Application Of Antisense Oligonucleotides Research Articles

The concept and application of antisense oligonucleotides.

Since the identification of the double-stranded DNA helix by Watson and Crick in 1953, the knowledge of nucleotide structure and function has been an important potential tool in the study and therapy of disease. There is recent clinical evidence that antisense oligonucleotides may be important therapeutic compounds in the clinical therapy of a range of diseases, including infection (viruses and bacteria), oncology, and inflammation. Our laboratory-based understanding of antisense oligonucleotide activity has provided a foundation for their use in several human diseases. Potentially relevant applications include inflammatory bowel disease therapy, psoriasis, transplantation, rheumatoid arthritis, cytomegalovirus retinitis, hepatitis C, and solid tumor therapy. Here we will outline these applications as well as our ongoing clinical trials for Crohn's disease.

The role of CRH in behavioral responses to stress

Corticotropin-releasing hormone (CRH) and urocortin in the central nervous system affect behavior and can enhance behavioral responses to stressors. The action of CRH-related peptides is mediated through multiple receptors that differ markedly in their pharmacological profiles and anatomical distribution. Comparative pharmacology of CRH receptor agonists suggests that CRH, urocortin, sauvagine and urotensin consistently mimic, and CRH receptor antagonists consistently lessen, functional consequences of stressor exposure. Recently, important advances have been made in understanding the CRH system and its role in behavioral responses to stress by the development of specific CRH receptor antagonists, application of antisense oligonucleotides and development of transgenic mice lacking peptides and functional receptors. This review summarizes recent findings with respect to components of the CRH system and their role in stress-induced behavioral responses.

The role of CRF receptor subtypes in stress-induced behavioural responses

The actions of corticotropin-releasing factor (CRF) and CRF-related peptides in the brain and periphery are mediated through multiple receptors. Two CRF receptor subtypes that differ markedly in their pharmacological profiles and anatomical distribution have been identified and characterized. Important advances have been made in understanding CRF and its actions through the development of specific CRF receptor antagonists, application of antisense oligonucleotides, and the production of transgenic mice lacking functional CRF 1 receptors. This chapter describes recent findings with respect to CRF-related peptides and CRF receptors and their role in stress-induced behaviours.

19] Pharmacokinetic properties of oligonucleotides in brain

The application of antisense oligonucleotides is becoming a popular technique with which to study the function of genes in the brain. These compounds can serve as a useful alternative when conventional pharmacological tools lack adequate specificity or are unavailable. Theoretically, the versatility of this technology is enormous. However, as with any genetic methodology, theoretical success is hindered by numerous practical difficulties. In this chapter we address several potential pitfalls that arise when using antisense oligonucleotides. Importantly, these include the biodistribution of the molecules within the region of delivery, as well as their potential transport to one or more projection nuclei. Also, determination of oligonucleotide pharmacokinetics is essential for effective experimental design and assessing the interval of maximal antisense effects. Furthermore, the stability of the oligonucleotides, which can be altered by biochemical modification (i.e., phosphorothioate substitution), may impact on the temporal development and duration of antisense effects. Finally, it may be important to assess the neuronal—glial interactions in a particular region and the effects of antisense oligonucleotides on gene expression in both cell types.

Antisense Oligodeoxynucleotide Complementary to CXCR4 mRNA Block Replication of HIV-1 in COS Cells

CXCR4 is both a chemokine receptor and an entry co-receptor for the T-cell line-adapted human immunodeficiency virus type 1 (HIV-1). To find a more efficacious therapeutic treatement of acquied immunodeficiency syndrome, we exmined the effects of antisense oligonucleotides on CXCR4 production. COS cells, stably expressing CXCR4 and CD4, were incubated with several kinds of oligonucleotides. Total human p24 antigen production was determined using an enzyme-linked immunosorbent assay system. An antisense phosphorothioate-modified oligonucleotide, complementary to the translation region of the CXCR4 mRNA, showed minimal inhibition of p24 antigen production at the high concentration of 2μM. On the other hand, the antisense phosphorothioate oligonucleotide, when used with transfection reagents, showed high efficiency at low concentrations, and confirmed the sequence-specific action. Interestingly, the oligonucleotide with the natual phosphodiester backbone, when used with the transfection reagents, also had high functional effects, comparable to the modified oligonucleotide. This defines the prerequisite criteria necessary for the design and the application of antisense oligonucleotides against HIV-1 in vivo.

Mechanisms of Neointima Formation—Lessons from Experimental Models

The lamina intima of an artery is the region between the endothelial cell surface and the internal elastic lamina, which forms the luminal margin of the media. In humans the intima of atherosclerosis-prone arteries becomes thicker due to accumulation of smooth muscle cells, which originate from the media. The introduction of percutaneous transluminal coronary angioplasty (PTCA) boosted scientific interest in intimal thickening, because restenosis remains an unresolved problem of this intervention. In order to unravel the mechanisms of intimal thickening there is a need for appropriate animal models. A brief overview of these models is given together with factors that control proliferation and/or migration. Despite intensive research on neointima formation, an effective therapy for restenosis has not emerged to date. This may be due to the fact that other processes, such as acute elastic recoil and chronic constrictive remodeling may contribute to lumen narrowing as well. Other limitations of neointima models are related to species and anatomical differences. Most studies are performed in arteries that are either lesion-free, or contain relatively mild plaques, in contrast to the complicated, stenotic lesions that are the substrate for human PTCA. Other differences are the severity of the injury and incorporation of a mural fibrin-rich thrombus. Nevertheless, studies based on superficial injury, like the frequently used balloon denudation model, are useful. There are similarities with angioplasty, such as endothelial cell damage and proliferation of medial and intimal smooth muscle cells. The use of techniques such as differential display, gene transfer and application of antisense oligonucleotides may provide new therapeutic approaches to reduce neointima formation.

Enhancement of neurite-sprouting by suppression of HPC-1/syntaxin 1A activity in cultured vertebrate nerve cells

HPC-1/syntaxin 1A is a C-terminal anchored neuronal membrane protein, of which all of the N-terminal regions are located on the intracellular side, and it interacts with presynaptic membrane proteins, synaptic vesicle proteins and soluble N-ethylmaleimide-sensitive fusion protein attachment proteins (SNAPs). HPC-1/syntaxin 1A has been proposed to act as a target SNAP receptor (t-SNARE) in the neuron and contributes to the vesicle docking/fusion process during the fast exocytosis at the presynaptic active zone. However, studies using an electron-microscope revealed that HPC-1/syntaxin 1A distributed not only at the presynaptic region but throughout the whole axonal membrane, and the functions of this axonal HPC-1/syntaxin 1A remain completely unknown. To investigate its physiological role, we attempted to inhibit the function of HPC-1/syntaxin 1A in cultured neural cells by following two methods. First, de novo synthesis of HPC-1/syntaxin 1A was inhibited by an application of antisense oligonucleotide in cultured adult rat dorsal root ganglion (DRG) neurons. Second, antibody against HPC-1/syntaxin 1A was applied intra-axonally in the cultured chick retinal ganglion neuron. Both treatments, which were expected to downregulate the function of HPC-1/syntaxin 1A, consistently elicited an enhancement of the axonal sprouting. These results suggest that the axonal HPC-1/syntaxin 1A would physiologically suppress the excess axon-collateral sprouting. Downregulation of HPC-1/syntaxin 1A expression may underlie the control of collateral sprouting and synapse formation during development and memory processes.

Antisense oligonucleotides: towards clinical trials

Antisense oligonucleotides have the ability to selectively block disease-causing genes, thereby inhibiting production of disease-associated proteins. The specificity and application of antisense oligonucleotides have been strongly validated in animal models for various disease targets. Based on the pharmacological, pharmacodynamic and pharmacokinetic profiles, the first generation of antisense oligonucleotides--phosphorothioates--have reached the stage of human clinical trials for various diseases. While ongoing human clinical trials are being carried out to further establishing the safety and efficacy of these oligonucleotides, the experience gained is providing a basis for designing a second generation of antisense oligonucleotides.

Specific suppression of human tumor necrosis factor-alpha synthesis by antisense oligodeoxynucleotides.

Recent clinical studies using neutralizing antibodies point to a key role for tumor necrosis factor-alpha (TNF-alpha) in chronic inflammatory diseases. Antisense technique is a recent approach aiming at inhibition of single proteins. Previously, we described nonspecific induction of TNF by phosphorothioate oligonucleotides. In this study, we established an in vitro model that allows specific inhibition of TNF synthesis, bypassing TNF induction. Freshly isolated human monocytes were incubated with oligonucleotides and the cationic lipid lipofectin in different ratios. TNF synthesis was stimulated with lipopolysaccharide and quantified by a specific radioimmunoassay (RIA). Among all sequences tested, one of the antisense oligonucleotides complementary to the translation initiation region of TNF mRNA (5'-CAT GCT TTC AGT CAT-3') revealed highest efficacy. At 2 microM, the antisense oligonucleotide inhibited TNF synthesis by up to 79%. A concentration as low as 250 nM of the antisense oligonucleotide was effective. Scrambled controls and controls with different, defined degrees of mismatches confirmed a sequence-specific action. Examination with confocal fluorescence microscopy showed a marked difference comparing lipofectin-mediated vs. spontaneous uptake. This study defines criteria that from the prerequisite necessary for design and application of antisense oligonucleotides against TNF in vivo.

Section Review: Biologicals & Immunologicals: Antisense oligonucleotides: Rational drug design for genetic pharmacology

During the seventeen years since the Zamecnik and Stephenson report [77] of antisense oligonucleotide activity, the literature describing efficacy in cell culture and animals has mushroomed. The technology has become far more sophisticated with the application of new methods for rapid oligonucleotide synthesis; for chemical modifications to improve pharmacological properties of the oligonucleotides; for detailed studies at the level of transcription and translation to define the antisense mechanisms of activity; for preclinical pharmacokinetic, pharmacology, and toxicology studies; for detailed and sensitive analysis to define oligonucleotide purity and metabolism; and for large scale synthesis to fill the needs of multiple animal studies and the human clinical studies. Clearly the application of antisense oligonucleotides has helped to validate gene targets for therapeutic intervention, and established that drug intervention can occur at the level of translation. This approach, like that of gene therapy, has heralded the new field of genetic pharmacology; and, like gene therapy, antisense oligonucleotide therapy is now being evaluated in an expanding array of clinical protocols.The focus of this review is to alert those concerned with pharmaceutical drug development to the fact that this technology has momentum and opens versatile avenues to disease control and prevention. The field is still in it infancy and many issues are yet to be addressed. However, by the nature of the opportunity to alter the expression of many genes heretofore unapproachable by traditional methods, antisense oligonucleotide therapy should have a major impact on drug development.

Potential for transcatheter application of antisense oligonucleotides for the treatment of vascular diseases.

Potential for transcatheter application of antisense oligonucleotides for the treatment of vascular diseases.