Therapeutic applications of aptamers

Aptamers constitute a new class of oligonucleotides that have gained therapeutic importance. With the approval of the first aptamer drug, pegaptanib, interest in this class of oligonucleotides, often referred to as ‘chemical antibodies’, has increased. This article discusses aptamers in relation to other oligonucleotide molecules such as antisense nucleotides, short inhibitory sequences, ribozymes and so on. The development of pegaptanib is looked at from the point of view of the challenges faced in converting aptamers into therapeutic molecules. Cases of other aptamers, which show promise as drugs, are discussed in slightly greater detail. Comparison with antibodies and small molecules, which have hitherto held monopoly in this area, is also made.     

2'-Modified oligonucleotides for antisense therapeutics

Chemically modified antisense oligonucleotides are currently progressing in multiple clinical trials. Among several chemical modifications made, modification of the 2'-position has proved most successful. Second generation antisense oligonucleotides incorporating these 2'-modifications exhibit high binding affinity to target RNA, enhanced metabolic stability, and improved pharmacokinetic and toxicity profiles. This is, in part, due to the enhanced biophysical properties of second generation antisense oligonucleotides. 2'-Modifications that influence the sugar to adopt a 3'-endo sugar pucker can improve properties such as affinity. 2'-Modifications that provide a gauche effect and/or a charge effect can play a significant role in the level of nuclease resistance. The heterocyclic base modifications such as 2-thiothymine provides additive effect on the affinity of 2'-F and 2'-O-MOE modifications. This review summarizes the structural and biophysical properties of selected 2'-modified nucleosides which are candidates for use in oligonucleotide therapeutics.     

Non-specific antiviral activity of antisense molecules targeted to the E1 region of human papillomavirus

Antisense phosphorothioate oligonucleotides (ODN1 0x5 OMe) directed against the E1 start region of human papillomavirus 11 (HPV11) can inhibit papillomavirus induced growth of implanted human foreskin in a mouse xenograft model. Administration of a mismatch control oligonucleotide (ODN9 0x5 OMe), in which guanine was replaced with adenine in the same model, had no effect on papilloma induced growth. However, the apparent antiviral activity of ODN1 0x5 OMe was also shown in a lethal mouse cytomegalovirus (CMV) model, in which the oligonucleotides are not expected to have antisense activity. To understand the mechanisms of action of these oligonucleotides, a mismatch oligonucleotide (ODN61 0x5 OMe) was prepared which retained the CpG motifs of ODN1 0x5 OMe. This was tested in the mouse xenograft model and shown to have moderate inhibitory activity. As a definitive experiment, a comparison was made between the efficacy of the active oligonucleotide ODN1 0x5 OMe against two papilloma viruses HPV11 and HPV40. Both these viruses cause benign genital warts, but differ by four bases in their E1 sequence that was the target for ODN1 0x5 OMe. Papillomavirus induced growth in the mouse xenograft model was inhibited by ODN1 0x5 OMe in both cases, suggesting that oligonucleotide molecules have a non-specific antiviral activity that is not directly related to their antisense sequence.     

Tritium labeling of full‐length small interfering RNAs

A simple procedure is described for full‐length single internal [³H]‐radiolabeling of oligonucleotides. Previous labeling strategies have been applied to large molecular weight compounds such as proteins and oligonucleotides, for example, iodination and ¹¹¹In labeling via covalently bounded chelators. However, a procedure has not yet been reported for single internal radiolabeling of oligonucleotides that preserves the molecular structure (³H replacing a ¹H). In following our strategy, the radiolabel can be strategically placed within a stable and predetermined internal position of the siRNA. This placement was accomplished by placing a 5‐bromouridine or 5‐bromo‐2′‐O‐methyluridine phosphoramidite building block into the middle of the antisense strand using standard phosphoramidite chemistry. The deprotected full‐length antisense strand was tritium labeled by bromine/tritium exchange, catalyzed by palladium on charcoal in the predetermined 5‐position of either uridine or 2′‐O‐methyluridine. Internal placement of the building block within the oligonucleotide sequence and label placement at 5‐position decreases the likelihood of the label to be readily cleaved from the oligonucleotide in vivo, and loss of the label by spontaneous tritium/hydrogen exchange. The tritiated single‐stranded and double‐stranded RNAs were also shown to be radio and chemically stable for at least 6 months at ?80 °C. This allows more than sufficient time to conduct pharmaceutical formulation and pharmacokinetic studies. Copyright © 2012 John Wiley & Sons, Ltd.     

Antisense oligonucleotides as emerging drugs

Virtually every major human disease can be characterised by the discordant over-expression of one or more genes whose protein products contribute to the underlying pathophysiology. Antisense oligonucleotides provide a direct means with which to attenuate such discordant gene expression and epigenetically modify disease. Antisense oligonucleotides have thus been called the pharmacology of the future and the next great wave of the biotechnology revolution'. Their attractiveness as pharmaceuticals derives from the fact that they can theoretically modify disease at a point more proximal to its cause - the messenger RNA (mRNA) that acts as the intermediary between the DNA code of the disease gene and its protein effector molecule - than do more traditional 'small molecule' drugs. Such 'small molecule' drugs target proteins already participating in the disease process. In contrast, antisense oligonucleotides prevent the very formation of the disease-associated protein. In addition, the potential specificity and binding avidity of antisense oligonucleotides are orders of magnitude greater than comparable traditional drugs. Their specificity derives from two facts: first, that a single base change is sufficient to destroy the hybridisation potential of an antisense oligonucleotide, making it possible to selectively target viral RNAs, mutant mRNAs (e.g., oncogenes) without affecting their wild-type isomers, or mRNAs of individual members of gene families without cross-reactivity (adenosine A1 receptors vs. A2A, A2B or A3 receptors, for example); and second, that, on a theoretical basis, an antisense oligonucleotide 14 nucleotides in length or longer will be specific for a single gene in human DNA. The avidity of binding of antisense oligonucleotides derives from the combinative strength of hydrogen bonding inherent in Watson-Crick base pairing. In terms of further comparison of antisense oligonucleotides to traditional pharmaceuticals, one might draw the following analogy. The disease-related protein can be considered to resemble a piece of malfunctioning machinery, the gears of which are turning too rapidly, out of control. Traditional 'small molecule' antagonists, then, can be compared to wrenches thrust into the machinery to stop the spinning of its gear mechanism. The antisense oligonucleotide approach is more akin to walking behind the machinery and switching it off. One might consider this a more physiological strategy - more the way nature itself might engineer a response to a disease process. It is at least, teleologically, a most elegant mechanism to intervene in the disease process, if such elegant theory can be reduced to practice. However, significant problems arose in the development of antisense therapeutics, which have only partially been answered to date. Among these, delivery of adequate amounts of material to the target tissue has been especially troublesome. When administered systemically, many applications of antisense oligonucleotides appear to require large doses. Such large doses permit a variety of toxicities that can occur with this class of molecules to become apparent, e.g., certain sequence-dependent, non-antisense effects related to specific sequence motifs within the oligonucleotide ('G strings' - CpG dinucleotide induction of immunoreactivity; [1,2]), as well as certain sequence-independent, non-antisense effects associated with chemical modifications employed to prevent nuclease degradation. Alternative routes of delivery, e.g., inhalation of respirable antisense oligonucleotides, or other means of local administration (topical, intravitreal, etc.) that deliver the drug directly to the target tissue, may permit the full potential of antisense oligonucleotides to be realised [3,4]. It was perhaps because of the fact that the antisense mechanism is so straightforward and understandable that an exaggerated significance was placed on problems that arose during initial attempts to reduce antisense theory to practice. Several observers wrongly inferred from initially confounding data that antisense oligonucleotides could not achieve selective ablation of gene function in vivo. However, with the passage of time, several well-controlled examples of powerful in vivo antisense effects have been published (e.g., [3,5-7]), and at least two antisense oligonucleotides have been proven to have clinical benefit in man, as discussed below. Therapeutic application of antisense oligonucleotides is a field still in its infancy. Yet very significant progress has been made, and at a rate in excess of that observed for most other therapeutic classes. Because there remain significant unmet medical needs that have not proven amenable to traditional drug design strategies, the future of therapeutic antisense oligonucleotides appears promising.     

Medicinal chemistry of antisense oligonucleotides--future opportunities.

The emerging future of antisense oligonucleotides depends on rational modifications of its nucleotide repeating units. Currently the most prevalent design features that are crucial for continued antisense development are nuclease resistance, cellular uptake, hybridization properties, and disruption of RNA functions through terminating events. Very few structure-activity relationship (SAR) studies have been directed to these problems and these have typically used binding affinities and nuclease sensitivities as activity end points rather than in vitro or in vivo biological activities. Further SAR studies may be approached by sequence selection SARs which hold a certain uniform modification type constant (e.g. phosphorothioates) while varying the sequence of the oligonucleotide. The more traditional approach is to modify the oligonucleotide while keeping the sequence constant. This review is concerned with the latter approach and summarizes modifications of the phosphorus atom, pentofuranosyl (sugar) linker, pentofuranosyl ring and its 2'-substituents, and the heterocycles. The review covers the 1989-91 literature of various modified oligonucleotides designed and synthesized to enhance pharmacokinetic and pharmacodynamic properties of antisense oligonucleotides.     

Antisense and/or immunostimulatory oligonucleotide therapeutics

Antisense technology, which is based on a simple and rational principle of Watson-Crick complementary base pairing of a short oligonucleotide with the targeted mRNA to downregulate the disease-causing gene product, has progressed tremendously in the last two decades. Antisense oligonucleotides targeted to a number of cancer-causing genes are being evaluated in human clinical trials. While the first-generation phosphorothioate antisense oligonucleotides are in clinical trials, a number of factors, including sequence motifs that could lead to unwanted mechanisms of action and side effects, have been identified. The severity of the side effects of first-generation antisense oligonucleotides is mostly dependent on the presence of certain sequence motifs, such as CpG dinucleotides. A number of second-generation chemical modifications have been proposed to overcome the limitations of the first-generation antisense oligonucleotides. The safety and efficacy of several second-generation mixed-backbone antisense oligonucleotides are being evaluated in clinical trials. The immune stimulation affects observed with CpG-containing antisense oligonucleotides are being exploited as a novel therapeutic modality, with several CpG oligonucleotides being evaluated in clinical trials. A number of medicinal chemistry studies performed to date suggest that the immunomodulatory activity of CpG oligonucleotides can be fine-tuned by site-specific incorporation of chemical modifications in order to design disease-specific oligonucleotide therapeutics.     

Central administration of small interfering RNAs in rats: a comparison with antisense oligonucleotides

To date there are only few reports of the use of small interfering RNA (siRNA) in whole animals and most of these are restricted to systemic application of siRNAs targeting the liver. In our present studies we have investigated whether siRNAs can be used against a central target after intracerebroventricular (i.c.v.) application and compared their effects with those of antisense oligonucleotides. For this purpose we designed different siRNA and antisense oligonucleotide molecules against the rat hypothalamic melanocortin MC(4) receptor and selected the siRNA and antisense oligonucleotide with the highest efficacy in vitro. We observed that siRNA, encompassing the same gene sequence as antisense oligonucleotides, induced a stronger inhibition of melanocortin MC(4) receptor expression than antisense oligonucleotides. When fluorescence-labeled siRNA were applied i.c.v. in rats no label was detected in brain tissue in spite of the use of cell detergents to improve the delivery. In contrast to these findings the i.c.v. administered fluorescence-labeled antisense oligonucleotides reached the brain structures expressing melanocortin MC(4) receptor and were taken up by the cells in these areas. In summary it seems as if 'naked' antisense oligonucleotides have an advantage over 'naked' siRNA for experiments in vivo. The development of optimized vector systems seems to be a prerequisite before siRNA can be regarded as a suitable experimental tool for in vivo studies.     

Toxicology of antisense therapeutics

Targeting unique mRNA molecules using antisense approaches, based on sequence specificity of double-stranded nucleic acid interactions should, in theory, allow for design of drugs with high specificity for intended targets. Antisense-induced degradation or inhibition of translation of a target mRNA is potentially capable of inhibiting the expression of any target protein. In fact, a large number of proteins of widely varied character have been successfully downregulated using an assortment of antisense-based approaches. The most prevalent approach has been to use antisense oligonucleotides (ASOs), which have progressed through the preclinical development stages including pharmacokinetics and toxicological studies. A small number of ASOs are currently in human clinical trials. These trials have highlighted several toxicities that are attributable to the chemical structure of the ASOs, and not to the particular ASO or target mRNA sequence. These include mild thrombocytopenia and hyperglycemia, activation of the complement and coagulation cascades, and hypotension. Dose-limiting toxicities have been related to hepatocellular degeneration leading to decreased levels of albumin and cholesterol. Despite these toxicities, which are generally mild and readily treatable with available standard medications, the clinical trials have clearly shown that ASOs can be safely administered to patients. Alternative chemistries of ASOs are also being pursued by many investigators to improve specificity and antisense efficacy and to reduce toxicity. In the design of ASOs for anticancer therapeutics in particular, the goal is often to enhance the cytotoxicity of traditional drugs toward cancer cells or to reduce the toxicity to normal cells to improve the therapeutic index of existing clinically relevant cancer chemotherapy drugs. We predict that use of antisense ASOs in combination with small molecule therapeutics against the target protein encoded by the antisense-targeted mRNA, or an alternate target in the same or a connected biological pathway, will likely be the most beneficial application of this emerging class of therapeutic agent.     

Intraocular delivery of oligonucleotides

Anti-mRNA and particularly antisense oligonucleotides are molecules able to inhibit gene expression after intracellular penetration being potentially very interesting for the treatment of ocular diseases where growth factors are involved such as ocular scarring diseases or for the inhibition of viral multiplication. In most cases, the site of action of oligonucleotides has shown to be the posterior segment of the eye and these molecules are injected mainly by the intravitreal route. However, oligonucleotides are poorly stable in biological fluids, have a low intracellular penetration and are quickly eliminated form the vitreous. These issues request repeated administration of oligonucleotides which are able to induce severe damages to the retina. This is the reason why drug delivery systems were developed to improve the stability and intracellular penetration of oligonucleotides and, by sustained release, to increase their long term activity in the treatment of ocular diseases.     

纯合子型家族性高胆固醇血症治疗药物-Mipomersen sodium

Mipomersen sodium是一种apo B-100合成的寡核苷酸抑制剂,是一种具有全新作用机制的降胆固醇药,临床上用于纯合子型家族性高胆固醇血症的辅助治疗。文中对Mipomersen sodium的作用机制、药效学、药代动力学、药物相互作用、临床评价和安全性等进行综述。     

Evaluation of synthetic oligonucleotides as inhibitors of West Nile virus replication

A series of synthetic oligonucleotide phosphorothioate 15-mers were generated against specific sequences in the West Nile virus RNA genome. These antisense oligonucleotides targeted (1) conserved features of the West Nile virus RNA genome that may be expected to lead to inhibition of virus replication since such features play essential roles in the virus lifecycle; (2) G-quartet oligonucleotides with potential facilitated uptake properties and that also targeted conserved sequences among a range of West Nile virus strains. Several formulations with significant in vitro antiviral activity were found. Among the active oligonucleotides were examples that targeted both C-rich RNA sequences of the West Nile RNA genome as well as recognized conserved sequences key to West Nile virus replication. Since the antiviral activity of the latter oligonucleotides diminished upon 2'-O-methyl substitution, it is likely that their activity involves RNase H-catalyzed RNA degradation. One G-rich oligonucleotide that did not target a West Nile virus RNA sequence also was found. These results suggest the potential of antisense strategies for the control of West Nile virus replication if the attendant problem of oligonucleotide delivery can be adequately addressed.     

Microwave accelerated 68Ga‐labelling of oligonucleotides

Oligonucleotides are extensively used for characterization of gene expression in vitro and have now been studied as inhibitors of gene expression in vivo in various diseases. Labelled antisense oligonucleotides are therefore of potential interest for possible in vivo imaging of gene expression, considering the biology of tumors and applications in designing novel molecule‐targeted therapies. In the present work a method of microwave accelerated ⁶⁸Ga‐labelling of oligonucleotides and analysis of the resulting tracers are described. Four modified and functionalized 17‐mer oligonucleotides with a hexylamine group in the 3′‐ or 5′‐position were studied. The oligonucleotides were conjugated to the bifunctional chelator, 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA), and then labelled with ⁶⁸Ga(T_1/2=68 min) using microwave activation. The isolated decay‐corrected radiochemical yields ranged from 30 to 52%. Labelled products were stable in water and ethanol for more than 4 h. The impact of the labelling procedure on the oligonucleotide probes was investigated using hybridization to a complementary 17‐mer sense oligonucleotide in solution. Chemical modification did not influence either the labelling or hybridization ability of the oligonucleotides. The radiolabelled oligonucleotides will be used for the further in vitro and in vivo biology studies. Copyright © 2003 John Wiley & Sons, Ltd.     

Down-regulation of a gene-expression by an antisense BNA oligonucleotide

We recently developed a novel nucleic acid analogue, Bridged Nucleic Acid (BNA), one of the most promising artificial nucleic acids for antisense and/or antigene methodology. The antisense effects of BNA modified oligonucleotides targeting the Internal Ribosomal Entry Site (IRES) in Hepatitis C Virus (HCV) RNA were evaluated. As a result, it was found that the antisense BNA oligonucleotides efficiently suppressed the targeted gene-expression in a sequence specific manner. Although the stem region in the mRNA is generally thought to be out of target for the antisense strategy, BNA oligonucleotide targeting the stem region in the HCV-IRES gave a positive antisense effect, also. It is quite noteworthy.     

Enhanced antisense efficacy of oligonucleotides adsorbed to monomethylaminoethylmethacrylate methylmethacrylate copolymer nanoparticles.

The purpose of this study was the investigation of cationic nanoparticles as drug delivery systems for antisense oligonucleotides. Cationic monomethylaminoethylmethacrylate (MMAEMA) copolymer nanoparticles were prepared from N-monomethylaminoethylmethacrylate hydrochloride and methylmethacrylate. Oligonucleotides were adsorbed onto MMAEMA nanoparticles. Cell penetration was investigated in vitro with fluorescently labeled oligonucleotides and nanoparticles. Antisense effects of oligonucleotides adsorbed to MMAEMA nanoparticles were evaluated by sequence specific inhibition of ecto-5'-nucleotidase expression. The amount of enzyme expressed in PC12 cells was detected and quantified by immunocytochemistry using fluorescein isothiocyanate-labeled antibodies. Oligonucleotides were adsorbed to MMAEMA nanoparticles by the formation of ion-pairs between the positively charged secondary amino groups located on the particle surface and the anionic phosphodiester or phosphorothioate backbones of the oligonucleotides. Adsorption to nanoparticles led to an increased cellular uptake of oligonucleotides and to a significantly enhanced antisense efficacy of unmodified phosphodiester oligonucleotides as well as phosphorothioates. The results of the cell penetration and the antisense assay demonstrated that MMAEMA nanoparticles are promising carriers for oligonucleotide administration.     

Hepatic Distribution and Clearance of Antisense Oligonucleotides in the Isolated Perfused Rat Liver

Purpose. This study was conducted to investigate the impact of backbone modifications on the hepatobiliary disposition of oligonucleotides. Methods. The disposition of backbone-modified antisense oligonucleotides [phosphorothioate (PS) and methylphosphonate (MP)] of the same base-length and sequence (5-TAC-GCC-AAC-AGC-TCC-3), complementary to the codon 12 activating mutation of Ki-ras, was investigated in the isolated perfused rat liver. Livers were perfused for 2 hr; perfusate and bile concentrations were analyzed by HPLC. Hepatocellular distribution was examined by measuring the amount of radiolabeled PS oligonucleotide associated with hepatocytes and Kupffer cells. Protein binding of the PS and MP oligonucleotides was determined in rat serum by ultrafiltration. Results. MP oligonucleotide perfusate concentrations remained constant during the 2-hour perfusion. In contrast, PS oligonucleotide was eliminated slowly by the isolated perfused liver [Cl = 1.05 ± 0.21 mL/min; extraction ratio = 0.06 ± 0.01]. Uptake of PS oligonucleotide by Kupffer cells appeared to exceed uptake by hepatocytes, based on standard cell separation techniques as well as confocal microscopy. The degree of protein binding in rat serum was greater for the PS oligonucleotide (79.9 ± 2.2%) than for the MP oligonucleotide (53.0 ± 4.7%). Conclusions. Backbone modifications significantly influence the hepatic clearance of oligonucleotides. Uncharged MP oligonucleotides are not extracted by the isolated perfused rat liver, whereas the charged PS oligonucleotide is processed more readily.     

Antisense technology: a selective tool for gene expression regulation and gene targeting

This review deals with the antisense technology that, together, forms a very powerful tool to inhibit gene expression and may be used for studying gene function (functional genomics) and for therapeutic purpose (antisense gene therapy). Antisense oligonucleotides block translation of target mRNAs in a sequence specific manner, either by steric blocking of translation or by destruction of the bound mRNA via RNase-H enzyme. For proper designing, accessible sites of the target RNA for binding antisense oligonucleotides have to be identified. Whether being used as an experimental reagent or pharmaceuticals, several problems or drawbacks have to be overcome for successful applications. Toward this direction, various modifications of sugar, bases and phosphate backbone of antisense oligonucleotides have been attempted. In recent years valuable progress has been achieved through the development of advanced cellular delivery systems and novel chemically modified nucleotides with improved properties such as enhanced serum stability, higher target affinity and low toxicity. These qualities and the specificity of binding make this technique a potentially powerful therapeutic tool for gene targeting and/ or expression regulation. This review discusses the basis of structural design, mode of action, chemical modification, enhanced cellular uptake, therapeutic application and future possibilities in the field of advanced antisense technology.