Oligodeoxynucleoside Phosphorothioates Research Articles

Malaria merozoite surface protein antisense oligodeoxynucleotides lack antisense activity but function as polyanions to inhibit red cell invasion

The effects on malaria parasite growth of antisense and sense oligodeoxynucleoside phosphorothioates based on a merozoite surface protein mRNA was examined. Specific antisense effects of the oligonucleotides could not be demonstrated over three cycles of schizogony or when added as a complex with cationic liposomes. Antisense and sense oligonucleotides however, inhibit merozoite invasion of red blood cells at similar concentrations to dextran sulphate, a polyanion, as determined by microscopy and [ 3H]hypoxanthine incorporation into DNA. Neutralisation of the negative charge on the oligonucleotides by binding to cationic lipid liposomes, prevented the inhibition of merozoite invasion. We postulate that oligonucleotides because of their polyanionic nature interfere with the binding of merozoites to receptors on red blood cells and that polyanions may be useful in malaria therapy.

Delivery to cancer cells of antisense L‐myc oligonucleotides incorporated in fusogenic, cationic‐lipid‐reconstituted influenza‐virus envelopes (cationic virosomes)

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75–150 μl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy. Int. J. Cancer 77:728–733, 1998. © 1998 Wiley-Liss, Inc.

Delivery to cancer cells of antisense L-myc oligonucleotides incorporated in fusogenic, cationic-lipid-reconstituted influenza-virus envelopes (cationic virosomes).

Antisense oligodeoxy-nucleoside phosphorothioates (OPTs) of L-myc were encapsulated into reconstituted influenza-virus-A envelopes (virosomes). The envelopes of the virosomes consisted of a single positively charged (cationic) lipid bilayer. Binding of cationic virosomes to cellular receptors that are membrane glycoproteins or glycolipids containing terminal sialic acid is mediated by the hemagglutinin glycoprotein (HA) of the influenza virus. After internalization through receptor-mediated endocytosis, cationic virosomes fuse efficiently with the membranes of the endosomal-cell compartment, and as a consequence the encapsulated OPT are delivered to the cell cytoplasma. Examination by fluorescence microscopy of the cellular uptake of cationic virosomes containing fluorescein-labeled OPT showed rapid and efficient incorporation of virosomes. Addition of cationic virosomes (75-150 microl) containing antisense L-myc OPT in the picomolar range to small-cell-lung-cancer (SCLC) cell cultures that expressed highly the L-myc oncogene led to strong inhibition of thymidine incorporation in a concentration-dependent manner. Virosome-entrapped sense L-myc OPT and random-order OPT had only minimal effects on the thymidine uptake. Cells of SCLC cell line NCI-H82 expressing a very low level of L-myc were not affected by antisense-L-myc virosomes. In Western-blot analysis, expression of L-myc protein was suppressed in the antisense-virosome-treated NCI-H209 cells but not in untreated control NCI-H209 cells. These results suggest that cationic virosomes may have great potential as an efficient delivery system for antisense oligonucleotides in cancer therapy.

Transformed and immortalized cellular uptake of oligodeoxynucleoside phosphorothioates, 3′-Alkylamino oligodeoxynucleotides, 2′- o-methyl oligoribonucleotides, oligodeoxynucleoside methylphosphonates, and peptide nucleic acids

Direct quantitative comparisons of cellular uptake across a wide variety of analogs and cell types are necessary for the design of oligonucleotide diagnostic and therapeutic applications. This work reports quantitative cellular uptake and nuclear localization of [ 14C]oligodeoxynucleoside phosphorothioates (PS), 3′-alkylamino Oligodeoxynucleoside phosphodiesters (PO-NH 2), 2′-O-methyl oligoribonucleoside phosphodiesters (2OM), peptide nucleic acids (PNA), and oligodeoxynucleoside methylphosphonates (MP) in several transformed or immortalized cell lines. All analogs demonstrated active cellular uptake in that intracellular concentrations greatly exceeded the extracellular 1 μM concentration within 1–3 hr. However, by 9–24 hr, cellular accumulations of PS exceeded those of PO-NH 2 and 2OM by 3- to 5-fold, PNA by 6- to 7-fold, and MP by 8- to 10-fold. Similar results were observed in two transformed cell lines, HL-60 leukocytes and H- ras transformed fibroblasts, using three different heterogeneous sequences. H- ras and IGF-1R transformed fibroblasts had a 2- to 5-fold higher uptake of all analogs than non-transformed immortalized fibroblasts. Nuclear levels of the PO-NH 2, PS, and MP analogs were approximately 25% of total cellular uptake, while nuclear percentages of 2OM and PNA were less than 20%, suggesting some differences in nuclear localization among the analogs. These observations provide a direct quantitative comparison of cellular uptake as a function of oligonucleotide modification, and imply that transformation enhances cellular uptake. From the perspective of therapy and diagnosis, clear trade-offs were apparent between efficiency of uptake on the one hand, and nuclease resistance and hybridization strength on the other.

Hybrid oligonucleotides: Synthesis, biophysical properties, stability studies, and biological activity

We have designed and synthesized hybrid oligonucleotides 2–5, as analogues of oligodeoxynucleoside phosphorothioates, in an effort to have agents with improved ‘antisense activity’ with reduced phosphorothioate content. The hybrid oligonucleotides contain segments of 2′- O-methyl ribonucleoside phosphoric diesters and oligodeoxynucleoside phosphorothioates. Thus, compared with the ‘all’ phosphorothioate analogues 1 and 6, the analogues 2–5 showed significantly reduced effect on complement activation. In addition, thermal denaturation studies with complementary RNA revealed that the analogues 2–5 had higher T m compared with that with oligodeoxynucleoside phosphorothioates. Additionally, the RNA component of the oligo/RNA duplex is efficiently cleaved by RNase H, the site of endonucleolytic cleavage being dictated by the length of the oligodeoxynucleoside phosphorothioate segment.

Isolation of Oligodeoxynucleoside Phosphorothioate Diastereomers by the Combination of DEAE Ion-Exchange and Reversed-Phase Chromatography

Abstract In antisense trials, oligodeoxynucleoside phosphorothioates (S-oligo) stereoisomers arise from the thiophosphate R/S configuration. We have explored effective separation of these isomers by the combination of DEAE ion-exchange (IELC) and reversed-phase (RPLC) liquid chromatography. Twenty-six S-oligos and some of the natural counterparts were examined. In the presence of DMTr at the 5′-end, isomers could be separated well by ELC, while RPLC gave good resolution in the absence of this protecting group. Combination of these two different modes leads to more effective isolation.

Anti-Sense Oligodeoxynucleoside Phosphorothioates Nonspecifically Inhibit Invasion of Red Blood Cells by Malaria Parasites

Anti-sense and sense oligodeoxynucleoside phosphorothioates, based on analysis of the secondary structure ofPlasmodium falciparumdihydrofolate reductase -thymidylate synthase mRNA, were synthesized. Their effects onP. falciparumgrowth in vitro were examined by microscopy and [3H] hypoxanthine incorporation. Both anti-sense and sense oligodeoxynucleoside phosphorothioates inhibit invasion of red blood cells by merozoites and this is interpreted as being caused by their polyanionic nature. Specific anti-sense effects of the oligonucleoside phosphorothioates could not however be demonstrated.

Sequence specificity on the growth suppression and induction of apoptosis of chronic myeloid leukemia cells by BCR-ABL anti-sense oligodeoxynucleoside phosphorothioates.

Sequence specificity of inhibitory effects of various BCR-ABL anti-sense oligodeoxynucleoside phosphorothioates (AS PS-ODN) on the proliferation of the chronic myeloid-leukemia cell line BV173 was examined. We confirmed that 26, 18, and 16mer B2A2 AS PS-ODN had strong inhibitory effects on the proliferation of BV173 cells with B2A2 mRNA expression, and that B3A2 AS PS-ODN were equally inhibitory when cultures were initiated at lower cell concentrations. However, at higher cell concentrations, the inhibitory effects by B3A2 AS PS-ODN were reduced and B2A2 AS PS-ODNs could suppress the proliferation of BV173 cells with much more relative sequence specificity. The 26mer B2A2 AS PS-ODN induced apoptosis of BV173 cells following reduction of BCR-ABL mRNA expression and p210 protein synthesis. Various sense (S), reverse order, and random sequences had no inhibitory effects except 16mer B2A2 S and B3A2 S that revealed significant suppressive effects. Furthermore, 26mer B3A2 AS also reduced B2A2 mRNA expression and p210 protein synthesis, while 16mer S sequences did not. These results suggest that B2A2 AS may be cross-reactive with B3A2 AS on the growth suppression of CML cells under certain culture conditions, possibly due to their partial hybridization to the ABL portion of the target mRNA, although other non-sequence-specific mechanisms are also possible.

A study on the use of phenylacetyl disulfide in the solid‐phase synthesis of oligodeoxynucleoside phosphorothioates

AbstractSulfurization of protected [i.e., 2‐cyanoethyl, methyl and 2‐(4‐nitrophenyl)ethyl] internucleosidic phosphite triesters can be conveniently executed with the easily accessible reagent, phenylacetyl disulfide (1). High‐quality oligodeoxynucleotides containing predetermined combinations of natural and phosphorothioate linkages can be obtained via a solid‐phase approach by using reagent 1 and 2‐(4‐nitrophenyl)ethyl‐protected phosphoramidites.

Oligodeoxynucleoside phosphorothioate stability in subcellular extracts, culture media, sera and cerebrospinal fluid

Degradation of a synthetic oligodeoxynucleoside phosphorothioate was studied in six systems used for antisense inhibition experiments. Oligodeoxynucleoside phosphorothioates were degraded very slowly at 37°C in all of the systems studied. Measured half-lives of pentadecamers were 12±1 h in rabbit reticulocyte lysate, 7±1 h in HeLa cell postmitochondrial extract, 14±2 h in RPMI 1640 with 10% fetal bovine serum, 8±1 h in undiluted fetal bovine serum, 9±1 h in adult human serum, and 19±7 h in rat cerebrospinal fluid.