to effect biochemical "microinjection" of these molecules directly into the cytoplasm. KY01 myelogenous leukemia cells treated in this way accumulated over 100-fold higher intracellular levels of oligodeoxynucleotides than in the absence of streptolysin O and, in contrast to the latter case, were observed to concentrate internalized molecules in their nuclei.