Current Pharmaceutical Biotechnology - Volume 5, Issue 4, 2004

Despite intensive researches aimed at the treatment of relevant human pathologies such as cancer, cardiovascular and virus mediated diseases, optimal drugs have not yet been identified. In this regards, an attractive and novel therapeutic strategy is based on the use of the so called “nucleic acid based drugs”. These molecules, among which the most actual are represented by ribozymes (Rzs), DNAenzymes (DNAzs) and small interfering RNAs (siRNAs), emerged as potential therapeutics in the last two decades with Rzs (described in Grassi et al. of this theme issue) being the first molecules studied, followed soon after by DNAzs (described in Achenbach et al.; Kachigan of this these issue) and finally by siRNAs (describe in Zentilin et al.; Heidenreich; Scherer et al.; Poliseno et al. of this theme issue), whose therapeutic potential become clear in the last three years. Despite the different mechanisms ruling their biological activities, all these molecules base their action on the ability to recognize, in a sequence specific fashion, a target RNA, triggering its destruction. Thus, they can be used to reduce the intracellular level of a specific RNA coding for a protein which affects cellular metabolism or environment, causing disease. As nucleic acid based drugs can be engineered to reduce the level of virtually any RNA, they have a very broad applicability as therapeutics in several human diseases as detailed in the reviews of this theme issue. Whereas the studies about the therapeutic potential of three classes of molecules, which all base their action on the destruction of deleterious RNAs, may appear redundant, the parallel comparison among them will significantly increase the chances to select the most suitable molecules for each specific disease to be treated. Ribozymes, made by ribonucleotides, have been shown to be effective to knock down gene expression in the cellular environment, however their high costs of synthesis and relative short half-life in the cells, suggested to explore the use of DNAzs which, on the contrary, are made by the less expensive deoxynucleotides. DNAzs, in turn, present the disadvantage of not having the possibility to be expressed from viral vectors, thus significantly limiting their delivery options. Finally, in the case of siRNAs, only limited information about their efficacy is available as investigation on their therapeutic potential just started in the last three years. Preliminary studies, however, indicate that they may work at concentrations significantly lower than Rzs and DNAzs, thus potentially conferring to these molecules higher efficacy. Further studies are required to prove these preliminary observations and to understand whether or not this powerful molecules will become the method of choice for sequence specific knockdowns, or they will just be another tool in the arsenal of nucleic acid based drugs. Another issue, which will require intensive investigation before the practical use of nucleic acid based drugs, deals with the individuation of optimal delivery systems. This consideration rises from the observation that, whereas in in vitro studies these molecules show high biological efficacy, in vivo they tend to have considerably lower effects. To overcome this problem, improvements in the amount of molecules delivered, in the permanence of the active molecule and improvement in the delivery specificity to the diseased cell / tissue, need to be further addressed. In this sense, lentiviral vectors as well as bio-compatible polymers are arising as novel and promising delivery systems for nucleic acid based drugs. Together with the appropriate delivery systems, safety concern related the use of nucleic acid based drug need to be carefully investigated. So far, only limited information are available about the induction of possible unwanted side effects as well as off targeting. In this regards, the evaluation of nucleic acid based drug effects on the expression level of as many as possible cellular genes in parallel, obtained by means of microchips, can contribute to clarify their biological impact on cell biology providing relevant information about their safety. In conclusion, whereas some major aspects about the use of nucleic acid based drugs still need to be investigated, the encouraging results displayed so far together with the broad diffusion and the relevant social impact of the pathologies which may be treated by these molecules, fully justifies further efforts, economic and scientific, to optimize their use and to bring them closer to the clinical practice.

DNAzymes, also known as deoxyribozymes or DNA enzymes, refer to single-stranded DNA molecules with catalytic capabilities. DNAzymes are generated de novo by in vitro selection - a powerful and yet simple technique that has been routinely used to isolate extremely rare DNA or RNA sequences with a function of interest (e.g. ligand-binding or catalysis) from an extraordinarily large population of single-stranded DNA or RNA molecules. Since the report of the first DNAzyme nearly ten years ago, hundreds of DNA sequences have been isolated in many research laboratories around the world to facilitate many chemical transformations of biological importance. In recent years, considerable efforts have been undertaken to assess a variety of DNAzymes for innovation-driven applications ranging from biosensing to gene regulation. This article provides a review on several key aspects of DNAzyme-related research. We will first review in vitro selection techniques used for DNAzyme creation as well as some DNAzymes created for a few representative chemical transformations. We will then discuss recent progresses in studying and developing DNAzymes as reporter molecules for detection-oriented applications, and as therapeutic agents to regulate gene expression at the RNA level. Future outlook on efforts aimed to bring the wonder of catalytic DNA from laboratory curiosity to real world application are also discussed.

DNA enzymes, or DNAzymes, are all-DNA molecules with inherent catalytic activity that bind and cleave at their complementary sequence in the target mRNA through Watson-Crick base pairing. These agents have been successfully used to tease out the role the targeted gene plays in both cellular systems and in a variety of animal models. DNAzymes have the potential to serve as novel nucleic acid-based therapeutic agents in pathologies involving aberrant smooth muscle cell growth and a range of other disorders.

RNA interference can induce potent gene silencing through degradation of complementary mRNA. Short double-stranded interfering RNAs are incorporated into an RNA-induced silencing complex that mediates the recognition and degradation of messenger RNAs in a very targeted manner. Though this phenomenon has been described in mammalian cells only a few years ago, there has been an explosion of interest in using small interfering RNAs to efficiently knockdown genes. Consequently, much effort has been put into the development of systems that allow chip and efficient delivery of these molecules into mammalian cells in vitro and in vivo. To overcome the transient inhibitory effects of transfected RNA molecule synthesis in vitro, expression plasmids, mostly based on RNA polymerase III promoters, have been designed to achieve long-term or stable inhibition of the target genes. Moreover, these expression cassettes have been incorporated into viral vectors to obtain gene silencing also in primary cells refractory to plasmid transfection, and to target specific genes in vivo in animal models. The rapid progression in the field of RNA interference has revolutionized the manner in which gene function is studied and, notably, pharmaceutical companies are already validating this technology for medical applications in the near future.

Almost all human cancers have accumulated multiple genetic lesions including oncogenes. It is often unknown whether an oncogene is continuously required for tumorigenesis. Furthermore, it is very difficult to target an essential oncogene with drugs without affecting the corresponding nonmutated protooncogene or related factors. The recent discovery of RNA interference and the application of small interfering RNAs in mammalian cell culture provide now tools to examine the role of oncogenes in tumor development. Furthermore, oncogene-specific siRNAs may become promising candidates for more cancer-specific therapeutic approaches. This review discusses the potential and the limitations of oncogene-targeting siRNAs and describes examples for the application of siRNAs in the functional analysis of oncogenes.

RNAi is a powerful cellular mechanism that involves targeted destruction of mRNAs. Although the phenomenon was first discovered in plants and lower eukaryotic organisms, it was later discovered as an important genetic regulatory mechanism in mammalian cells. RNAi is triggered by double stranded RNAs that are cleaved into short 21-23 base pair duplexes by an RNAse III type enzyme called Dicer. The short RNAs, termed small interfering RNAs (siRNAs), act as triggers for targeted RNA degradation. One of the two strands is selectively incorporated into a complex of proteins called the RNA induced silencing complex, or RISC. The incoroporated small RNA guides the complex to the complementary target sequence, and this event is followed by endonucleolytic cleavage of the target and recycling of RISC. In mammalian cells, siRNAs do not activate interferon pathway genes, thereby making these powerful tools for sequence specific knockdown of RNAs. In this article we review the methods for programming mammalian cells with siRNAs, and overview a number of applications ranging from targeting oncogenes to inhibiting viral replication. The article also summarizes some important biological conclusions that can be drawn from selective downregulation of certain mRNA targets and addresses potential uses of RNAi as a new thereapeutic modality.

RNA interference consists of a sequence specific post-transcriptional gene silencing phenomenon triggered by a double strand RNA molecule homologous to the silenced gene. The dsRNA is cleaved by DICER enzyme in small dsRNA pieces, named short interfering RNAs (siRNAs). These fragments are thereafter associated to RISC complex where the cleavage of target RNA occurs. The observation that siRNAs can trigger the RNA interference mechanism in mammalian cells represents a fundamental discovery that discloses new horizons in genetic researches in that theoretically each gene can be silenced. The relative simplicity by which active short interfering RNAs can be designed and synthesized explains their widespread use in basic and applied researches, even if appropriate controls that exclude offtarget effects are strictly required. The findings that siRNAs are active even when expressed in viral vectors open the possibility that they can be very soon used for gene therapy of several human diseases.

The limited efficacy of current therapeutic approaches for a number of socially relevant human diseases such as cancer and cardiovascular pathologies, has required the exploration of alternative and more effective therapeutic strategies. In the last two decades, nucleic acid based drugs have emerged as an attractive and novel alternative with great therapeutic potential. Among these molecules, hammerhead ribozymes were the first to be extensively studied and predicted to be of potential practical utility. Hammerhead ribozymes are catalytic RNA molecules capable of inducing the site-specific cleavage of a phosphodiester bond within an RNA molecule. Thus, they can be used to reduce the intracellular level of a specific mRNA coding for a protein which affects cellular metabolism or environment, causing disease. As hammerhead ribozymes can be engineered to reduce the level of virtually any mRNA, they have a very broad applicability. Among the several pathological conditions amenable for a hammerhead ribozyme based therapeutic approach, we focused our attention on pathologies sustained by a dis-regulated and excessive cellular proliferation, being sure to properly demonstrate their usefulness. Trying to be as objective as possible in regard to the feasibility of hammerhead ribozyme employment as therapeutics, a technical section, describing some of the unresolved problems in this field, has been also included. Although some aspects of hammerhead ribozymes as therapeutics can and should be optimized, the encouraging results displayed so far fully justifies further efforts, economic and scientific, to bring them closer to the clinical practice.

To understand the pathogenesis of a given ion channel disorder, knowledge of the mutation alone is insufficient, instead, the description of the associated functional defect is decisive. The patch clamp technique enables to achieve this both in native tissue as well as heterologous expression systems. By this technique, structure-function relationships of ion channels were elucidated that not only support the homology already suggested by amino acid alignments of different channel types, but that also pointed to regions important for gating, ion selectivity, or subunit interaction. Currently, effort is being made to develop automation of the technique which will result in a cost-effective, fast, and highly accurate method to test for drug actions on high throughput scales. This review contains an overview of channel structures, channel diseases, and methods to study channel function by the patch clamp technique.

The purpose of this review article is to describe the history of photodynamic therapy (PDT), its current medical applications, the mechanism of action, contraindications of the method, and different types of photosensitizers used. The second part of the article deals with applications for gastrointestinal diseases. The treatment of obstructing oesophageal cancer, early-stage oesophageal cancer, Barrett's esophagus, hilar cholangiocarcinoma, stomach-, colon- and pancreatic cancer are discussed. The final part focuses on future directions of PDT like certain innovative ideas, which are currently under investigation.