Current Pharmaceutical Biotechnology, Vol. 5, No. 4, 2004
Contents
Nucleic
Acid Based Drug as Novel Therapeutics in the Treatment of Human Disease
Guest
Editors: G. Grassi and M. Grassi
DNAzymes:
From Creation In Vitro to Application In Vivo Pp. 321-336
J.C.
Achenbach, W. Chiuman, R.P.G. Cruz and Y. Li
[Abstract]
Deoxyribozymes
as Inhibitors of Vascular Smooth Muscle Cell Growth Pp. 337-339
Levon
M. Khachigian
[Abstract] [Purchase Issue/Articles]
In
Vivo Transfer and Expression of Genes Coding for Short Interfering RNAs Pp. 341-347
Lorena
Zentilin and Mauro Giacca
[Abstract]
Oncogene
Suppression by Small Interfering RNAs Pp. 349-354
Olaf
Heidenreich
[Abstract]
Recent
Applications of RNAi in Mammalian Systems Pp. 355-360
Lisa
Scherer and John J. Rossi
[Abstract]
RNA-Based
Drugs: From RNA Interference to Short Interfering RNAs Pp. 361-368
L.
Poliseno, A. Mercatanti, L. Citti and G. Rainaldi
[Abstract]
Therapeutic
Potential of Hammerhead Ribozymes in the Treatment of Hyper-Proliferative
Diseases Pp. 369-386
G.
Grassi, P. Dawson, G. Guarnieri, R. Kandolf and M. Grassi
[Abstract]
The
Patch Clamp Technique in Ion Channel Research Pp. 387-395
K. Jurkat-Rott and F. Lehmann-Horn
[Abstract] [Purchase Issue/Articles]
General
Principles of Photodynamic Therapy (PDT) and Gastrointestinal Applications Pp. 397-408
Marcus W. Wiedmann and Karel Caca
Abstracts
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DNAzymes: From Creation In Vitro to Application In Vivo
J.C. Achenbach, W. Chiuman, R.P.G. Cruz and Y. Li
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.
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Deoxyribozymes as Inhibitors of Vascular Smooth Muscle Cell Growth
Levon
M. Khachigian
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.
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In Vivo Transfer and Expression of Genes Coding for
Short Interfering RNAs
Lorena Zentilin and Mauro Giacca
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.
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Oncogene Suppression by Small Interfering RNAs
Olaf
Heidenreich
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.
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Recent Applications of RNAi in Mammalian Systems
Lisa Scherer and John J. Rossi
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.
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RNA-Based Drugs: From RNA Interference to Short Interfering RNAs
L. Poliseno, A. Mercatanti, L. Citti and G. Rainaldi
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 off-target 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.
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Therapeutic Potential of Hammerhead Ribozymes in the Treatment of Hyper-Proliferative
Diseases
G.
Grassi, P. Dawson, G. Guarnieri, R. Kandolf and M. Grassi
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.
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The Patch Clamp Technique in Ion Channel Research
K. Jurkat-Rott and F. Lehmann-Horn
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.
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General Principles of Photodynamic Therapy (PDT) and Gastrointestinal Applications
Marcus W. Wiedmann and Karel Caca
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.