Antisense oligonucleotides targeting angiotensinogen: insights from animal studies.

Angiotensinogen (AGT) is the unique substrate of all angiotensin peptides. We review the recent preclinical research of AGT antisense oligonucleotides (ASOs), a rapidly evolving therapeutic approach. The scope of the research findings not only opens doors for potentially new therapeutics of hypertension and many other diseases, but also provides insights into understanding critical physiological and pathophysiological roles mediated by AGT.

High Boron-loaded DNA-Oligomers as Potential Boron Neutron Capture Therapy and Antisense Oligonucleotide Dual-Action Anticancer Agents.

Boron cluster-modified therapeutic nucleic acids with improved properties are of interest in gene therapy and in cancer boron neutron capture therapy (BNCT). High metallacarborane-loaded antisense oligonucleotides (ASOs) targeting epidermal growth factor receptor (EGFR) were synthesized through post-synthetic Cu (I)-assisted "click" conjugation of alkyne-modified DNA-oligonucleotides with a boron cluster alkyl azide component. The obtained oligomers exhibited increased lipophilicity compared to their non-modified precursors, while their binding affinity to complementary DNA and RNA strands was slightly decreased. Multiple metallacarborane residues present in the oligonucleotide chain, each containing 18 B-H groups, enabled the use of IR spectroscopy as a convenient analytical method for these oligomers based on the diagnostic B-H signal at 2400-2650 cm-1. The silencing activity of boron cluster-modified ASOs used at higher concentrations was similar to that of unmodified oligonucleotides. The screened ASOs, when used in low concentrations (up to 50 µM), exhibited pro-oxidative properties by inducing ROS production and an increase in mitochondrial activities in HeLa cells. In contrast, when used at higher concentrations, the ASOs exhibited anti-oxidative properties by lowering ROS species levels. In the HeLa cells (tested in the MTT assay) treated (without lipofectamine) or transfected with the screened compounds, the mitochondrial activity remained equal to the control level or only slightly changed (±30%). These findings may be useful in the design of dual-action boron cluster-modified therapeutic nucleic acids with combined antisense and anti-oxidant properties.

Preclinical evaluation of the toxicological effects of a novel constrained ethyl modified antisense compound targeting signal transducer and activator of transcription 3 in mice and cynomolgus monkeys.

ISIS 481464 is a constrained ethyl (cEt) modified phosphorothioate antisense oligonucleotide (ASO) targeting signal transducer and activator of transcription 3 (STAT3) studied in mice and monkey to support oncology clinical trials. Six-week toxicology studies were performed in mice and cynomolgus monkey (up to 70 and 30 mg/kg/week respectively). Reduction in STAT3 protein up to 90% of control was observed in monkey. Cynomolgus monkey was considered the most relevant species to human with respect to pharmacokinetic properties, but mice are useful in their relative sensitivity to the potential proinflammatory and hepatic effects of oligonucleotides. In monkeys, there was no impact on organ function at doses up to 30 mg/kg/week for 6 weeks. Minimal to slight proximal tubular epithelial cell degeneration and regeneration within the kidney was observed, which had no impact on renal function and showed reversibility at the end of the treatment-free period. Additionally, mild and transient activated partial thromboplastin time elevations and mild increases in complement Bb were observed at the higher doses by intravenous dosing only. In mice, the alterations at 70 mg/kg/week included spleen weight increase up to 1.4-fold relative to control, increases in alanine aminotransferase and aspartate aminotransferase up to 1.8-fold over control, interleukin-10 increases up to 3.7-fold, and monocyte chemoattractant protein-1 increase up to 1.9-fold over control. No significant clinical pathology or histopathology changes were seen in mice at 20 mg/kg/week or less. The toxicity profile of ISIS 481464 is consistent with effects observed with phosphorothioate ASOs containing 2'-O-methoxyethylribose modifications instead of cEt.

The interactions of amphiphilic antisense oligonucleotides with serum proteins and their effects on in vitro silencing activity.

Antisense oligonucleotides (AONs) are a class of compounds with high therapeutic potential. One of the challenges facing this platform is the development of effective techniques to achieve cellular delivery. AON conjugates, in which traditional AONs are attached to certain biomolecules, can exhibit improved intracellular bioavailability in the absence of delivery systems. In this study, the lipophilic moieties docosahexaenoic acid, cholesterol, and docosanoic acid (DSA) were conjugated to various phosphorothioated DNA and chemically-modified 2'-fluoro-arabinonucleic acid AONs via an amino-hexanol-linker added to the 5'-end of the molecule. The gene silencing potential of these compounds was evaluated in vitro in the absence or presence of a transfecting agent (polyion complex micelle). Incubation with sub-micromolar concentration of DSA-conjugates could, in the absence of serum proteins, downregulate more than 60% of the targeted mRNA under carrier-free and carrier-loaded delivery methods. Gene silencing activity of carrier-free DSA-conjugates was, however, decreased in a dose-dependent fashion by adding albumin in the transfection medium. Supplementing the medium with free fatty acid prevented the interaction of the DSA-conjugate with albumin, and restored its silencing activity. These findings suggest that strategies aiming at preventing the association of hydrophobized AONs to serum proteins at the site of action may improve their activity.

PRO-051, an antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy.

PRO-051 (GSK-2402968), being developed by GlaxoSmithKline plc, under license from Leiden University Medical Center and Prosensa Therapeutics BV, is a 2'-O-methyl phosphorothioate antisense oligonucleotide for the potential treatment of Duchenne muscular dystrophy (DMD). The PRO-051 oligonucleotide sequence induces skipping of exon 51 of the dystrophin gene by binding to a sequence within the dystrophin pre-mRNA and masking the exon inclusion signals that are used for splicing. Removal of exon 51 from an exon 45 to 50, 47 to 50, 48 to 50, 49 to 50, 50, 52 or 52 to 63 deleted transcript allows restoration of the open reading frame and synthesis of an internally truncated, semi-functional dystrophin protein. By targeting exon 51, approximately 13% of patients with DMD could be treated, the largest proportion of patients that could benefit from targeting a single dystrophin exon. A proof-of-concept clinical trial of PRO-051 in patients with DMD demonstrated that a single intramuscular administration of PRO-051 induced exon skipping within muscle fibers adjacent to the injection site, while biopsies revealed dystrophin expression in treated but not control muscle fibers. At the time of publication, a phase I/IIa trial to evaluate subcutaneous delivery of PRO-051 had been completed, although full results were yet to be published.

Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases.

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A(16)). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.

Fine tuning of electrostatics around the internucleotidic phosphate through incorporation of modified 2',4'-carbocyclic-LNAs and -ENAs leads to significant modulation of antisense properties.

In the antisense (AS) and RNA interference (RNAi) technologies, the native single-stranded 2'-deoxyoligonucleotides (for AS) or double-stranded RNA (for RNAi) are chemically modified to bind to the target RNA in order to give improved downregulation of gene expression through inhibition of RNA translation. It is shown here how the fine adjustment of the electrostatic interaction by alteration of the substituents as well as their stereochemical environment around the internucleotidic phosphodiester moiety near the edge of the minor grove of the antisense oligonucleotides (AON)-RNA heteroduplex can lead to the modulation of the antisense properties. This was demonstrated through the synthesis of various modified carbocyclic-locked nucleic acids (LNAs) and -ethylene-bridged nucleic acids (ENAs) with hydroxyl and/or methyl substituents attached at the carbocyclic part and their integration into AONs by solid-phase DNA synthesis. The target affinity toward the complementary RNA and DNA, nuclease resistance, and RNase H elicitation by these modified AONs showed that both the nature of the modification (-OH versus -CH(3)) and their respective stereochemical orientations vis-a-vis vicinal phosphate play a very important role in modulating the AON properties. Whereas the affinity to the target RNA and the enzymatic stability of AONs were not favored by the hydrophobic and sterically bulky modifications in the center of the minor groove, their positioning at the edge of the minor groove near the phosphate linkage resulted in significantly improved nuclease resistance without loss of target affinity. On the other hand, hydrophilic modification, such as a hydroxyl group, close to the phosphate linkage made the internucleotidic phosphodiester especially nucleolytically unstable, and hence was not recommended. The substitutions on the carbocyclic moiety of the carba-LNA and -ENA did not affect significantly the choice of the cleavage sites of RNase H mediated RNA cleavage in the AON/RNA hybrid duplex, but the cleavage rate depended on the modification site in the AON sequence. If the original preferred cleavage site by RNase H was included in the 4-5nt stretch from the 3'-end of the modification site in the AON, decreased cleavage rate was observed. Upon screening of 52 modified AONs, containing 13 differently modified derivatives at C6' and C7' (or C8') of the carba-LNAs and -ENAs, two excellent modifications in the carba-LNA series were identified, which synergistically gave outstanding antisense properties such as the target RNA affinity, nuclease resistance, and RNase H activity and were deemed to be ideal candidates as potential antisense or siRNA therapeutic agents.

Bioconjugation of oligonucleotides for treating liver fibrosis.

Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is urgently needed to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remain the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of alpha1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in-depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed.

Technology evaluation: AVI-4126, AVI BioPharma.

AVI BioPharma is developing AVI-4126, an antisense oligonucleotide targeted to c-myc mRNA for the potential treatment of restenosis, cancer and polycystic kidney disease. AVI-4126 is currently undergoing phase II clinical trials.

Terminally modified oligodeoxynucleotides directed against p53 in an orthotopic xenograft model: a novel adjuvant treatment strategy for pancreatic ductal carcinoma.

OBJECTIVES: Investigation of a terminally modified oligodeoxynucleotide (ODN) directed against p53 mRNA (p53-3' polyethylene glycol-5' tocopherol ODN as a novel drug for pancreatic ductal carcinoma therapy in vitro and in vivo. METHODS: The impact of lipophilic modifications at the 5' end of p53-directed ODNs on cellular uptake was analyzed in vitro using proliferation assays, fluorescence-activated cell sorting analysis, and confocal laser scanning microscopy. The in vivo effects of p53-PT-ODN on the growth of orthotopically xenografted human pancreatic ductal carcinoma cells (PancTuI) were studied in SCID beige mice. Distribution was examined in vitro and in vivo using Cy3-labeled ODNs. RESULTS: Terminally modified p53-PT-ODN showed excellent cellular uptake without using transfection reagents. Microscopically detectable levels of p53-PT-ODN were reached in vivo within 3 hours after intraperitoneal injection, even in extraperitoneal organs. At this time, Cy3-labeled p53-PT-ODN was found in solid tumor formations. We observed a significant inhibition of tumor growth (50%) in vivo at low doses of p53-PT-ODN, whereas at high doses, 2 of 9 animals had no detectable tumors at necropsy. When p53-PT-ODN was injected on the day of tumor cell inoculation, the growth inhibition of solid tumors was significantly stronger compared with that with delayed treatment. CONCLUSIONS: p53-Directed modified ODNs might be of therapeutic value in pancreatic ductal carcinoma, particularly as adjuvant therapy after pancreatic tumor resection.

Efficiency of antisense oligonucleotide drug discovery.

The costs for discovering and developing new drugs continue to escalate, with current estimates that the average cost is more than $800 million for each new drug brought to the market. Pharmaceutical companies are under enormous pressure to increase their efficiency for bringing new drugs to the market by third-party payers, shareholders, and their patients, and at the same time regulators are placing increased demands on the industry. To be successful in the future, pharmaceutical companies must change how they discover and develop new drugs. So far, new technologies have done little to increase overall efficiency of the industry and have added additional costs. Platform technologies such as monoclonal antibodies and antisense oligonucleotides have the potential of reducing costs for discovery of new drugs, in that many of the steps required for traditional small molecules can be skipped or streamlined. Additionally the success of identifying a drug candidate is much higher with platform technologies compared to small molecule drugs. This review will highlight some of the efficiencies of antisense oligonucleotide drug discovery compared to traditional drugs and will point out some of the current limitations of the technology.

Is antisense an appropriate nomenclature or design for oligodeoxynucleotides aimed at the inhibition of HIV-1 replication?

We have evaluated the specificity and the variation in activity against human immunodeficiency virus (HIV) infection of antisense oligodeoxynucleotides (ODNs) with regard to factors such as dose-response range, number and choice of experimental controls, backbone modifications of the ODNs, type of cell infection, length of assays, and delivery approach. The highest level of inhibition was achieved in our long-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free phosphorothioate-modified ODNs (PS-ODNs). The highest level of specificity was observed in our short-term assay with MOLT-3 cells acutely infected with HIV-1 (IIIB) and treated with free PS-ODNs. The highest potency (IC50 level) was observed in our short-term chronic-infection model with (DLS)-delivered ODNs in which the DLS delivery improved the ODN activity up to 106 times compared to the activity of free ODNs. Thus, the near blocking of HIV replication obtained when using PS-ODNs appears because of the addition of extracellular and/or membrane effects. The higher efficacy of PS-ODNs compared to unmodified ODNs, when both are delivered with the DLS system, was demonstrated solely in our short-term assay with MOLT-3 cells. Important variations in the level of sequence specificity were observed and depended on the type of control used and the type of cell assay employed. It seems that all 3 groups of control-tested, random, sense sequence, and non-antisense T30177 ODNs might have distinct activity and, consequently, different modes of action in inhibiting HIV replication. Our data buttress the notion that the contribution of the sequence-specific mediated mode of action is minor compared to the other mechanisms involved in ODN antiviral activity.

Liposomal delivery of a 30-mer antisense oligodeoxynucleotide to inhibit proopiomelanocortin expression.

An oligodeoxynucleic sequence of 30 bases (30-mer ODN), complementary to a region of beta-endorphin mRNA, was synthesized to have an antisense effect with regard to the expression of this oligopeptide. Following the solid-phase synthesis of the oligodeoxynucleotide, the 30-mer ODN was encapsulated within liposomes to provide a higher resistance against DNases and an improved entrance into cells. The most suitable liposome formulation as a 30-mer ODN carrier consisted of small unilamellar vesicles (50 nm) with an encapsulation capacity of 4.76 microL/micromol. The liposomal formulations containing dipalmitoyl-DL-alpha-phosphatidyl-L-serine presented fusogenic properties, which are of great importance for the delivery of antisense compounds. The antisense activity of 30-mer ODN-loaded liposomes was evaluated by the determination of beta-endorphin levels in AtT-20 cells. The free 30-mer ODN did not provide any lowering of the beta-endorphin production, whereas the liposomally entrapped compound elicited a concentration-dependent inhibition. The inhibition was determined by a sequence-specific binding of the 30-mer ODN with the target mRNA.

Kinetics for hybridization of peptide nucleic acids (PNA) with DNA and RNA studied with the BIAcore technique.

The binding of a mixed-sequence pentadecamer PNA (peptide nucleic acid) containing all four nucleobases to the fully complementary as well as various singly mismatched RNA and DNA oligonucleotides has been systematically investigated using thermal denaturation and BIAcore surface-interaction techniques. The rate constants for association (k(a)) and dissociation (k(d)) of the duplex formation as well as the thermal stability (melting temperature, T(m)) of the duplexes have been determined. Upon binding to PNA tethered via a biotin-linker to streptavidin at the dextran/gold surface, DNA and RNA sequences containing single mismatches at various positions in the center resulted in increased dissociation and decreased association rate constants. T(m) values for PNA x RNA duplexes are on average 4 degrees C higher than for PNA x DNA duplexes and follow quantitatively the same variation with mismatches as do the PNA x DNA duplexes. Also a faster k(a) and a slower k(d) are found for PNA x RNA duplexes compared to the PNA x DNA duplexes. An overall fair correlation between T(m), k(a), and k(d) is found for a series of PNA x DNA and PNA x RNA duplexes although the determination of k(a) seemed to be prone to artifacts of the method and was not considered capable of providing absolute values representing the association rate constant in bulk solution.

Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Regulation of transforming growth factor-beta 1 (TGF-beta 1) expression with a novel TGF-beta 1 complementary DNA.

We describe here a novel TGF-beta 1 complementary DNA (antisense oligomer) that is specific for TGF-beta 1 genomic DNA. The TGF-beta 1 antisense oligomer, complementary to the nucleotides flanking the first transcription start site of the human TGF-beta 1 gene and phosphorothioate modified, was efficacious in: a) constraining TGF-beta 1 promoter activity; b) reducing TGF-beta 1 secretion; and c) preventing TGF-beta 1 dependent inhibition of DNA synthesis in TGF-beta sensitive A-549 human adenocarcinoma cells. The biologic activities of the TGF-beta 1 antisense oligomer were sequence specific since neither the TGF-beta 1 sense oligomer nor the TGF-beta 1 missense oligomer prevented TGF-beta 1 expression. Our findings, in addition to demonstrating the efficacy and specificity of the TGF-beta 1 antisense oligomer, suggest that the oligomer might be of value for the treatment of diseases in which TGF-beta 1 overexpression might play a pathogenetic role (e.g., diabetic renal disease, AIDS).

Biotinylated antisense methylphosphonate oligodeoxynucleotides. Inhibition of spliceosome assembly and affinity selection of U1 and U2 small nuclear RNPs.

Methylphosphonate (PC) backbone oligodeoxynucleotides complementary to the 5'-terminal nucleotides of U1 and U2 small nuclear (sn) RNAs do not elicit RNase H action under conditions in which natural (phosphodiester) oligodeoxynucleotides yield extensive RNase H cleavage. We show here that antisense PC oligonucleotides can mask sites in U1 and U2 snRNPs that are required for spliceosome formation. We further report that biotinylated derivatives of antisense PC oligos can be used for affinity selection of U1 and U2 snRNPs.

Antisense oligonucleotide derivatives as gene-targeted drugs.

The strategies and problems involved in designing oligonucleotide derivatives as gene-targeted drugs are discussed. Experiments with isolated and cellular nucleic acids, studies with infected cell cultures, and preliminary animal tests all demonstrate that various derivatives of complementary oligonucleotides (antisense oligonucleotide derivatives) can act as extremely specific and potent inhibitors of gene expression. The design and synthesis of more stable oligonucleotide analogues that can enter mammalian cells and efficiently affect preselected nucleic acids will result in the development of a new generation of drugs, including those with antiviral and anticancer properties.