Current Drug Targets - Volume 5, Issue 8, 2004

Ribozymes are catalytically active nucleic acids capable of site-specific cleavage of target mRNAs. They have widely been employed as tools in functional studies and for therapeutic purposes. Different classes of ribozymes distinguished by size and mechanism of action have been discovered in natural systems or obtained by in vitro selection. After an introduction to different types of ribozymes with a special focus on the hammerhead and hairpin ribozyme, major challenges in the process of developing ribozymes for medical purposes will be described in the present review. Subsequently, examples of ribozyme applications in animal models for various diseases including cancer, viral infections, rheumatoid arthritis and cardiovascular diseases will be given. The course of phase I and II clinical trials with ribozymes designed to treat patients with virus infections or cancer will be outlined. Finally, the current significance of ribozymes will be discussed in the light of the emergence of new powerful anti-mRNA strategies, particularly RNA interference (RNAi).

Among the technologies available for gene knockdown RNase H-dependent antisense oligonucleotides and RNAi are very popular. Both offer specificity and efficient knockdown of the genes; both are useful tools to study gene functions. Antisense and RNAi methods share many practical problems such as site selection, toxicity at high concentration, and the difficulty of transfection in certain cell types. We will focus in this review on the most important issues in the development of both methods and their possible use in gene-silencing therapy.

The down regulation of gene expression is a promising strategy for molecular medicine and experimental biology. Molecules that bind to the DNA double helix may interfere with gene expression and, in addition to potential therapeutic applications, can be helpful for the investigation of DNA processing, chromatin package, or associated biological processes. Triplex-forming oligonucleotides (TFOs) bind to specific sequences in the DNA double helix via hydrogen bonding interactions. TFOs have been shown to down-regulate gene expression, to induce targeted genomic DNA modifications, to stimulate DNA recombination, and to modulate chromatin organization. Additionally, they may be used as carriers to position DNA-modifying agents to selected sequences. TFO-mediated effects have been mostly described in cell culture, but one study reported TFO activity in a mouse model. Critical issues regarding TFO-based technologies are the development of new oligonucleotide analogues with improved binding affinity, better target selectivity, and sufficient stability in the intracellular environment. A prerequisite for the development of such DNA-binding molecules is the availability of appropriate methods to assess their binding properties quantitatively at the desired target sequence in the genome. This review focuses on recent results regarding gene-inhibitory effects of TFOs in cell culture and methods to evaluate TFO-binding to the desired target sequence in the context of the human genome.

Thioaptamers are thiophosphate ester modified nucleic acids that are isolated via in vitro or bead-based thioaptamer selection against a target molecule such as a protein. Thioaptamers offer advantages over traditional aptamers in their enhanced affinity and specificity and higher stability, largely due to the properties of the sulfur backbone-modifications. An in vitro thioaptamer selection procedure that simultaneously selects for sequence and optimized hybrid phosphoromonothioate or phosphate backbone substitutions is outlined. A novel bead-based thioaptamer selection protocol that can produce mixed phosphorodithioate, phosphoromonothioate or phosphate hybrid backbones is also described. Several examples of thioaptamers targeting specific protein are provided. Such thioaptamers are shown to modulate protein activity in vivo.

Recent progress in cellular and molecular research has provided a new technique to inhibit target gene expression based on DNA technology such as antisense oligodeoxynucleotides (ODN), small interfering RNA (siRNA), ribozyme and decoy ODN. Especially, recently, a successful ODN-based approach termed decoy ODN has used synthetic ODN containing an enhancer element that can penetrate cells, to bind to sequence-specific DNA-binding proteins and interfere with transcription in vitro and in vivo. Transfer of cis-element double-stranded decoy ODN has been reported as a new powerful tool in a new class of anti-gene strategies to treat various diseases as gene therapy or as a research tool to examine the molecular mechanisms of expression of a specific gene. Transfer of double-stranded ODN corresponding to the cis-sequence will result in attenuation of the authentic cis-trans interaction, leading to removal of trans-factors from the endogenous cis-elements with subsequent modulation of gene expression. To date, we have chosen several target transcription factors such as nuclear factor-κB (NF-κB) and E2F to prevent the progression of several diseases including renal diseases. As other targets, we focused on negative regulatory element (NRE) for the renin gene and angiotensinogen gene-activating element (AGE) for the angiotensinogen gene to examine the molecular mechanisms of gene expression, AP-1 and ets-1. In this paper, we introduce the decoy strategy and demonstrate examples of application of decoy ODN approach targeting E2F and NF-κB in renal diseases.

Peptide nucleic acids (PNAs)-DNA chimeras have been recently described as DNA mimics constituted of a part of PNA and of a part of DNA. We have demonstrated that double stranded molecules based on PNA-DNA chimeras bind to transcription factors in a sequence-dependent manner. Accordingly, these molecules can be used for transcription factor decoy (TFD) pharmacotherapy. Effects of double stranded PNA-DNA chimeras targeting NF-kappaB and Sp1 were determined on in vitro cultured human cells and were found to be comparable to those observed using double-stranded DNA decoys. The TFD molecules based on PNA-DNA chimeras can be further engineered by addition of short peptides facilitating cell penetration and nuclear localization. Therefore, these engineered molecules could be of great interest for in vivo experiments for non-viral gene therapy of a variety of diseases, including neoplastic and viral diseases, for which the TFD approach has been already demonstrated as a very useful strategy.

Double-stranded oligodeoxynucleotides (ODN) containing the consensus binding sequence of a transcription factor are valuable tools for the manipulation of gene expression at the transcriptional level by means of the decoy strategy. The approach involves flooding the cells with enough ODN decoy to compete for binding of the transcription factor with its consensus sequence in target genes. The technique has been proven effective in vitro and in vivo, suggesting its use in therapy. Therefore, great attention has been recently focused on chemical modifications enhancing biostability of the oligonucleotides so as to improve their pharmacological properties. Unfortunately, benefits in terms of nuclease resistance have been often negated by a decrease in the affinity and / or specificity of transcription factor binding. To circumvent these problems, circular dumbbell and chimeric decoy oligonucleotides, the latter comprising a central stretch of transcription factor recruiting deoxynucleotides flanked by modified nucleotides, have been introduced. Nevertheless, these approaches have the limitation of leaving the ODNs still sensitive to endonuclease cleavage. To address these concerns, we have recently investigated the use of a new class of nucleotide analogs termed locked nucleic acids (LNA) in the designing of decoy molecules for the transcription factor κB (NF-κB). In this article, we review the advantages of LNA in the design of ODN decoys pointing to the feasibility of introducing modifications in the transcription factor binding sequence in order to obtain molecules with increased stability compared to end-capped ODNs, while remaining efficiently recognized by NF-κB.

Positron emission tomography (PET) and single photon emission tomography (SPECT) are high-resolution, sensitive, molecular and functional imaging techniques that permit repeated, noninvasive assessment and quantification of specific biological and pharmacological processes in humans. PET and SPECT are also the most advanced technologies currently available for studying in vivo molecular interactions and therefore can advantageously play a key role in both drug discovery and development of pharmaceuticals, by assessing their in vivo distribution, pharmacokinetics, and dynamics, once labeled with a positron or γ-emitter. Recent advances in positron and γ-emitter labeling of bioconjugates allow the design and development of complex high-molecular-weight bioactive chemical structures as radiopharmaceuticals including single-stranded oligonucleotides. Besides, the introduction of high-resolution tomographic devices for imaging the distribution of radioactivity in small animal models such as mice and rats offers a unique opportunity to study the biological behavior of labeled compounds in integrated, unperturbed living systems. The present review illustrates the current technologies for labeling oligonucleotides with various radioisotopes, and their use as in vivo probes of oligonucleotide distribution and molecular interactions in target tissues. Emphasis will be also given to the role which could be played by these technologies in the development of oligonucleotide-based drugs.