Mixed Backbone Oligonucleotides Research Articles

Electron transfer followed by collision‐induced dissociation (NET‐CID) for generating sequence information from backbone‐modified oligonucleotide anions

Oligonucleotides with 2'-modifications and/or phosphorothioate (PS) backbones are prone to undergo limited backbone fragmentation upon ion trap collision-induced dissociation (CID). For better identification and characterization of chemically modified oligonucleotides, a more universal fragmentation method is desirable. Gas-phase dissociation of various 2'-position-modified oligonucleotides and mixed-backbone oligonucleotides (MBOs) has been studied by ion trap CID of the radical anion species formed via electron transfer ion/ion reactions. For 2'-modified mix-mer radical anions, complete sequence information was generated with non-complementary d/w-ion series, while a/z-ions were observed randomly with relatively low intensity. The 2'-position modification, which has been observed to affect CID patterns of oligonucleotide anions, did not exhibit any observable influence on the dissociation patterns of oligonucleotide radical anions. For MBOs comprised of DNA nucleotides, ion trap CID of even-electron species generated complementary a-B/w-type ions and multiple fragment types at the phosphorothioate (PS) linkages. For MBOs comprised of 2'-OMe-modified nucleotides, only PS bond cleavage was observed for ion trap CID of doubly deprotonated precursor ions. Negative electron transfer reaction with or without supplemental activation of MBOs gave rise to a/d/w-type fragments similar to those of the 2'-modified mix-mers. PS bonds were observed to be more fragile under the electron detachment process, and phosphodiester (PO) bond cleavages were noted upon further collisional activation. NET-CID proved to be an efficient method of generating full sequence information for 2'-modifications and/or mixed-backbone oligonucleotides.

Pharmacokinetics of oligonucleotides.

The effectiveness of antisense oligonucleotides as therapeutic agents depends on their pharmacokinetics, tissue disposition, stability, elimination and safety profile. Pharmacokinetic data allow one to determine the frequency of administration and any potential toxicity associated with chronic administration. Phosphorothioate oligonucleotides degrade from the 3' end, the 5' end, and both the 3' and 5' ends in a time- and tissue-dependent manner. After intravenous administration in mice, rats and monkeys, phosphorothioate oligonucleotides are detected in plasma; they distribute rapidly and are retained in the majority of tissues. The major route of elimination is the urine. The pharmacokinetic profile is similar following subcutaneous, intradermal or intraperitoneal administration, but with lower maximum plasma concentrations. Phosphorothioate oligonucleotides have a short plasma half-life in humans. End-modified, mixed-backbone oligonucleotides (MBOs) contain nuclease-resistant 2'-O-alkylribonucleotides or methylphosphonate internucleotide linkages at both the 3' and 5' ends of phosphorothioate oligonucleotides. These end-modified MBOs have pharmacokinetic profiles similar to those of the parent phosphorothioate oligonucleotides, but they are significantly more stable in vivo and they can be administered orally. Centrally modified MBOs contain modified RNA or DNA in the centre of a phosphorothioate oligonucleotide. They show controlled degradation and elimination following administration in rats. The pharmacokinetics of antisense oligonucleotides depends on the sequence, the nature of the oligonucleotide linkages and the secondary structure.

Synthesis and RNA Binding Selectivity of Oligonucleotides Modified with Five-Atom Thioacetamido Nucleic Acid Backbone Structures

Convenient chemical synthesis and incorporation of dithymidine and thymidine-cytidine dimer blocks connected with a five-atom amide linker N3'-CO-CH2-S-CH2 into oligonucleotides (ONs) are reported. The UV-Tm experiments for binding affinities of these mixed backbone ONs with complementary DNA and RNA sequences revealed important results such as significantly higher RNA-binding selectivity as compared with complementary DNA. NMR studies of the dimer blocks suggested a marginal increase in the N-type sugar conformations over that of the native DNA.

Novel Antisense Anti-MDM2 Mixed-Backbone Oligonucleotides: Proof of Principle, In Vitro and In Vivo Activities, and Mechanisms

The MDM2 oncogene has been suggested as a novel target for cancer therapy, based on the following observations: 1) MDM2 is overexpressed in many human cancers, including breast, colon, and prostate cancer; 2) high MDM2 levels are associated with poor prognosis in patients with cancer; 3) MDM2 overexpression is associated with advanced cancer phenotypes such as metastatic tumors and hormone-independent tumors; 4) MDM2 overexpression is associated with tumor resistance to chemotherapy and radiation therapy; and 5) inhibiting MDM2 expression or function results in tumor growth inhibition and regression. There are many options for inhibiting MDM2 function, including the use of gene silencing technologies, antibodies, peptides and small molecules. Considering the complexity of MDM2 functions, we have chosen to use gene silencing technologies including antisense oligonucleotides and RNA interference. In this article, we summarize the investigation of the antisense technology for inhibiting MDM2 expression. Antisense mixed-backbone oligonucleotides (MBO) specifically inhibit MDM2 expression in a dose- and time-dependent manner, resulting in significant anti-tumor activity in vitro and in vivo. The MBO also potentiates the therapeutic effects of chemotherapeutic agents and radiation therapy in various tumors, through both p53-dependent and p53-independent mechanisms, indicating that MDM2 inhibitors have a broad spectrum of anti-tumor activity in human cancers, regardless of p53 status. These results provide a basis for clinical evaluation of antisense anti-MDM2 oligonucleotides as chemosensitizers and radiosensitizers. In addition, the MBO has been successfully used to identify novel functions of MDM2.

An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the h-Phosphonate approach

The mixture of carbon tetrachloride, N-methyl morpholine (NMM), pyridine and water in acetonitrile has been exploited for the oxidation of dinucleoside H-phosphonate diesters to the corresponding phosphates. The system is found to be inert to the phosphoramidate (P-N) and the phosphorothioate (P-S) linkages and has successfully been applied to the solid phase synthesis of mixed-backbone oligonucleotides (MBOs).

Oligonucleotides comprised of alternating 2′-Deoxy-2′-fluoro-β- d-arabinonucleosides and d-2′-deoxyribonucleosides (2′F-ANA/DNA ‘Altimers’) induce efficient RNA cleavage mediated by RNase H

Chimeric oligonucleotides comprised of alternating residues of 2′-deoxy-2′-fluoro- d-arabinonucleic acid (2′F-ANA) and DNA were synthesized and evaluated for an important antisense property—the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2′F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.

Antisense anti-MDM2 mixed-backbone oligonucleotides enhance therapeutic efficacy of topoisomerase I inhibitor irinotecan in nude mice bearing human cancer xenografts: In vivo activity and mechanisms

Antisense oligonucleotides have been investigated as anticancer agents administered alone or in combination with conventional chemotherapeutics. In the present study, we demonstrated synergistic effects between anti-MDM2 antisense oligonucleotides and the clinically used anticancer agent irinotecan, using nude mouse models of human colon cancers (LS174T and DLD-1). Surprisingly, a 5-base mismatch oligonucleotide also showed similar effects. To elucidate the underlying mechanisms, in vitro and in vivo pharmacokinetic and pharmacodynamic studies were performed. In LS174T cells, the antisense oligonucleotide, but not the mismatch oligonucleotide, specifically inhibited MDM2 expression, resulting in a significant increase in irinotecan-associated p53 activation and p21 induction. In DLD-1 cells, the antisense oligonucleotide specifically inhibited MDM2 expression, resulting in a significant increase in irinotecan-associated p21 induction although mutant p53 levels remained unchanged. Both oligonucleotides increased tissue uptake of irinotecan and the conversion of irinotecan to its active metabolite SN-38. These results suggest that oligonucleotides have a role in irinotecan metabolism and action, providing a basis for future development of antisense oligonucleotides as a sensitizer for irinotecan-based therapy.

Vascular endothelial growth factor (VEGF) is an autocrine growth factor for VEGF receptor-positive human tumors.

Angiogenesis is required for the progression of tumors from a benign to a malignant phenotype and for metastasis. Malignant tumor cells secrete factors such as vascular endothelial growth factor (VEGF), which bind to their cognate receptors on endothelial cells to induce angiogenesis. Here it is shown that several tumor types express VEGF receptors (VEGFRs) and that inhibition of VEGF (VEGF antisense oligonucleotide AS-3) or VEGFRs (neutralizing antibodies) inhibited the proliferation of these cell lines in vitro. Furthermore, this effect was abrogated by exogenous VEGF. Thus, VEGF is an autocrine growth factor for tumor cell lines that express VEGFRs. A modified form of VEGF AS-3 (AS-3m), in which flanking 4 nucleotides were substituted with 2-O-methylnucleosides (mixed backbone oligonucleotides), retained specificity and was active when given orally or systemically in vitro and in murine tumor models. In VEGFR-2-expressing tumors, VEGF inhibition may have dual functions: direct inhibition of tumor cell growth and inhibition of angiogenesis.

Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines.

We have constructed a series of 22 phosphorothioate 20-mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose-dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage-independent growth, however, varied among the EGFR antisense sequences with an IC(50) ranging between 0.5 and 3.5 microM. Two of these antisense oligonucleotides targeting the regions between 2457-2476 and 614-4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs.

Simultaneous blockage of different EGF-like growth factors results in efficient growth inhibition of human colon carcinoma xenografts.

A majority of human colon carcinomas coexpress the epidermal growth factor (EGF)-related peptides transforming growth factor alpha (TGFalpha), amphiregulin (AR) and CRIPTO-1 (CR). We have synthesized novel, antisense mixed backbone oligonucleotides (AS MBOs) directed against TGFalpha, AR and CR. We screened the EGF-related AS MBOs for their ability to inhibit the anchorage independent growth of GEO human colon carcinoma cells. The MBOs that showed a high in vitro efficacy were then used for in vivo experiments. TGFalpha, AR and CR AS MBOs were able to inhibit the growth of GEO tumor xenografts in nude mice in a dose-dependent manner. Furthermore, the AS MBOs were able to specifically inhibit the expression of the target mRNAs and proteins in the tumor xenografts. A more significant tumor growth inhibition was observed when mice were treated with a combination of the three AS MBOs as compared to treatment with a single AS MBO. Finally, tumors from mice treated with TGFalpha, AR and CR AS MBOs showed a significant reduction of microvessel count, as compared with tumors from untreated mice or from mice treated with a single AS MBO. These data suggest that combinations of AS oligonucleotides directed against different growth factors might represent a novel, experimental therapy approach of colon carcinomas.

The impact of molecular medicine upon early cardiovascular drug development.

Heart disease remains the leading cause of death and morbidity in the western world despite significant advances in cardiovascular (CVS) therapeutics. Since the 1960s clinical pharmacologists have been very much involved in bringing forward new classes of CVS drugs. This started with the introduction of β-adrenoceptor blockers in the early 1960s and has continued with calcium antagonists, ACE inhibitors and more recently endothelin antagonists. The principles for classical drug design have been based upon interaction with: receptor sites (β-adreno-ceptor blockers), enzymes (thrombolytics and anticoagulants) and ion channels (calcium antagonists, antiarrhythmics). The advent of molecular biology has added a fourth principle for CVS drug development – that of molecular therapeutics. While classical approaches will still provide new CVS drugs (e.g. platelet IIb/IIIa receptor antagonists and directly acting thrombin inhibitors), there is now anticipation that molecular therapeutics will provide a very specific means of intervention thus potentially avoiding some of the problems associated with the non specificity of conventional approaches, e.g. side-effects associated with lack of cardioselectivity with β-adrenoceptor blockers, risk of bleeding with anticoagulants, cough with ACE inhibitors, controversy following retrospective analysis of the use of calcium antagonists. In the broader sense molecular therapeutics includes both gene therapy and oligonucleotide therapy. However the advances in genetic analysis (identification of polymorphisms associated with diseases) mean that there may be a further impact upon drug development – namely the use of known gene polymorphisms to select patient populations at risk. We will review the status of each of these and prospects for the future.

Mixed-backbone oligonucleotides as second generation antisense drugs

Mixed-backbone oligonucleotides as second generation antisense drugs

Oligonucleotide transport in rat and human intestine Ussing chamber models

Cellular and intestinal absorption of naked oligonucleotides (ONs) is limited and still remains a developmental challenge. A previous report in the literature suggests that ON absorption occurs via a paracellular mechanism. The aim of this study was to test this hypothesis using rat and human intestine in a Ussing chamber and in Caco-2 cells. Transport of a 35S-labelled mixed backbone ON (MBO) across human or rat intestinal tissue or across Caco-2 cells was measured after a 2-h incubation in the presence or absence of increasing MBO concentrations or with uptake inhibitors and enhancers. MBO intestinal absorption was compared with an internal standard, mannitol. 35S-MBO demonstrated very little absorption (<1%) across rat and human intestinal tissues. Transport appeared to be unsaturable up to 500 μM, and relatively insensitive to compounds that opened tight junctions or inhibited P-glycoprotein. However, preliminary studies with Caco-2 cells suggest a possible saturable mechanism at higher ON concentrations. Confocal fluorescence microscopy studies show that fluorescein isothiocyanate (FITC)-MBO was internalized into intestinal cells. Although some differences in ON transport were observed as a function of the transport model, MBO transport was mostly consistent with a transcellular, rather than a paracellular, absorption mechanism. Copyright © 1999 John Wiley & Sons, Ltd.

Oligonucleotide transport in rat and human intestine ussing chamber models.

Cellular and intestinal absorption of naked oligonucleotides (ONs) is limited and still remains a developmental challenge. A previous report in the literature suggests that ON absorption occurs via a paracellular mechanism. The aim of this study was to test this hypothesis using rat and human intestine in a Ussing chamber and in Caco-2 cells. Transport of a (35)S-labelled mixed backbone ON (MBO) across human or rat intestinal tissue or across Caco-2 cells was measured after a 2-h incubation in the presence or absence of increasing MBO concentrations or with uptake inhibitors and enhancers. MBO intestinal absorption was compared with an internal standard, mannitol. (35)S-MBO demonstrated very little absorption (<1%) across rat and human intestinal tissues. Transport appeared to be unsaturable up to 500 microM, and relatively insensitive to compounds that opened tight junctions or inhibited P-glycoprotein. However, preliminary studies with Caco-2 cells suggest a possible saturable mechanism at higher ON concentrations. Confocal fluorescence microscopy studies show that fluorescein isothiocyanate (FITC)-MBO was internalized into intestinal cells. Although some differences in ON transport were observed as a function of the transport model, MBO transport was mostly consistent with a transcellular, rather than a paracellular, absorption mechanism.

Properties of mixed backbone oligonucleotides containing 3′-O-methyl ribonucleosides

Oligonucleotides containing 3′-O-methyl ribonucleosides were synthesized, and their thermal stabilities and global conformations with RNA and DNA have been studied. The duplexes displayed lower T m values as compared to the unmodified ones, and adopted A-conformations. Furthermore, they are not a substrate for RNase H, are slightly resistant to snake venom phosphodiesterase, and are not cleaved by nuclease S 1

Mixed-Backbone Oligonucleotides Containing Segments of Deoxynucleosides Phosphorothioate and 2'-O-Methylribonucleosides Methylphosphonate: Synthesis and Properties

Synthesis of mixed-backbone oligonucleotides containing segments of deoxynucleoside. phosphorothioate and 2'-O-methylribonucleoside methylphosphonate have been achieved by using N-Pent-4-enoyl (PNT) heterocyclic base-protected 2'-O-methylribonucleoside methylphosphonamidites. Use of PNT for heterocyclic base-protection allows the deprotection to be carried out under milder conditions without affecting base labile methylphosphonate internucleotide linkages. New MBOs have reduced phosphorothioate internucleotide linkages while maintaining affinity with target RNA and are stable towards nucleases.

Mixed-Backbone Oligonucleotides Containing Segments of Deoxynucleosides Phosphorothioate and 2'-O-Methylribonucleosides Methylphosphonate: Synthesis and Properties

Synthesis of mixed-backbone oligonucleotides containing segments of deoxynucleoside. phosphorothioate and 2'-O-methylribonucleoside methylphosphonate have been achieved by using N-Pent-4-enoyl (PNT) heterocyclic base-protected 2'-O-methylribonucleoside methylphosphonamidites. Use of PNT for heterocyclic base-protection allows the deprotection to be carried out under milder conditions without affecting base labile methylphosphonate internucleotide linkages. New MBOs have reduced phosphorothioate internucleotide linkages while maintaining affinity with target RNA and are stable towards nucleases.

Mixed-Backbone oligonucleotides as second-generation antisense agents with reduced phosphorothioate-related side effects

Mixed-backbone oligonucleotides containing alternative phosphorothioate and phosphodiester linkages in the 2′-O-methylribonucleosides segment show increased affinity with complementary targets, increased stability towards nucleases in vitro and in vivo, and reduced phosphorothioate-related prolongation of partial thromboplastin time compared to phosphorothioate oligodeoxynucleotides, thereby providing antisense agents with reduced side effects.

Impact of mixed-backbone oligonucleotides on target binding affinity and target cleaving specificity and selectivity by Escherichia coli RNase H

All phosphorothioate mixed-backbone oligonucleotides (MBOs) composed of deoxyribonucleotide and 2′-O-methylribonucleotide segments were studied for their target binding affinity, specificity, and RNase H activation properties. The 2′-O-methylribonucleotide segment, which does not activate RNase H, serves as a high affinity target-binding domain and the deoxyribonucleotide (DNA) segment, which binds to the target with a lower affinity than the former domain, serves as an RNase H-activation or target-cleaving domain. In order to understand the influence of the size and position of the DNA segment of MBOs on RNase H-mediated cleavage of the RNA target, we designed and synthesized a series of 18-mer MBOs with the DNA segment varying from a stretch of two to eight deoxyribonucleotides in the middle, at the 5′-end, or at the 3′-end of the MBOs. UV absorbance melting experiments of the duplexes of the MBOs with the complementary and singly mismatched RNA targets suggest that the target binding affinity of the MBOs increases as the number of 2′-O-methylribonucleotides increases, and that the binding specificity is influenced by the size and position of the DNA segment. Analysis of RNase H assay results indicates that the minimum substrate cleavage site and cleavage efficiency of RNase H are influenced by the position of the DNA segment in the MBO sequence. RNA cleavage efficiency decreases as the position of the DNA segment of the MBO·RNA heteroduplex is changed from the 3′-end to the middle and to the 5′-end of the target strand. Studies with singly mismatched targets indicate that the RNase H-dependent point mutation selectivity of the MBOs is affected by both the position and size of the DNA segment in the MBO sequence.

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.

Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo.