Antisense ribozymes

The development of nucleic acid-based therapeutics is not as straightforward as researchers had initially anticipated. Stability, toxicity, specificity, and delivery of the compounds continue to be challenging issues that need further optimization. In recent years, researchers have come up with intricate solutions that have greatly improved the efficacy of potential antisense, ribozyme, as well as RNAi-based therapeutics. Clinical trials for all these types of nucleic acid-based therapeutics are underway. So far, data from several trials and studies in animal models look promising, in particular, the therapies that trigger the RNAi pathway. However, history has shown that compounds that do well in phase I or phase II clinical trials may still fail in phase III. A striking example is the nonspecific suppression of angiogenesis by siRNA via toII-Iike receptor 3 (Kleinman et al. 2008). It will become clear in the near future which compounds will make it as a new class of antiviral therapeutics. 

Horster, A., Teichmann, B., Homes, R., Grimm, D., Kleinschmidt, J. and Sczakiel, G. (1999) Recombinant AAV-2 harboring gfp-antisense/ribozyme fusion sequences monitor transduction, gene expression, and show anti-HIV-1 efficacy. Gene Then, 6,1231-1238. 

Gene silencing techniques such as antisense, ribozyme, and RNA interference (RNAi) are also powerful tools to determine the transport activity of a specific protein. Flagenbuch et al. [162] have investigated the effect of coinjection of transporter (Ntcp or OatplalJ-specific antisense oligonucleotide on the uptake of BSP and taurocholate... 

Antisense Oligonucleotides. Table 1 Malignant disorders as potential targets for ribozyme gene therapy... 

Kleimnan et al. 2008). In addition, synthetic siRNAs are also subject to degradation in vivo by nuclease activity. Besides side effects and instability, the efficient and specific delivery of the RNAi indncers to the target cell still requires optimization. Here we snmmarize the cnrrent statns of nncleic acid-based antiviral therapentics. The focns will be on antiviral strategies nsing antisense and RNAi technology. Additionally, antiviral ribozymes and aptamers will be discussed briefly, with a focus on recent studies. Gene therapy approaches and delivery systems are the subject of Chapter 11 of this book. 

Antisense RNAs, RNA decoys, ribozymes, small interfering RNAs, and RNA ap-tamers are potential tools in antiviral gene therapy. The application of the antiviral RNAs is described in detail in Chap. 9 of this volnme by J. Haasnoot and B. Berkhont. 

RNAi and ribozymes represent two additional approaches to gene silencing/down-regulation with therapeutic potential. RNAi is an innate cellular process that achieves silencing of selected genes via an anti-sense mechanism. It shares many characteristics with the antisense-based approach described above, but also some important differences, e.g. in the exact mechanism by which the antisense effect is achieved. 

Hughes, M.D., Hussain, M., Nawaz, Q., Sayyed, P., and Akhtar, S. 2001. The cellular delivery of antisense oligonucleotides and ribozymes. Drug Discovery Today 6(6), 303-315. 

Alvarez-Salas, L.M., Benitez-Hess, M.L., and DiPaolo, J.A. 2003. Advances in the development of ribozymes and antisense oligodeoxynucleotides as antiviral agents for human papillomaviruses. Antiviral Therapy 8(4), 265-278. 

For gain-of-function mutations gene-blocking therapy, using antisense oligonucleotides or ribozymes to block gene expression... 

Antigene sequences and ribozymes form two additional classes of antisense agents. However, the therapeutic potential of these agents is only now beginning to be appraised. Certain RNA sequences can function as catalysts. These so-called ribozymes function to catalyse cleavage at specific sequences in a specific mRNA substrate. Many ribozymes will cleave their target mRNA where there exists a particular triplet nucleotide sequence G-U-C. Statistically, it is likely that this triplet will occur at least once in most mRNAs. 

The contraindications to and tolerance of interferon-based therapies in the treatment of HCV infection are similar to those described for patients with HBV infection. As for HBV infection, considerable efforts are being made to develop new agents to improve response rates in patients with HCV infection. Direct antiviral strategies with antisense oligonucleotides, ribozymes, and inhibitors of the viral enzymes— polymerase, helicase, and protease—are under investigation. However, it is likely that interferons will continue to serve as the foundation of therapy for HCV infections, with new agents serving as adjuncts. 

The induction of a loss or reduction of specific function by transgene expression is not trivial and has found restricted applications. However, a few interesting concepts have been realized overexpression of a receptor to reduce free ligand (Hofmann et al., 1988) expression of transgenes encoding dominant negative mutations expression of antisense RNA (or a ribozyme) or expression of a toxic polypeptide. These latter concepts are discussed in more detail. 

In most cases, the DNA inserted into the MCS for expression is not a genomic gene with the original exon/intron configuration, but a cDNA. In exceptional cases, antisense RNA or ribozymes may be expressed. cDNA lacks intronic sequences, but still has the 5 and 3 UTR sequences. As discussed above, the presence of the 3 UTR may reduce the stability of mRNA transcribed from the cDNA insert. Furthermore, the length and secondary structure of the 5 UTR may influence the efficiency of translation. Therefore, it is generally recommended to use cDNA with only short 5 and 3 UTR sequences for expression with expression vectors. 

Cagnon, L. and Rossi, J.J. (2000) Downregulation of the CCR5 beta-chemokine receptor and inhibition of HIV-1 infection by stable VAl-ribozyme chimeric transcripts. Antisense Nucleic Acid Drug Dev., 10, 251-261. 

---