Steffen Schubert

Ribozyme- and deoxyribozyme-strategies for medical applications.

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).

Deletion analysis in the catalytic region of the 10-23 DNA enzyme

In this study, the functional relevance of the core nucleotides of the RNA cleaving 10–23 DNA enzyme (DNAzyme) was investigated. Systematic deletion studies revealed that DNAzymes lacking thymine at position 8 (T8) retain catalytic activity comparable to that of the wild‐type enzyme. Deletion of the adjacent cytosine at position 7 (C7) also resulted in a highly active enzyme and even the double deletion mutant C7/T8 displayed cleavage activity, although the catalytic rate under multiple turnover conditions was found to be reduced by one order of magnitude. The identification of non‐essential nucleotides in the catalytic core might help to stabilize the DNAzyme against nucleolytic degradation and to overcome problems in elucidating its three‐dimensional structure.

Developing an effective RNA interference strategy against a plus-strand RNA virus: silencing of coxsackievirus B3 and its cognate coxsackievirus-adenovirus receptor

Abstract Coxsackievirus B3 (CVB-3) is a plus-strand RNA virus that is believed to be the most common causal agent of viral myocarditis. Since no specific treatment for CVB-3 infections is available to date, we and others have recently started to develop RNA interference (RNAi) approaches to prevent virus propagation. Here we describe our strategy for the development of efficient small interfering RNAs (siRNAs) against viral genomes. Initially, fusion constructs of a reporter (green fluorescent protein) and viral subgenomic fragments were employed to select active siRNAs against the virus. Moreover, in an attempt to achieve sustained virus silencing and reduce the risk of generating escape mutants, only highly efficient siRNAs directed against regions of the viral genome that are unlikely to tolerate mutations were considered for virus inhibition. Two siRNAs directed against the 3D RNA-dependent RNA polymerase were found to inhibit virus propagation by 80–90%. The protective effect of the efficient siRNAs lasted for several days. Furthermore, we have first evidence that inhibition of the cellular coxsackievirus-adenovirus receptor (CAR) by RNAi also reduces the virus titre. Our strategy is likely to be applicable to other (RNA) viruses as well.

Oligonucleotide-Based Antiviral Strategies

In the age of extensive global traffic systems, the close neighborhood of man and livestock in some regions of the world, as well as inadequate prevention measures and medical care in poorer countries, greatly facilitates the emergence and dissemination of new virus strains. The appearance of avian influenza viruses that can infect humans, the spread of the severe acute respiratory syndrome (SARS) virus, and the unprecedented raging of human immunodeficiency virus (HIV) illustrate the threat of a global virus pandemic. In addition, viruses like hepatitis B and C claim more than one million lives every year for want of efficient therapy. Thus, new approaches to prevent virus propagation are urgently needed. Antisense strategies are considered a very attractive means of inhibiting viral replication, as oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The ensuing targeted destruction of viral RNA should interfere with viral replication without entailing negative effects on ongoing cellular processes. In this review, we will give some examples of the employment of antisense oligonucleotides, ribozymes, and RNA interference strategies for antiviral purposes. Currently, in spite of encouraging results in preclinical studies, only a few antisense oligonucleotides and ribozymes have turned out to be efficient antiviral compounds in clinical trials. The advent of RNA interference now seems to be refueling hopes for decisive progress in the field of therapeutic employment of antisense strategies.

Adenovirus vector-mediated RNA interference for the inhibition of human parvovirus B19 replication.

Human parvovirus B19 (B19V) has been considered to cause acute and chronic myocarditis, which is accompanied by endothelial dysfunction. Currently, no causative treatment option for B19V-infections is available. Since RNA interference (RNAi) has proven to be a highly potent antiviral approach, the aim of the current study was to develop an RNAi-based strategy to inhibit B19V replication. Three B19V-VP2-specific short hairpin RNAs (shRNAs) were designed and tested for their silencing activity in reporter assays and the expression cassette of the best one was introduced into an adenoviral shuttle vector (Ad5). B19V-permissive UT7/Epo-S1 cells were infected with B19V and the RNAi triggers were delivered by the adenoviral vector (Ad5shVP2) 24h thereafter. The shRNA targeting the B19V-VP2 gene significantly suppressed VP2 mRNA levels as determined by quantitative RT-PCR. Additionally, also the expression levels of the non-targeted non-structural B19V-NS1 mRNA were strongly reduced. Our results demonstrate that vector-mediated delivery of shRNA expression cassettes targeting the structural B19-VP2 gene is a suitable approach to inhibit B19V replication.

Inhibition of picornaviruses by means of RNA interference

Picornaviruses are a class of RNA viruses with a single-stranded genome in positive orientation. Since the prospects of treatment are limited, we employ RNA interference (RNAi) as an antiviral tool to inhibit different picornaviruses. We identified small interfering RNAs (siRNAs) against the 3D RNA dependent RNA polymerase of coxsackievirus B3 that were capable of efficiently inhibiting the virus. Targeting of the conserved 5 UTR of the virus turned out to be a challenging task since stable structures of this region are detrimental to silencing. We developed a rational strategy to solve this problem and found an siRNA containing locked nucleic acids (LNAs) to possess high antiviral potency. To analyse the mechanism of virus inhibition in more detail, LNAs were incorporated into the siRNA to inactivate either of the siRNA strands. These experiments clearly revealed that only the genomic plus-strand but not the intermediary synthesised minus-strand can be targeted by siRNAs. Furthermore, siRNAs were employed to silence the virus receptor on the host cell and thus prevent viral spread.

Inhibition of coxsackievirus B3 by RNA interference (RNAi)

Here, we demonstrate the potential of RNAi to inhibit a picornavirus, coxsackievirus B3 (CVB3), a major causes of myocardial diseases. Targeting the 3D RNA dependent RNA polymerase (RdRP) with virus specific small interfering RNAs (siRNAs) resulted in a reduction of the virus titre by up to 90%. A detailed analysis of the mechanism of virus silencing with strand-specific siRNAs revealed that only the viral plus-strand can be efficiently targeted in an RNAi approach, whereas the minus-strand is resistant to siRNA-mediated silencing. CVB3 is known to have a high error-rate during replication and can thus be expected to escape RNAi-silencing upon prolonged treatment. We have therefore developed an siRNA double expression vector (SiDEx), which generates two independent siRNAs simultaneously. This vector maintained its silencing capacity against the target RNA with an artificially introduced mutation in a reporter assay, whereas single siRNA-expression vectors lost their capacity to silence their respective target after substitution of a base in the centre of the target site. We will present a recently developed method to drastically facilitate and accelerate the generation of SiDEx vectors. Delivery of the siRNA double expression cassette into neonatal rat cardiomyocytes by an adenovirus-associated virus vector resulted in up to 370 fold reduction of the CVB3 titre. As an alternative approach to cope with the problem of viral escape, we silenced the coxsackievirus-adenovirus receptor on the host cell and achieved a significant reduction of CVB3 propagation.