Application of Ribozymes

Because of their ability to cleave a suitable RNA target in trans, ribozymes have great potential as gene inhibitors. Concepts for using ribozymes in gene therapy were developed over the past decade and have been transformed into a number of clinical trials, although success is still limited. In general, ribozyme-based strategies focus on suppression of pathological gene products to treat diseases caused by viral infections or malignancies. The major aim is to block gene expression by interfering with RNA transcription (Figure 5.2.1).

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Fig. 5.2.1. Schematic representation of ribozyme based inhibition of gene expression.

Ribozymes recognize their substrates by Watson-Crick base pairing. By altering substrate recognition sequences target RNA molecules can therefore be cleaved in a highly sequence-specific manner. Among the natural RNA catalysts the hammerhead and the hairpin ribozyme, in particular, are used for this purpose. Because of their simple structure and small size both species are particularly well suited to be engineered for cleavage of a desired RNA (Figure 5.2.2).

In addition to the hammerhead and hairpin ribozyme, nature harbors several other catalytic RNAs with useful activities. Group I introns, in particular, have been studied as tools for therapeutic RNA manipulation. The activity of group I introns includes a two step transesterification with guanosine as co-factor (cis-splicing, Figure 5.2.3). Natural cis-splicing introns can be engineered to perform this reaction in trans and thus gain importance as therapeutic agents to correct genetic disorders (trans-splicing, Figure 5.2.3).

Trans-Splicing ribozymes have been generated initially to repair mutant lacZ transcripts in bacteria [5] and mammalian cells [6], and more recently to amend mutant transcripts associated with myotonic dystrophy [7] and many cancers in mammalian cell lines [8], and with sickle cell anemia in erythrocyte precursors isolated from patients with sickle cell disease [9]. Trans-Splicing group I introns can be designed virtually against any RNA target, because the only conserved nu-cleotide is a uridine 5' to the splice site. Because of the short recognition sequence (6-9 bases), however, non-specific splicing has been detected [6]. Furthermore, the whole 3' fragment which should replace the cleaved mutated sequence (Figure 5.2.3) has to be attached to the approximately 350-nts-long group I intron. De-

Fig. 5.2.2. Consensus sequence of (a) the hammerhead and (b) the hairpin ribozyme for therapeutic RNA cleavage.

pending on the lengths of the transcript and the location of the mutation very large constructs (~500 nts) have to be used.

We sought to develop a system for correction of genetic disorders based on small ribozymes. As discussed above, the endonucleolytic activity of hammerhead and hairpin ribozymes has been used in gene therapeutic approaches. Both ribozymes are small, synthetically available, and easy to handle. Whereas the hammerhead ribozyme cleaves its substrate 200 times faster than it ligates the formed products [10], the hairpin ribozyme is a twofold better ligase than an endonuclease [11]. Because repair requires a tool with both cleavage and ligation activity, the hairpin ribozyme is an appropriate basis for the design of an RNA repair ribozyme. Our strategy involved combination of two catalytic modules in one molecule (twin ri-bozyme) to generate a ribozyme with two processing sites at the target RNA. After removal of a mutated RNA sequence by a two-site cleavage in the first step, a new fragment carrying the correct sequence could be incorporated in the second step, now using the ligation activity of the ribozyme (Figure 5.2.4). If it is possible to combine and control cleavage and ligation activity, a hairpin-derived twin ribozyme might be a potential tool for RNA manipulation in vitro with the possibility in future of applying a similar method in vivo.

Construction of a hairpin ribozyme targeting more than one site within the substrate RNA also yields further insight into the structural limits of hairpin ribozyme-catalyzed RNA processing and can provide information on the structural and mechanistic requirements of catalysis.

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