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Human Molecular Genetics Pages 1649-1653  

Ribozymes as therapeutic tools for genetic disease
Introduction
Ribozymes Can Be Used To Down-Regulate Gene Expression
   Trans-cleaving ribozymes
   Utilization of trans-cleaving ribozymes
   Hammerhead ribozymes can be targeted to dominant mutations
   Future directions
Ribozymes Can Be Used To Repair Mutant RNA Molecules
   Group I intron ribozymes can trans-splice RNA
   Ribozyme-mediated repair of mutant RNA
   Group I intron ribozymes are promising tools for RNA repair
Conclusions
Acknowledgements
References

Ribozymes as therapeutic tools for genetic disease

Ribozymes as therapeutic tools for genetic disease

Leonidas A. Phylactou*, Michael W. Kilpatrick1 and Matthew J.A. Wood

Department of Human Anatomy, Oxford University, South Parks Road, Oxford OX1 3QX, UK and 1Department of Pediatrics, University of Connecticut Health Center, Farmington, CT 06030, USA

Received April 24, 1998

The discovery that RNA can act as a biological catalyst, as well as a genetic molecule, indicated that there was a time when biological reactions were catalysed in the absence of protein-based enzymes. It also provided the platform to develop those catalytic RNA molecules, called ribozymes, as trans-acting tools for RNA manipulation. Viral diseases or diseases due to genetic lesions could be targeted therapeutically through ribozymes, provided that the sequence of the genetic information involved in the disease is known. The hammerhead ribozyme, one of the smallest ribozymes identified, is able to induce site-specific cleavage of RNA, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Its ability to down-regulate gene expression through RNA cleavage makes the hammerhead ribozyme a candidate for genetic therapy. This could be particularly useful for dominant genetic diseases by down-regulating the expression of mutant alleles. The group I intron ribozyme, on the other hand, is capable of site-specific RNA trans-splicing. It can be engineered to replace part of an RNA with sequence attached to its 3[prime] end. Such application may have importance in the repair of mutant mRNA molecules giving rise to genetic diseases. However, to achieve successful ribozyme-mediated RNA-directed therapy, several parameters including ribozyme stability, activity and efficient delivery must be considered. Ribozymes are promising genetic therapy agents and should, in the future, play an important role in designing strategies for the therapy of genetic diseases.

INTRODUCTION

According to the RNA-world view, there was a time when all biological reactions were catalysed by RNA. This theory first received support with the identification of natural catalytic RNA molecules, called ribozymes. RNA catalysis was first described by Altman and Cech with the discovery of RNase P and the group I intron respectively (1,2). This makes RNA the only molecule with information-carrying capacity and inherent catalytic activity. So far, several natural ribozyme motifs have been identified and their physical structure, biological and biochemical properties have been the subject of reviews (3,4). In general, however, natural ribozymes have been divided into groups based on their specialized catalytic properties. The hammerhead, hairpin and hepatitis delta virus (HDV) ribozyme motifs can be characterized by their ability for self-cleavage of a particular phosphodiester bond. These motifs typically are found in virus or viroid RNAs. Group I and group II intron ribozymes, which are found in lower eukaryotes and also in some bacteria, can be characterized by their capacity for self-splicing, by cleavage and ligation of phosphodiester bonds. Finally, the RNase P ribozyme, first identified in Escherichia coli, in conjunction with a protein cofactor is characterized by its ability to cleave a phoshodiester bond of a variety of cellular tRNA precursors. Apart from the RNA activities identified in nature, many others, including amino acid transfer and RNA polymerization, were identified by the process of in vitro evolution (5). This was achieved through random selection of RNA molecules able to catalyse particular reactions.

Although the importance of ribozymes in early evolution is now acknowledged, there has been a considerable effort to study the structure and function of natural ribozymes and convert them into tools for manipulation of RNA. This review will concentrate on the application of trans-acting ribozymes, mainly of hammerhead and group I intron types, to the regulation of gene expression.

RIBOZYMES CAN BE USED TO DOWN-REGULATE GENE EXPRESSION

Trans-cleaving ribozymes

The hammerhead ribozyme, the smallest ribozyme identified, is composed of ~30 nucleotides and is capable of site-specific cleavage of a phosphodiester bond (4). The demonstration that the self-cleaving hammerhead ribozyme can be resolved into a substrate and a catalytic strand (ribozyme) (6) led to much attention being paid to the application of antisense hammerhead ribozymes to biological systems (7-12). The hammerhead ribozyme has been studied extensively to understand the relationship between structure and function, since it provides a very valuable tool for genetic therapy through its RNA-mediated inhibition of gene expression. Such trans-acting ribozymes consist of a catalytic core, flanked by two arms that share complementarity with the RNA sequence to be cleaved (6) (Fig. 1). It is possible that any RNA molecule can be cleaved by a hammerhead ribozyme in trans, provided that it contains a putative cleavage site XUY (where X is any base and Y is any base except G) (13). The Watson-Crick base pairing between ribozyme and substrate, forming helices I and III (Fig. 1), makes the cleavage of target RNA molecules sequence specific and its convenient size and high reactivity (6) makes the hammerhead ribozyme the most widely applied ribozyme in living systems. Moreover, the absence of any conserved bases in helices I and III gives the hammerhead ribozyme the flexibility to target, theoretically, any RNA molecule.


Figure 1. Cleavage of an RNA molecule by a hammerhead ribozyme. The hammerhead ribozyme (bottom strand), bound to its target RNA (top strand), forms a typical three-stem structure (numbered I-III) which leads to the cleavage of the target RNA at the site (indicated with an arrow). The absence of conserved bases in arms I and III of the ribozyme strand provides great flexibility for the selection of appropriate targeted sequence. The only sequence requirement for target RNA selection is the presence of an XUY cleavage site, where X is any base and Y is any base but G. The rest of the conserved bases, necessary for cleavage, are situated in the ribozyme strand.

Similarly to the hammerhead ribozyme, the hairpin ribozyme has been shown to act in a trans-acting fashion. RNA molecules can be targeted by hairpin ribozymes via Watson-Crick base pairing (14). However, substrate requirements are more complex than in the case of hammerhead ribozymes. A GUC triplet flanked by other conserved bases is necessary in the substrate. Additionally, the hairpin ribozyme contains four helices and five loops.

Utilization of trans-cleaving ribozymes

Hammerhead and hairpin ribozymes both have been used extensively to down-regulate cellular and viral gene expression (15,16). Different approaches have been taken to ensure that ribozymes were delivered and expressed appropriately in target cells. Several viral vectors have been developed for gene therapy purposes that can also be used to deliver ribozyme-encoding genes. These include adenoviral, retroviral and adeno-associated viral vectors (17,18). Ribozymes have also been delivered to their target RNAs with other systems such as cationic lipids or transferrin-polylysine conjugates (19-21). More particularly, much progress has been achieved in designing trans-cleaving ribozymes against the human immunodeficiency virus (HIV). Retroviral delivery systems, which contain an RNA genome, have been used extensively to make hammerhead and hairpin ribozymes against HIV RNA targets. Reducing HIV infection by ribozyme cleavage of the HIV RNA is an extremely attractive clinical goal. Inhibition of HIV viral gene expression (e.g. through the down-regulation of gag, rev and env genes) has been shown in a variety of infected cell lines (7,22-25).

Hammerhead ribozymes can be targeted to dominant mutations

Control of gene expression by trans-cleaving ribozymes has the potential to interfere with the expression of undesirable gene products. This could be particularly advantageous for the therapy of dominant-negative genetic disorders where the product of the mutant allele interferes with normal function. Deletions, insertions or point mutations could be targeted using ribozymes. The flexibility in the design of the hammerhead ribozyme arms I and III can be used to cleave the mutant RNA preferentially. Discrimination between mutant and normal RNAs can be more difficult in the situation where the mutation is a single base change. Attempts to design mutation-specific ribozymes have utilized situations where the mutation creates a putative ribozyme cleavage site that is not present in the normal allele (26,27). However, such an approach is limited to those mutations that create a suitable cleavage site. Little attention has been paid to the alternative approach of targeting a mutation via the ribozyme-binding arms rather than the cleavage site. Such an approach depends on the effect of base mispairing between ribozyme and target RNA on the overall cleavage efficiency of the ribozymes (22,28-30).

One of the dominant genetic diseases that can be targeted via this approach is Marfan syndrome (MFS), the most common connective tissue disorder (31). Mutations in the fibrillin gene (FBN1 gene) on chromosome 15 (32) have been identified as the primary lesions in MFS, most of those mutations being base substitutions that seem to act via a dominant-negative mechanism (33-35). The base specificity that the hammerhead ribozyme provides suggests that it might be possible to utilize this system specifically to down-regulate the expression of mutant FBN1 alleles, for example by designing hammerhead ribozymes specific for the mutant allele. Many FBN1 gene mutations result in the synthesis of reduced levels of mutant transcripts (36,37) that exert their effect via a dominant-negative mechanism. It has been shown that a hammerhead ribozyme, targeted to the 5[prime] end of the FBN1 mRNA can reduce the extracellular deposition of the fibrillin protein (fibrillin-1) in cultured fibroblasts (20). Therefore, it may be possible to apply this method to down-regulate preferentially the production of mutant protein and thus restore normal fibrillin-1 function. Alternatively, it might be possible to combine delivery of a ribozyme to ablate completely all endogenous FBN1 expression with delivery of a construct for exogenous expression of normal FBN1. The exogenous FBN1 mRNA could be rendered resistant to ribozyme cleavage whilst retaining the ability to code for wild-type fibrillin-1, by taking advantage of the degeneracy of the genetic code.

Future directions

It is just over 10 years since Cech, Altman and colleagues identified the first catalytic RNA molecules, and since then there has been an increasing interest in using them as agents for regulating gene expression. However, despite the wide application that these molecules have, there are challenges that need to be overcome in order for them to be considered safe in the clinic. As mentioned above, an efficient delivery and expression system is always important to ensure that sufficient ribozyme is delivered and produced inside the cell. As an alternative to the delivery systems described above, pre-synthesized ribozymes can be applied to the cells or animals. In this situation, the ribozymes which are synthesized chemically can be modified so that they are protected from nucleolytic degradation, ensuring that the catalytic efficiency is not reduced (38). The chemical characteristics of these modified ribozymes recently have been reviewed in detail (39,40). Furthermore, the interactions between ribozyme and target should be optimized. For example, co-localization of the ribozyme with the target and accessibility of the ribozyme for binding to the target may influence the overall effect.

Nevertheless, the trans-cleaving ribozymes have a great advantage over proteins for gene therapy since they are less likely to induce a host immune response and they are small, enabling them to be incorporated easily into gene therapy vectors. Their exploitation as tools for RNA-directed therapy should be continued not only for classical genetic diseases but also for those with more complex genetic background. It might be possible that down-regulation of a certain gene may be beneficial for certain polygenic or poorly understood genetic diseases.

RIBOZYMES CAN BE USED TO REPAIR MUTANT RNA MOLECULES

Group I intron ribozymes can trans-splice RNA

An alternative way to interfere with the expression of faulty genes, using ribozymes, is to repair the mutant RNA. Although the group I intron ribozyme was the first catalytic RNA molecule to be identified (2), it was only recently that its natural self-splicing ability was exploited to trans-splice RNA targets. The self-splicing reaction of the Tetrahymena group I intron ribozyme can be characterized as a two-step transesterification reaction resulting in the excision of the intron and the subsequent ligation of the two exons (41). As with the hammerhead and hairpin ribozymes, the group I intron ribozyme can be modified to perform the reaction in trans (42). It can trans-splice an exon joined to its 3[prime] end (`new part' RNA) onto a separate 5[prime] exon (target RNA) (Fig. 2A). For trans-splicing to occur, the group I intron ribozyme must possess a target RNA-binding site in its 5[prime] end, which will allow binding of the ribozyme to its target RNA via base pairing. The only sequence requirement in the binding site is the formation of a U:G base pair (Fig. 2A). The catalytic nucleotides in the ribozyme are all present between the binding site and the 3[prime] exon (41). Therefore, the presence of a uridine is the only sequence requirement for choosing the RNA target to be trans-spliced. Binding of the ribozyme to its target causes cleavage of the latter at the 3[prime] end of uridine, followed by the release of the 3[prime] exon (`new part' RNA) and the ligation of the two exons. Therefore, it is possible to design a group I intron ribozyme to repair a mutant RNA by replacing the faulty genetic information with the wild-type, engineered to be attached to the ribozyme. Moreover, repair of RNA can be widely applied since the only sequence requirement in the two exons is a uridine preceding the 5[prime] splice site in the target RNA.

   A

   B

Figure 2. (A) Group I intron ribozyme-mediated repair of mutant RNA molecules by trans-splicing. Following binding via Watson-Crick base pairing, the ribozyme cleaves its target RNA at a specific site (5[prime] splice site). A second cleavage then occurs at the 3[prime] end of the ribozyme strand (3[prime] splice site) which releases the `new part' RNA. This results in the replacement of the mutation (shown as a grey rectangle) in the mutant RNA with the wild-type sequence contained in the `new part' RNA. The only sequence requirement for the selection of appropriate target sites is a uridine preceding the 5[prime] splice site. (B) Ribozyme-mediated repair of DM mutations. Group I intron ribozymes can repair the CUG trinucleotide repeat expansion in the 3[prime] end of the mutant DMPK mRNA, by trans-splicing. The ribozyme can bind upstream of the mutation area (CUGm) and replace it with the wild-type sequence (CUGn), engineered to be carried in the ribozyme.

Ribozyme-mediated repair of mutant RNA

Ribozyme-mediated RNA trans-splicing was first demonstrated in an RNA fragment of the bacterial lacZ, both in E.coli (42) and in mouse fibroblasts (43). This clearly showed that group I intron ribozymes are capable of revising genetic information in a cellular environment. Recently, a tailor-made group I intron ribozyme has been created which was able to edit the 3[prime] end of the myotonic dystrophy protein kinase (DMPK) mRNA (44). Mutations in the DMPK gene are thought to be responsible for myotonic dystrophy (DM). DM is an autosomal dominant genetic trait and is the most common inherited neuromuscular disease in adults (45,46). DM belongs to a group of diseases characterized by a change in the size of the genomic fragment due to amplification of a repeated unit. The DM mutation is an amplification of a CTG repeat in the 3[prime]-untranslated region of the DMPK gene on chromosome 19 (47-50). The CTG repeat is highly polymorphic in the normal population (5-35 repeat units) and DM alleles have >50 repeat units rising up to 2000 in patients with the classical or congenital form of the disease (51). The pathogenesis of DM is thought to be complicated; however, there is good evidence that the mutant DMPK mRNA is trapped in the nucleus and that the CUG expansion alters binding of RNA-binding proteins to the molecule (52,53). The ribozyme was designed to bind just upstream of the mutation area in the DMPK mRNA and replace its 3[prime] end with the wild-type RNA sequence (Fig. 2B). It was shown that the ribozyme could edit the endogenous DMPK mRNA from human fibroblasts. As a next step, the authors need to demonstrate that group I introns are capable of repairing mutant DMPK mRNA molecules containing large CUG trinucleotide repeat expansions.

Group I intron ribozymes are promising tools for RNA repair

One of the advantages of RNA repair over the usual gene supplementation approach is that the revised gene is still present in its natural regulatory environment. Moreover, ribozyme-mediated trans-splicing is applicable to inherited diseases and, compared with other methods, it is probably ideally suited to dominant genetic diseases where it is difficult to abolish the function of the mutant allele completely. One of the current drawbacks of the system is the low sequence specificity which arises from the relatively short (six nucleotides) ribozyme-binding site. However, derivatives of the Tetrahymena group I intron ribozyme have been synthesized with increased specificity for their target in vitro (54). So far, there is good evidence that group I intron ribozymes will be powerful tools for RNA repair. However, as in all gene therapy protocols, high catalytic efficiency should be combined with good delivery systems that will transport the ribozymes safely, accurately and efficiently to their target RNA molecules.

CONCLUSIONS

The discovery that certain RNA molecules possess catalytic properties has opened new avenues for genetic therapy. Moreover, ribozymes can also be considered excellent tools to study gene expression. A better understanding of the three-dimensional structure of ribozymes and the relationship between their structure and function will prove invaluable for the design of more efficient RNA catalytic molecules. There has been considerable progress especially with the trans-cleaving ribozymes, but there are still areas which need to be exploited. Ribozymes are therapeutic tools which may prove to be extremely useful components of future strategies for the therapy of many inherited diseases.

ACKNOWLEDGEMENTS

Work in the authors' laboratories was financially supported by the Muscular Dystrophy Group of Great Britain and Northern Ireland, the Medical Research Council, UK, the A.G. Leventis Foundation, the March of Dimes Birth Defects Foundation and the Coles Family Foundation.

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*To whom correspondence should be addressed. Tel: +44 1865 272196; Fax: +44 1865 272420; Email: leonidas.phylactou@anat.ox.ac.uk

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