Function and biological applications of catalytic nucleic acids

Biochimica et Biophysica Acta 1445 (1999) 1^20

Review

Function and biological applications of catalytic nucleic acids Derval J. Gaughan, Alexander S. Whitehead * Department of Pharmacology, University of Pennsylvania, School of Medicine, 153 Johnson Pavilion, 3620 Hamilton Walk, Philadelphia, PA 19104-6084, USA Received 28 September 1998; received in revised form 21 December 1998; accepted 28 January 1999

Keywords: Hammerhead ; Gene expression; Ribozyme; Therapeutics

Contents 1.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Background to ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3.

Hammerhead ribozymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Consensus hammerhead ribozyme structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Mechanism of ribozyme cleavage and the role played by metal ions . . . . . . . . . . . . . .

2 3 4

4.

Generation of `engineered' ribozymes . . . . . . . . . . . . . . . . 4.1. In£uence of secondary structure on ribozyme activity . 4.2. Co-localization of ribozyme and target mRNA . . . . . 4.3. Cis-cleaving ribozymes . . . . . . . . . . . . . . . . . . . . . . . 4.4. Multi-target ribozymes . . . . . . . . . . . . . . . . . . . . . . . 4.5. Protein enhancement of ribozyme activity . . . . . . . . . 4.6. Chemically modi¢ed ribozymes . . . . . . . . . . . . . . . . .

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5 5 7 7 8 9 10

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Ribozyme delivery to target cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Applications of ribozymes . . . . . . . . . . . . . . . . 6.1. Human immunode¢ciency virus type 1 . . . . 6.2. Ras and BCR/ABL oncogenes . . . . . . . . . . 6.3. Multiple drug resistance (MDR) . . . . . . . . 6.4. Genetic disorders . . . . . . . . . . . . . . . . . . . 6.5. Ribozymes as tools to study gene function .

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Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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* Corresponding author. Fax: (215) 5739135; E-mail: [email protected] 0167-4781 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 7 - 4 7 8 1 ( 9 9 ) 0 0 0 2 1 - 4

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1. Introduction Ribozymes (RNA enzymes) are RNA molecules with catalytic activity. Since their discovery 17 years ago much research has focused on de¢ning the mechanism of their action and in assessing their potential as investigative and therapeutic agents in human disease. The prototypic ribozyme binds to its target RNA in a sequence-speci¢c manner and, after e¡ecting cleavage, dissociates from the resulting fragments. Ribozymes remain intact and unmodi¢ed following cleavage reactions and can therefore bind to, and cleave, additional RNA targets. This activity, which is independent of protein co-factors, only requires the presence of a divalent metal ion, typically magnesium. The sequence requirements in the target RNA are such that substrate-speci¢c ribozymes can readily be designed and synthesized. This has led to basic and applied research aimed at the exploitation of ribozymes as therapeutic tools in diseases characterized by the endogenous production of diseasecausing, or disease-associated proteins, i.e. the pathogenic overproduction of a normal protein, the production of an intrinsically pathogenic protein from a dominant mutant allele or, as in the case of HIV, the production of proteins essential for viral replication. Ribozymes can be designed to speci¢cally cleave the mRNA encoding such disease-causing proteins which are consequently synthesized at reduced, less pathogenic concentrations. This review will focus on the mechanism of ribozyme action, features of ribozyme design and the application of ribozymes as therapeutic agents in disease management and as tools to study gene function. 2. Background to ribozymes The ¢rst naturally occurring ribozyme activity, described in 1981 by Cech and colleagues, was discovered in the self-splicing group I intron of Tetrahymena thermophila [1]. The intron folds to form an autocatalytic unit which e¡ects its excision, an event that precedes ligation of the £anking exons to form a mature mRNA. This excision reaction occurs in the complete absence of any protein co-factor. Subsequently, Altman and colleagues discovered the second naturally occurring ribozyme, the RNase P en-

zyme puri¢ed from Escherichia coli in which the 400 nucleotide RNA component of RNase P was shown to cleave its substrate (precursor tRNA) in the absence of its protein subunit [2]. The identi¢cation of these self-cleaving RNAs was quickly followed by the discovery of numerous other catalytic RNAs which have been divided into six main groups: (1) ribozymes derived from self-splicing of Tetrahymena group I introns, (2) RNA component of RNase P, (3) RNAs of the hepatitis N virus [3], (4) RNA transcripts of the mitochondrial DNA plasmid of Neurospora [4], (5) hammerhead ribozymes and (6) hairpin ribozymes. Among the six ribozyme classes, the hammerhead ribozyme is the simplest in terms of size and structural requirements. Consequently there have been many studies that have de¢ned the parameters that govern the performance of hammerhead ribozymes. A sophisticated understanding of the importance of sequence to the structure and function of hammerhead ribozymes has permitted the design and synthesis of reagents that have been successfully used to down-regulate gene expression. Such modulation of gene expression by hammerhead ribozymes has been reported in plant cells [5,6], mammalian cells [7], transgenic Drosophila embryos [8], zebra¢sh embryos [9] and Xenopus oocytes [10,11]. This review will focus mainly on the structure and mechanism of hammerhead ribozyme action as well as on the design and generation of synthetic hammerhead ribozymes and their application as therapeutic reagents and genetic tools. The application of other catalytic nucleic acids, such as the self-splicing group I intron and the recently described DNAzymes, will also be discussed. Many of the points discussed in this review are also applicable to antisense technology. 3. Hammerhead ribozymes Hammerhead ribozymes have been found in plant and animal pathogenic RNAs (such as the tobacco ringspot virus (TRSV) and the avocado sunblotch virus (ASBV)) where it has been proposed that they function during the rolling circle pathway of viral RNA replication, a process which requires highly speci¢c cleavage of concatameric RNAs. The cleavage reaction mediated by naturally occurring hammerhead ribozymes is usually an intramolecular

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reaction in which a single molecule contains both the target and catalytic sequences. However, Uhlenbeck [12] noted that the self-cleavage reaction of ASBV requires the participation of two sequences which are widely separated on the same RNA strand and therefore proposed that an intermolecular transcleavage reaction in which two independent RNA strands could interact to form an active hammerhead structure should be possible. This hypothesis was validated in experiments in which a 19 nucleotide ribozyme and a 24 nucleotide substrate, both based on the ASBV sequence, were transcribed in vitro: co-incubation of these two separate RNA molecules resulted in rapid and e¤cient cleavage of the substrate. 3.1. Consensus hammerhead ribozyme structure The consensus sequence for hammerhead ribozymes is shown in Fig. 1. Hammerhead ribozymes were so called because of their characteristic `hammerhead' shaped secondary structure which consists of three double helical stem regions (I, II and III), two single-stranded loop regions (residues 3^9 and 12^14) and one bulged residue (residue 17 which is immediately 5P to the cleavage site) [13] (Fig. 1). It was subsequently shown that, in three dimensions, the structure of the ribozyme is more of a `wishbone' structure than a hammerhead in which helices I and

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II diverge from the core at an acute angle, while helix III points in the opposite direction [14,15]. The hammerhead ribozyme structure can be divided into two components, a substrate and an enzyme. The catalytic component binds to the substrate component through helices I and III. It is also possible, though less usual, to form a catalytic hammerhead ribozyme by hybridizing two RNA molecules through helices I and II as well as through II and III. The ¢rst hammerhead ribozyme consensus nucleotide sequence was described by Haselho¡ and Gerlach in 1988 [16]. It has since been sequentially mutated to identify the minimum number of nucleotides required for cleavage and the positions where variations are tolerated [17,18]. To date no conserved nucleotides have been identi¢ed in helix I or helix III. Therefore it is theoretically possible to design ribozymes whose £anking regions are complementary to, and capable of binding, any sequence in a target RNA. The catalytic core contains two stretches of highly conserved sequence: 5P-CUGANGA and 5PGAAA. All except one of the naturally occurring satellite and viroid RNAs cleave at a GUC target site [19]. As the sequence of this trinucleotide therefore appeared to dictate (and limit) the selection of cleavable target sites on substrate mRNAs, mutagenesis studies were performed by a number of investigators to establish the extent to which particular substitutions could be

Fig. 1. Consensus hammerhead ribozyme structure. N represents any nucleotide; H represents nucleotides C, U or A. The nucleotides are numbered according to Hertel et al. [13].

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made without compromising cleavage activity [16,17,19,20,21]. It was ¢rst established that U in the central position is an absolute requirement for cleavage. Additional mutagenesis studies revealed that any nucleotide in the ¢rst position and any nucleotide except G in the last position is tolerated. This has led to the generally accepted NUH rule (where N = A, G, C or U and H = A, C or U) which states that any substrate with an NUH triplet can be cleaved by a hammerhead ribozyme designed to target that site. The early studies were superseded by more detailed analyses [22] in which the nucleotides in the ¢rst and last positions of the GUC triplet were systematically mutated and the relative catalytic e¤ciency of each mutant measured. All mutants targets (including double mutants) that deviated from the naturally occurring GUC were cleaved with much lower e¤ciency. Recently a novel AUG-cleaving hammerhead ribozyme has been described [23]. This ribozyme, identi¢ed using an in vitro selection procedure [24], has a secondary structure similar to that of the conventional hammerhead ribozyme. However, it has an altered core and helix II where all the nucleotides, except one, are essential for activity. This type of ribozyme cleaves 3P to an AUG or an AUA. AUU and AUC triplets are not cleaved and thus the ribozyme is active only when there is a purine at the third position in the triplet. This recently de¢ned triplet speci¢city increases the number of triplets in a given substrate that are potentially cleavable. 3.2. Mechanism of ribozyme cleavage and the role played by metal ions Ribozymes are considered to be a distinct class of metalloenzymes as the presence of a divalent metal ion is essential for their cleavage of substrate [25]. Extensive crystallographic and biochemical studies of the hammerhead ribozyme reaction have helped to elucidate the mechanism of ribozyme catalysis and de¢nes the role played by magnesium ions. A considerable body of evidence suggests that they play both a functional and a structural role [26,27]. The ¢rst two X-ray crystallographic structures that were reported for hammerhead ribozymes were nearly identical with respect to tertiary fold and conformation [14,15]. The fact that two independently

derived structures showed only minor di¡erences strongly suggested that both were good approximations to the theoretical structures [28]. These crystal structures provided a detailed model of the catalytic center but they also raised some important questions about the mechanism of cleavage. For example, in the hammerhead ribozyme reaction, the target phosphodiester bond is known to be cleaved by a process called the in-line attack mechanism. However, in the two crystallographic structures reported, the phosphate at the active site is in a conformation which is maximally incompatible with this mechanism of cleavage. Also, mutation of G residue at position 5 (G5) to an A [17] or its deletion [29] reduces the hammerhead cleavage reaction rate by 4 orders of magnitude, thus identifying G5 as a critical residue for catalysis. However, in the crystal structures, this î from the cleavage site and does guanosine is 10 A not make any direct hydrogen bonding contacts with the rest of the RNA molecule. In order to reconcile the above incompatibilities it has been proposed that ribozymes undergo a conformational change to reach a transition state [14,15,29,30]. Such a conformational rearrangement would reposition the labile bond during catalysis, bringing the phosphate into the conformation required for the execution of the in-line attack mechanism. A major technical impediment to resolving the above has been the di¤culty in generating a stable `late' transition-state intermediate. Scott et al. [30] obtained an `early' transition state intermediate which suggested that such a conformational change does occur. More compelling evidence for such a change was subsequently obtained when Murray et al. [31] succeeded in trapping a `late' transition-state intermediate. The latter investigators synthesized a hammerhead ribozyme with a methyl group on the 5P carbon atom adjacent to the phosphorus at the cleavage site. This modi¢cation slows down the catalytic rate; the modi¢ed hammerhead ribozyme cleaved 300-fold more slowly than the unmodi¢ed ribozyme which was su¤cient to allow time-course experiments to be performed in the crystals. X-Ray di¡raction data, combined with electron-density mapping, revealed that in this `late' intermediate the cytosine base and ribose located 5P to the cleavage site had rotated 60³ away from their original ground-state positions. This base rotation positions

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the phosphate in a conformation more compatible with the presumptive in-line attack mechanism of cleavage. These latest results suggest that large conformational changes relative to the ground state structure are not required for hammerhead ribozyme catalysis, and that local conformational changes around the scissile bond are probably su¤cient. Feig et al. [32] have recently proposed that binding of a metal ion to the catalytic center of hammerhead ribozymes may act as a switch to alter its conformational state. These investigators studied binding of magnesium (Mg2‡ ) to a hammerhead ribozyme by competition with terbium (Tb3‡ ). Tb3‡ was shown to e¡ectively inhibit the hammerhead ribozyme reaction by competing directly with a single magnesium ion. Crystallographic studies indicated that the Tb3‡ binding site was localized adjacent to G5. These results suggest a role for the conserved G5 residue in the catalytic core and it was therefore proposed that a magnesium ion binds to the ribozyme at this site in the ground state conformation and must be released before the ribozyme undergoes conformational rearrangement to form a transition state intermediate. By binding more tightly than Mg2‡ at this site, Tb3‡ locks the ribozyme in the ground state conformation and this prevents the ribozyme from adopting an active conformation. The hairpin ribozyme, in common with the hammerhead ribozyme, requires the presence of a divalent metal ion co-factor (typically Mg2‡ ) for cleavage to occur. However, Young et al. [33] have shown that metal ions play a passive role in reactions catalyzed by hairpin ribozyme and that they are probably only required for structural purposes. This contrasts with the role of the metal ion in the hammerhead ribozyme and therefore categorizes the hairpin ribozyme as a member of a di¡erent mechanistic class. 4. Generation of `engineered' ribozymes Mutational analysis together with structural studies and other biochemical experiments have de¢ned the consensus sequence, as well as the structural and kinetic characteristics of hammerhead ribozymes. The theoretical knowledge gained has made it possible to design and synthesize ribozymes that can, in principle, speci¢cally cleave a particular mRNA tar-

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get. However, if ribozymes are to be used therapeutically to down-regulate pathogenic gene expression in vivo, several important factors need to be considered. Binding of the ribozyme to its target mRNA is the ¢rst critical step in the ribozyme reaction. Features of ribozyme design that can optimize this step will be described in this section. 4.1. In£uence of secondary structure on ribozyme activity Ribozyme-mediated inhibition of gene expression requires that a ribozyme e¤ciently cleaves a speci¢c site within a large target mRNA. To test the in£uence of the secondary structure of the substrate on ribozyme activity, Bertrand et al. [34] examined the cleavage kinetics of two test substrates, a full-length 950 nt Pit-1 (pituitary-speci¢c positive transcription factor) mRNA and a 60 nt Pit-1 mRNA fragment, both of which contained the same 17 base target sequence. The e¤ciency with which the 950 nt Pit-1 mRNA could be cleaved in vitro was 5-fold lower than that of the 60 nt fragment, indicating that cleavage of a long mRNA is much less e¤cient than cleavage of a short substrate due to reduced accessibility of target sites embedded in the center of the RNA molecule. As most cellular RNAs are at least 500 nt in length the possible impact of complex secondary structures on the accessibility of target sites is a critical design parameter. Computer-generated predictions of target mRNA secondary structures, which can readily be generated using the RNAPlotFold program based on the algorithm of Zuker and Steigler [35], are therefore a useful aid in identifying the potential target sites that are likely to be most accessible. The output is usually represented as a `squiggle plot', a graphical representation of secondary structure that is easy to interpret. A typical squiggle plot depicting the predicted secondary structure of human acute phase serum amyloid A2 (A-SAA2) mRNA is shown in Fig. 2. Plots typically consist of double stranded stems, which represent regions where base pairing over two stretches of residues is possible, and circular, single-stranded, open loop regions. Each tenth residue of the mRNA transcript is numbered in the plot to allow ready reference to positions within the structure. The ideal location for a ribozyme target site is in an open-loop region, particularly one

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Fig. 2. Squiggle plot of the human A-SAA2 mRNA secondary structures predicted using the RNAPlotFold program. The eight potential GUH sites are indicated (*) and the GUU target site (residues 107^109) on the exposed loop is shown.

that is located at the end of a long stem and therefore most likely to be exposed. We used the mRNA structure depicted in Fig. 2 to identify candidate target sites for an anti-SAA2 hammerhead ribozyme [36]. The eight potential GUH cleavage sites that are present in the A-SAA2 mRNA sequence were located on the predicted secondary structure. One of these, residues 107^109, was selected as it ful¢lled the criteria for the `ideal' target site, i.e the target triplet and £anking sequences were all fully exposed on an open loop region. In contrast, a potential target site at residues 59^61 is embedded in a highly structured region of the mRNA and would, in theory, not be accessible to a ribozyme. As expected from the above, a hammerhead ribozyme targeted to triplet 107^109 could e¤ciently cleave A-SAA2 mRNA in vitro. In another example, L'Huillier et al. [37] used a predicted secondary structure for K-lactalbumin mRNA to select an K-lac ribozyme target site; two ribozymes targeted to the `unstructured' regions of the K-lac mRNA proved much more e¡ective in cultured cells than a

ribozyme targeted to a central region predicted to be within a complex region of the molecule. Several experimental approaches to identify cleavable sites on a target mRNA have also been described [38^40]. One method consists of binding the target RNA to randomized oligonucleotides and subsequent digestion with RNase H. The extent of RNase H digestion in certain regions of the RNA molecule can be correlated to the degree of secondary structure within these regions. Birikh et al. [38] used this technique to identify accessible sites on the human acetylcholinesterase mRNA and the most active ribozyme was 150-fold more e¡ective than one selected on the basis of the RNAPlotFold program. The oligonucleotide screening technique gives a broad indication of annealing sites where ribozyme cleavage could be targeted. A similar but more speci¢c approach used a ribozyme library to identify cleavable sites on human growth hormone (hGH) mRNA [39]. This involves screening the whole RNA molecule using in vitro transcribed ribozymes containing randomized sequences in the annealing

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arms, a procedure that is experimentally time-consuming but is e¡ective in identifying speci¢c ribozyme target sites. Seven potential accessible sites could be identi¢ed using this method and its utility was subsequently demonstrated by successfully using an adenoviral-expressed ribozyme targeted to one of these sites to signi¢cantly reduce hGH mRNA expression in mice [40]. 4.2. Co-localization of ribozyme and target mRNA Speci¢c mRNAs may be di¡erentially sub-localized within the cell (e.g. the nucleus, the cytoplasm or one of the subcellular compartments in the cytoplasm) and it is intuitively obvious that, to be optimally e¡ective, ribozymes need to be expressed in the same location. Sullenger and Cech [41] were the ¢rst to demonstrate directly that co-localization of a ribozyme with its target within the cell can indeed substantially increase its e¡ectiveness. They co-expressed two retroviral vectors, one encoding a hammerhead ribozyme and another encoding a target lacZ mRNA, inside retroviral packaging cells. In such a system, some of the mRNA transcripts will be translated by the host cell while the remainder are used as `genomic' RNA for viral replication. As viral RNA that is to be replicated is sorted to sites of viral budding on the surface of packaging cells both the lacZ mRNA and the ribozyme mRNA that are destined for replication will co-localize to this region. If such co-localization of a ribozyme and its target has a signi¢cant impact on cleavage e¤ciency then the reduction in the lacZ mRNA and consequent decrease in L-gal viral titer in the packaging cells should be greater than the reduction of the lacZ mRNA within the cell. Indeed, such was the case: Sullenger and Cech [41] reported a reduction in the L-gal viral titer of around 90% compared with a minimal reduction in L-gal activity within the cell. These results strongly suggested that co-localization of a ribozyme and its mRNA target is important, perhaps critical, for optimizing cleavage. The majority of synthetic ribozymes studied to date have been directed to the cytoplasm where their mature mRNA targets accumulate. However, ribozymes that are retained in the nucleus may achieve relatively high local concentrations and have the opportunity to cleave newly transcribed RNA prior to

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its export to the cytoplasm and subsequent distribution to particular subcellular compartments. Bertrand et al. [42] examined the in£uence of cytoplasmic versus nuclear localization on the e¤ciency of a ribozyme directed against HIV. Using several di¡erent expression cassettes to generate both nuclear and cytoplasmically localized ribozymes, they demonstrated that a ribozyme was only e¡ective when expressed as capped, polyadenylated RNA that co-localized with its target to the cytoplasm. This result suggested that, in this instance, the cytoplasm is the more suitable location for ribozyme activity. However, other studies have achieved very e¡ective inhibitions of gene expression using ribozymes expressed in the nucleus [6,43]. It is therefore likely that some ribozymes have greater activity in the nucleus while others are more active in the cytoplasm. Which of these pertains in any given case is likely to depend on parameters speci¢c to the target RNA such as the concentration, the mode of processing and the transit time from the nucleus to the cytoplasm. 4.3. Cis-cleaving ribozymes Ribozyme transcripts produced from expression vectors in vivo often contain additional 5P and 3P £anking sequences. These include vector-derived sequence that is functionally required for proper transcription initiation and termination and, in some instances, a poly(A) tail added to the 3P end of the ribozyme. Alternative, more complex, folding of ribozymes may be mandated by such extra sequences and may reduce activity, either by masking the catalytic center or by forming a larger structure which might simply have reduced access to the target sequence. Sequences £anking the core ribozyme sequence can be removed post-transcriptionally and several studies have described the design of expression vectors that produce ribozymes with little or no extraneous sequence [44^46]. These vectors consist of a promoter that drives the synthesis of three ribozymes in tandem: a core trans-acting ribozyme sequence inserted between two cis-acting ribozyme sequences (Fig. 3). During transcription, the two cisacting ribozymes e¡ect self-cleavage reactions to liberate the trans-acting ribozyme. An example of a ciscleaving ribozyme system is that reported by Ventura et al. [46] who designed a ribozyme to cleave the R

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Fig. 3. The basic cis-cleaving ribozyme cassette. Following transcription, the two cis-cleaving ribozymes fold back and form hammerhead ribozymes using the 5P and 3P ends of the trans-acting ribozyme. This results in cleavage of the trans-acting ribozymes at the 5P and 3P ends.

region of HIV-1 RNA. Studied in vitro using the T3 RNA polymerase promoter, it failed to cleave its speci¢c target sequence under any condition tested. To reduce the in£uence of potential cis-inhibitory T3 RNA sequences, a cis-acting ribozyme, designed to remove these sequences, was inserted upstream from the anti-HIV ribozyme. This e¡ected the release of a shortened transcript containing only the core ribozyme. When incubated with three distinct HIV-1 transcripts in vitro, this modi¢ed ribozyme was catalytic, thereby demonstrating that the addition of a cis-cleaving ribozyme could activate a previously non-catalytic ribozyme by removing £anking inhibitory sequences. 4.4. Multi-target ribozymes Although computer-predicted mRNA secondary structures are used to locate target sites positioned on theoretically exposed regions of the mRNA, such sites may not in fact be similarly accessible in a cellular environment. They may be compromised as target sites by the post-transcriptional and translational events to which cellular mRNAs are subjected and may, for example, be physically associated with the cellular proteins that mediate these processes, thereby masking or reducing ribozyme access. An e¡ective strategy to increase the likelihood of successfully selecting an accessible target site is to target di¡erent regions of a substrate mRNA simultaneously. This

can be achieved by using the so-called `connected approach' in which a `multi-target' ribozyme that contains several tandemly arranged ribozymes, each of which is speci¢c for a di¡erent target site, is generated (Fig. 4a). To evaluate the `connected' approach, Bertrand et al. [34] compared the activity of a pentaribozyme with that of a mixture of the ¢ve constituent monoribozymes directed against the 950 nt Pit-1 mRNA. Although the pentaribozyme was active it was less e¤cient than the monoribozyme mixture and it was concluded that additional structures surrounding each ribozyme in the pentaribozyme were limiting catalytic activity. In a subsequent study by Ohkawa et al. [47] using the so-called `shotgun' approach, multi-target ribozymes, each £anked by a 5P and 3P cis-acting ribozyme, were connected in tandem (Fig. 4b) such that multiple target-speci¢c ribozymes, trimmed at both the 5P and 3P ends, would be liberated post-transcriptionally [47]. When tested in vitro, the `shotgun' approach proved to be more e¡ective than the `connected' approach. The `shotgun' approach was re¢ned to establish whether the cis-acting ribozymes that had trimmed the 5P and 3P ends of each ribozyme could themselves be designed to have a further biological function, rather than simply await degradation by RNases. Several trans-activator proteins are essential for viral replication of HIV-1 and the TAR and RRE recognition sequences (which bind two such trans-activa-

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tor proteins) were incorporated into the Stem II region of each cis-acting ribozyme. When tested in vitro each resulting cis-acting ribozyme was able to trap Tat or Rev protein successfully [48]. In addition they were found to retain almost full trimming activity. Thus cis-acting ribozymes can ¢rstly trim the 5P and 3P ends of each trans-acting ribozyme and then be available to trap the Tat and Rev activator proteins. Consequently, the reduction in production of HIV RNA that is achieved by sequestering the transactivator proteins theoretically provides the transacting ribozymes with a greater chance of eliminating the remaining HIV RNA. 4.5. Protein enhancement of ribozyme activity The activities of three proteins, the nucleocapsid protein of HIV-1 (NCp7), the heterogeneous nuclear ribonucleoprotein A1 (A1) and the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been shown to enhance ribozyme cleavage in vitro [49^53]. In a detailed study, Bertrand and Rossi [49] used a previously characterized set of hammerhead ribozymes to establish that two prototypical RNA binding proteins (A1 and NCp7) e¡ected the binding, cleavage and product dissocia-

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tion steps of the hammerhead ribozyme cleavage reaction. Overall the results obtained in this study and others [50,51] indicate several ways in which A1 and NCp7 might be used to optimize ribozyme activity in vivo. Long recognition sequences, necessary to form a duplex that is uniquely base-paired with a single cellular RNA, result in ribozymes that are generally characterized by strong binding and slow product release. Furthermore, strong binding can result in cleavage of both matched and mismatched RNA substrates. A1 and NCp7 can help overcome these problems: both proteins can enhance turnover by facilitating release of the cleaved RNA fragments from the ribozyme. Speci¢city was shown to be enhanced by the accelerated rate of duplex dissociation which facilitates binding of the ribozyme to the correct RNA. Ribozyme cleavage of long substrate mRNAs is often inhibited when the substrate RNA is highly structured with inaccessible target sites; the strand unwinding activities of A1 and NCp7 help reduce this problem. It is suggested that strategies which take advantage of the activities of these proteins might be devised [50]. For example it may be possible to target ribozymes to locations containing high concentrations of these proteins, such as the nucleus for A1 and the viral capsid for NCp7. It

Fig. 4. Multi-target cis-cleaving ribozymes. (a) The `connected' ribozyme in which the three trans-acting ribozymes are released from the transcript as a joint molecule and (b) the `shotgun' ribozyme in which the three trans-acting ribozymes are released as separate molecules.

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might also be possible to link the ribozyme to a high a¤nity binding site for A1 or NCp7, so that the interaction between the protein, ribozyme and target will be greatly facilitated in vivo. In another study, Sioud et al. [52] reported that the activity of a ribozyme directed against tumor necrosis factor-K (TNF-K) mRNA could be improved by a protein factor which was subsequently identi¢ed as GAPDH [53] and shown to enhance in vitro cleavage rates by 25-fold. GAPDH is thought to act through a mechanism that is similar to A1 and NCp7 and it has been proposed that the addition of a sequence with high a¤nity for GAPDH would improve the e¤ciency of ribozymes in vivo. However, no reports have yet been published using ribozymes modi¢ed to incorporate A1, NCp7 or GAPDH binding elements. 4.6. Chemically modi¢ed ribozymes The stability of a ribozyme in vivo is a major factor in£uencing its e¤ciency under physiological conditions. The more stable the ribozyme, the more likely it is that large amounts will accumulate in the cell thereby increasing the probability that it will bind to the target mRNA. In addition, ribozymes with longer half-lives should theoretically cleave more substrate RNA molecules over time. Ribozymes expressed endogenously from a transfected vector normally have a 5P guanosine cap and a 3P poly(A) tail that are added by post-transcriptional cellular mechanisms. The presence of both of these modi¢cations have been shown to act as mRNA stability elements [54]. However, ribozymes that are chemically synthesized and delivered to the cell exogenously do not contain such elements and are therefore rapidly degraded by cellular nucleases. To overcome this problem various chemical modi¢cations have been made to exogenously produced ribozymes to improve their stability. The substitution of ribonucleotides by their corresponding 2P-amino or 2P-£uoro analogs in a hammerhead ribozyme confers resistance to RNase digestion and can increase ribozyme stability in rabbit serum [55]. The e¡ect of another chemical modi¢cation, the introduction of phosphorothioate groups, has also been assessed. It is well documented that the presence of phosphorothioate groups at certain positions in an oligonucleo-

tide protects them against various nucleases. Initial studies of phosphorothioate-substituted ribozymes indicated that they have dramatically reduced catalytic activity [17]. However, Shaw et al. [56] subsequently reported that a 3P exonuclease activity is predominantly responsible for degradation of oligonucleotides in fetal calf serum and cell supernatants. Therefore, it was postulated that phosphorothioate substitutions at the 3P end of a ribozyme should be su¤cient to protect against nuclease degradation, but have a minimum negative e¡ect on catalytic activity. Heidenreich and Eckstein [57] therefore investigated the in£uence of these chemical modi¢cations on the activity of a hammerhead ribozyme directed against the LTR RNA of HIV-1. When incubated in cell culture supernatant, 80% of unmodi¢ed ribozyme was degraded within 2 min. However, the chemically modi¢ed ribozyme showed no degradation during a 1 h incubation and, although there was a 7-fold decrease in catalytic activity, the concomitant more than 50-fold increase in ribozyme stability outweighed this disadvantage. Another approach is the use of DNA-RNA hybrid hammerhead ribozymes, where some ribonucleotides outside the catalytic core are replaced with 2P-deoxynucleotides [58,59]. One such study in which the RNA in the hybridizing arms of a ribozyme directed against the HIV-1 gag RNA were replaced with DNA yielded a DNA/RNA ribozyme with 6-fold greater catalytic activity than its all RNA counterpart [59]. These chimeric ribozymes, when transfected by cationic liposomes into human T-lymphocytes, are also more stable than their all-RNA counterparts. Shimayama et al. [60] combined the above two approaches by synthesizing a DNA/RNA chimeric ribozyme with phosphorothioate substitutions and demonstrated that such a thio-DNA/RNA chimera has a 7-fold higher cleavage activity than an allRNA ribozyme. Their results also suggested that thio-DNA/RNA ribozymes are more resistant to attack by nucleases in vivo. In yet another version of the combined chemical modi¢cation strategy, Scherr et al. [61] examined the e¡ect of adding two phosphorothioate substitutions to the 3P end of a chimeric DNA/RNA ribozyme. They showed that the phosphoro-modi¢ed ribozyme could markedly inhibit the in vivo expression of its substrate mRNA at a dose

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at which the unmodi¢ed ribozyme was completely ine¡ective. 5. Ribozyme delivery to target cells The method of ribozyme delivery to target cells must be e¡ective for ribozymes to be used successfully in gene therapy studies. E¤cient cellular uptake, speci¢c mRNA targeting, sustained ribozyme expression, long half-life, and safety are all desirable characteristics. There are essentially two ways to deliver ribozymes to their target cells: ribozymes can be synthesized chemically and delivered to target cells exogenously or they can be synthesized endogenously in the target cells following the introduction of ribozyme expression vectors. The main disadvantage of exogenous delivery, as well as non-integrated endogenous delivery, is that stable expression cannot be achieved and therefore multiple administrations are necessary if long-term presence of the ribozyme is required. The principal advantage of exogenous delivery is that it permits the use of chemically modi¢ed ribozymes or DNA-RNA hybrid ribozymes that are relatively resistant to degradation by nucleases. There are several methods for the delivery of presynthesized ribozymes including chemical techniques such as calcium phosphate precipitation (for cell cultures), mechanical techniques such as microinjection, membrane fusion-mediated transfer via liposomes, and direct DNA uptake and receptor-mediated DNA transfer [62]. Endogenous delivery, in which the ribozyme is expressed from a ribozyme-encoding gene driven by a promoter of choice, can result in either stable or transient expression depending on whether the vector carrying the above is incorporated into the host cell genome. Several viral vectors including retroviruses, adenoviruses and adeno-associated viruses have been exploited for endogenous delivery [62]. Each viral delivery system has its advantages and disadvantages. For example, a major advantage of retroviruses is the stable integration of their genetic material directly into the chromosome of the host cell which results in sustained gene expression; however, their potential application is limited as they can only transduce dividing cells, have a low vector titer and lack speci¢c integration sites. Their restricted ca-

11

pacity would nevertheless be an advantage in the treatment of cancer where tumor cells would be selectively transduced. Adenoviral vectors are more widely used than retroviral vectors as they can both transduce non-dividing cells and be grown to high titers. However, the main disadvantage of adenoviral vectors is that they generally remain episomal, do not replicate, and are therefore eventually lost. Adenoviral-associated vectors (AAVs) are a promising alternative, the main advantage of which is that their usual integration site on a short region of the long arm of chromosome 19 [63,64] ensures that they persist, but is not associated with any known pathogenic consequences. Like adenoviral vectors, AAVs can transduce both dividing and non-dividing cells. However, among the disadvantages of this type of vector are that 40^80% of adults have pre-existing immunity to AAV and the virus is not always incorporated into chromosome 19 which leaves open the possibility of insertional mutagenesis when it integrates into other sites. The choice of the promoter used to express a ribozyme is an important factor and depends on whether inducible, tissue-speci¢c or constitutive expression is required. An example of inducible ribozyme expression [65] is the use of the GRP94 promoter to express an anti-GRP ribozyme. GRP94 is a glycoprotein in the endoplasmic reticulum (ER) that has important Ca2‡ binding and protein chaperoning properties. While constitutively expressed in most cell types under normal growth conditions, it is highly induced in stress conditions, such as when intracellular Ca2‡ is depleted or when N-linked protein glycosylation is inhibited during which ER function is disrupted. During such stress conditions there is a 10-fold increase in transcriptional activation of GRP94. A ribozyme, targeted against GRP mRNA and under the control of the stress-inducible GRP94 promoter, could signi¢cantly reduce GRP mRNA and protein levels, thereby permitting studies on the role of GRP94 in the ER during stress conditions to be undertaken [66]. In some cases, tissue-speci¢c expression can potentially increase ribozyme e¡ectiveness. Tyrosinase, a key enzymes in melanogenesis, is synthesized almost exclusively in the melanocytic system and Ohta et al. [67] used this promoter to speci¢cally express an antiH-ras ribozyme in human melanoma cells. They

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compared the e¤ciency of the tissue-speci¢c tyrosinase promoter to express an anti-ras ribozyme with that of a viral promoter. The ribozyme controlled by the tyrosinase promoter achieved greater suppression of the human melanoma phenotype in vitro as characterized by changes in growth, melanin synthesis, morphology and H-ras expression. In another example, a ribozyme targeted against pancreatic L-cell glucokinase was placed under the control of the insulin promoter to develop an animal model for maturity onset diabetes [68]. The use of the insulin promoter ensured that expression of the ribozyme was pancreatic-speci¢c. Constitutive ribozyme expression can be achieved using a variety of promoters including those of RNA polymerase II (pol II), RNA polymerase III (pol III) and cytomegalovirus (CMV). tRNA genes transcribed by the pol III promoter have been exploited as ribozyme expression cassettes for several reasons including (i) their small size (less than 200 bp including the ribozyme coding sequence), (ii) their high rate of transcription, and (iii) their ubiquitous expression in most tissues. In addition, it was anticipated that a ribozyme embedded in a tRNA structure might be more stable than a linear form. In the ¢rst use of this approach, a ribozyme targeted against the 5P leader sequence of U7 small nuclear RNA (U7 snRNA) was embedded in the Xenopus tRNAmet gene [11]. When injected into Xenopus oocytes, the tRNA-ribozyme complex (ribtRNAmet ) e¤ciently cleaved its U7 snRNA substrate. There was no direct evidence that placing the ribozyme structure within the tRNA structure enhanced its stability since ribtRNAmet decayed more rapidly than tRNAmet ; however, ribtRNAmet was more stable than the linear ribozyme when assayed in nuclear extracts. Thompson et al. [43] increased the level of RNAs transcribed from human tRNAmet pol III promoters by modifying the tRNA structure such that the 3P terminus of the transcript hybridized with the 5P end, forming an intramolecular duplex that might be protected from degradation. This modi¢cation improved by 100-fold the accumulation of recombinant RNAs expressed from a human tRNAmet pol III promoter. An anti-HIV ribozyme expressed from this modi¢ed expression cassette accumulated to very high levels in cells stably transfected with the ribozyme.

Overall there are many di¡erent ribozyme delivery systems available, the choice of which depends on the desired therapeutic role of the ribozyme. For example if stable long-term expression is required then endogenous delivery is the obvious choice. If endogenous delivery is the selected method, then careful consideration as to the kind of ribozyme expression required, i.e. inducible, constitutive or tissue-speci¢c expression, will mandate selection of the most suitable expression system. 6. Applications of ribozymes The hammerhead and hairpin ribozymes have been exploited for use as therapeutic agents and genetic tools. The fact that these RNA enzymes have a high speci¢city for their target RNAs which they functionally inactivate via cleavage, and can be chemically and biochemically synthesized, make them attractive as potential therapeutic agents. The remaining section of this review will describe the application of ribozyme therapy in diseases such as human immunode¢ciency virus (HIV), multi-drug resistance (MDR) and cancer, and in the treatment of dominant genetic disorders. The use of ribozymes as a research tool to study gene function will also be discussed. 6.1. Human immunode¢ciency virus type 1 The role of ribozymes as therapeutic agents for the treatment of HIV infection has been extensively investigated. Sarver et al. [69] demonstrated that when human HeLa CD4‡ cells expressing an anti-HIV-1 gag ribozyme were challenged with HIV-1, gag gene expression, as well as p24 antigen levels, were lower in ribozyme-expressing cells compared to controls. Several approaches have been used to improve the e¤cacy of ribozymes targeted against HIV-1. One such is to target the ribozyme to relatively conserved regions such as the 5P leader sequence, a particularly attractive target due to the high degree of homology among most known HIV-1 isolates and its presence in both early and late viral gene products [70^72]. Other targeted regions include the 5P TAR region [73] and the retroviral packaging region [74] which has been conserved among 18 published HIV isolates.

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The use of multi-target ribozymes is another way to improve the e¡ectiveness of ribozymes against HIV, which has a high mutation rate due to the error-prone nature of reverse transcriptase [75,76]. A single point mutation at a ribozyme target site will be su¤cient to preclude cleavage. These considerations led to the development of the previously described multi-target ribozymes, capable of cleaving HIV RNA at multiple positions; thus, if one or more target sites were to mutate and thereby become resistant to cleavage by the polyribozyme, the remaining unchanged sites would still be subject to cleavage by the other individual ribozymes contained in the polyribozyme. Chen et al. [77] tested several multitarget ribozymes designed to cleave HIV-1 RNA at up to nine conserved sites. When co-expressed in HeLa cells with the infectious HIV-1 clone pNL4-3, the nona-ribozyme under the control of the HIV-1 LTR, dramatically inhibited HIV-1 replication. Ohkawa et al. [47] cleaved HIV-1 RNA at ¢ve di¡erent positions simultaneously using the shotgun approach. Recently, Ramezani et al. [78] designed a monomeric and a multimeric HIV ribozyme targeting one and nine highly conserved regions respectively within the HIV envelope (env) coding region. Both ribozymes speci¢cally cleaved the target RNA in vitro. Following transfection into a human CD4‡ T lymphocyte-derived MT4 cell line the monomeric ribozyme delayed virus replication although virus production eventually occurred and reached values observed in control cells lacking the ribozyme. In contrast, HIV infection was inhibited in cells transfected with the multimeric ribozyme for the duration of the study (60 days). These results suggest that such multimeric ribozymes targeted to multiple sites within HIV-1 may prove useful in anti-HIV gene therapy. A hairpin ribozyme targeted against the 5P leader sequence of HIV-1 was transiently transfected into HeLa cells where it inhibited HIV replication and p24 antigen synthesis by 90% and 95% respectively, causing a 10 000-fold decrease in virus production [72]. These encouraging in vitro results have led to a phase I clinical trial to test the safety and function of ribozymes in transduced human peripheral blood lymphocytes. The results of this and other clinical trials will provide important information on the practical aspects of using ribozymes as therapeutic agents for this disease.

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6.2. Ras and BCR/ABL oncogenes The growth and di¡erentiation of a cell depend on a number of signal transduction pathways. Proteins encoded by the three ras genes (H-ras, N-ras and Kras) are involved in these signal transduction pathways. Mutations of the ras oncogene family have been detected in a wide variety of tumors such as pancreatic carcinomas and tumors of the stomach and breast [79]. The ras proteins are members of a supergene family of small GTP/GDP binding proteins and studies of ras oncogenes in tumors have revealed several point mutations in codons 12, 13, 59 or 61 which cause structural changes in the GTP binding site. Mutant ras proteins, which remain in an active state due to a reduced ability to hydrolyze GTP to GDP, stimulate cell growth or di¡erentiation autonomously. Therefore speci¢c inhibition of the ras oncogene, such as might be achieved by ribozymes, may lead to e¡ective anticancer therapies. Ribozymes, by virtue of the sequence-speci¢c constraints governing their activity, can discriminate between the products of a normal ras gene and a mutated ras gene in which only a single base is di¡erent. For example a hammerhead ribozyme targeted against H-ras mutated at codon 12 (GGC to GUC, which fortuitously creates a GUC ribozyme target sequence) has been shown to discriminate between the normal H-ras mRNA and the mutated mRNA in vitro [80]. When transfected into EJ bladder carcinoma cells the ribozyme could not only decrease Hras expression but could also reverse the metastatic, invasive and tumorigenic properties of the cells. In another study, the N-ras oncogene was the target of ribozyme therapy [61]. Two hammerhead ribozymes targeted to a point mutation in codon 13, a GC transversion at position 763 that generates a GUC cleavage site that is not present in the normal gene, could cleave only the mutant transcript thereby demonstrating their speci¢city for the oncogenic form of N-ras. Phosphorothioate-substituted ribozymes of the same design were transfected into HeLa cells and their cleavage activity was measured using a luciferase-based assay system in which the ¢rst 452 bp of the N-ras gene was fused upstream and in frame with the gene for ¢re£y luciferase. In this system, ribozyme-mediated cleavage of the N-ras portion of the target mRNA should decrease lucifer-

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ase activity since the downstream luciferase portion of the chimeric mRNA is no longer translated into protein product. This permits luciferase activity, measured readily using a luminometer, to be used to directly measure ribozyme activity. In the above experiment luciferase activity was reduced by about 60% in HeLa cells transfected with the anti-N-ras ribozyme. Cleavage activity was restricted to the mutated N-ras species as luciferase activity in HeLa cells expressing a wild-type N-ras/luciferase transcript could not be reduced by targeting cells with the ribozyme. Chimeric RNAs, transcribed from chimeric genes generated genomic rearrangement and specifying abnormal pathogenic products, are obvious candidates for ribozyme targeting. Ribozymes can be designed so that their sequence recognition arms interact with junctional mRNA regions derived from each of the genes of the chimera. Therefore only the chimeric mRNA will be bound by both arms and held in the hammerhead conformation that promotes cleavage of the target NUX triplet, but neither of the normal non-chimeric mRNA products, will be cleaved. Chronic myelogenous leukemia (CML) is an ideal candidate for ribozyme therapy. CML is a disorder of hematopoietic stem cells associated with the Philadelphia chromosome which is the result of a chromosomal translocation between chromosomes 22 and 9 that brings the BCR gene (on 22) into juxtaposition with the ABL gene (on 9) [81]. This generates a BCR/ ABL chimeric gene which encodes a novel tyrosine kinase with transforming activity [82]. The chromosomal translocations that occur in CML can be divided into two types: K28 translocations and L6 translocations [83]. These translocations result in the formation of two di¡erent mRNA products: the b3a2 mRNA product (consisting of mRNA derived from BCR exon 3 and ABL exon 2) and the b2a2 mRNA product (consisting of mRNA derived from BCR exon 2 and ABL exon 2) respectively. Ribozymes designed to cleave the junction sequence of the BCR/ABL fusion mRNA have been tested as a potential therapy for the treatment of CML. The b3a2 mRNA product is a perfect ribozyme target as a GUU cleavage site is located just 3 nt upstream from the BCR/ABL junction. There are several examples of ribozymes that have been de-

signed to cleave at this GUU site in a speci¢c catalytic manner [84^86]. In contrast, the b2a2 mRNA product is not as suitable a target as there are no triplet sequences that are potentially cleavable by hammerhead ribozymes within 2 or 3 nt of the BCR/ABL junction. Cleavage of a chimeric mRNA is more speci¢c when the target site is located very close to the junction site as each of the £anking complementarity regions that are bound by the ribozyme are speci¢ed by a di¡erent partner in the chimera. This precludes binding to, and cleavage of, the transcribed products of the wild-type genes. The ribozyme cleavage site closest to the junction of the b2a2 transcript are located 7, 8, 9 and 19 nt into the ABL portion. Studies that de¢ne the activities of ribozymes targeted to these sites and containing a long substrate arm complementary to the BCR mRNA [87,88] have been published. As the cleavage site is in ABL mRNA, the only molecules containing BCR sequence that should be cleaved are those which also contain the ABL-speci¢c cleavage site (i.e. the chimeric BCR/ABL mRNA). The ability of such ribozymes to cleave BCR-ABL mRNA as well as wild-type ABL and BCR mRNAs was tested in vitro. Although there was e¤cient ribozyme-mediated cleavage of the BCR-ABL mRNA, a small amount of cleavage of the wild-type ABL mRNA also occurred. The use of an alternative ribozyme approach for targeting the b2a2 mRNA, based on DNA enzymes that can cleave RNA molecules at any sequence, has recently been described by Kuwabora et al. [89]. DNA enzymes, ¢rst described by Santoro and Joyce [90], are similar to conventional hammerhead ribozymes and consist of a 15 deoxynucleotide catalytic domain, the activity of which is magnesium dependent. The catalytic domain is £anked by two recognition domains of 7 or 8 deoxynucleotides. There are two types of DNA enzymes: type I DNA enzymes cleave between an A and a G residue while type II DNA enzymes cleave between a pyrimidine and a purine residue. The b2a2 mRNA contains several potential cleavage sites for DNA enzymes and three such enzymes were designed to cleave at nucleotides which are only 1, 2 and 3 nt from the junction. Each DNA enzyme could cleave the b2a2 mRNA at the expected site in vitro. No cleavage of the normal ABL or BCR substrate was observed thus demon-

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strating the high substrate speci¢city, and potential as therapeutic agents, of these DNA enzymes. 6.3. Multiple drug resistance (MDR) A signi¢cant problem in the treatment of many tumors with chemotherapeutic protocols is the development of MDR. This is most often caused by the overexpression of P-glycoprotein (P-Gp), a transmembrane protein that can act as a drug e¥ux pump. P-Gp is encoded by a small group of closely related genes termed MDR genes. In one approach to reversing multi-drug resistance, ribozymes have been targeted to the mdr1 mRNA. Kobayashi et al. [91] designed two ribozymes to cleave the mdr1 mRNA at codons 179 and 196. The two cleavage sites were selected because of their close proximity to codon 185, an important site for substrate preference and the consequent likelihood that the resulting mRNA fragments, even if they were translated into polypeptides, would specify non-functional products. Initial in vitro experiments demonstrated that the MDR1 ribozyme targeted to codon 196 was more e¡ective. Following stable transfection with this ribozyme, the human T-cell leukemia cell line MOLT3 exhibited a 35-fold increase in sensitivity towards the drug vincristine that was accompanied by decreases in MDR1 expression and the amount of P-Gp. Using a similar strategy to reverse the MDR phenotype, Holm et al. [92] used a hammerhead ribozyme to target codon 880 of the mdr1 mRNA. This target site is between two ATP binding sites which may be important for P-Gp to function as an ATPdependent pump. The ribozyme was expressed in a human pancreatic carcinoma cell line which was resistant to the drug daunorubicin and expressed the MDR phenotype. Expression of the ribozyme in these cells decreased the level of MDR1 expression, inhibited the formation of P-Gp and reduced by 300fold the resistance of the cells to daunorubicin. These results indicate that ribozyme-based therapies have considerable potential for reversing drug resistance during anti-cancer chemotherapy. 6.4. Genetic disorders The application of ribozymes to the treatment of dominant genetic disorders has recently been de-

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scribed [93,101,102]. While most gene therapy protocols aimed at correcting recessive genetic disorders are designed to replace the missing gene product in a speci¢c tissue, a quite di¡erent approach is required for the treatment of dominant genetic disorders. As such disorders are often characterized by the expression of a defective pathogenic protein from a mutant allele, the introduction of an additional copy of the normal allele would clearly have little or no e¡ect. One possible strategy is to speci¢cally reduce the level of mutant mRNA transcripts using antisense RNA techniques; however, such methods are not speci¢c enough to clearly distinguish between mutant and normal transcripts and can therefore cause large decreases in the level of the normal mRNA and its gene product. Ribozymes, with their high degree of speci¢city, o¡er a means to selectively reduce the intracellular level of a mutant mRNA transcript. Grassi et al. [93] have described a ribozyme-based strategy for the treatment of osteogenesis imperfecta (OI), a dominant genetic condition caused by a mutation in the type I collagen gene. This generalized disorder of the connective tissues is characterized by susceptibility of a¡ected individuals to bone fractures from very mild trauma (`brittle bone' syndrome). Five hammerhead ribozymes were designed to cleave synthetic transcripts of two naturally occurring human collagen mutations and to cleave the transcript speci¢ed by a construct containing a portion of the mouse COLA1 gene into which a point mutation had been introduced. In all of these cases the mutation creates a ribozyme cleavage site that is present only in the mutant transcript and, as expected, each of the ribozymes could cleave their speci¢c transcript in vitro. There was no cleavage of the wild-type transcripts indicating that each ribozyme was speci¢c for the mutant transcripts. The competitive e¡ect of the presence of both total RNA and the normal COLA1 transcript on ribozyme activity was also studied. There was no decrease in the e¡ectiveness of mutant mRNA cleavage in the presence of total RNA thereby con¢rming the ability of the ribozyme to localize and bind to its target even in the presence of a large excess of unrelated RNA. However, in the presence of a non-cleavable target with an identical binding site (i.e. normal allele transcript) cleavage of the mutant mRNA was greatly reduced. This indi-

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cates that the wild-type mRNA can sequester ribozyme and prevent it from binding to and cleaving its target. To address this problem, Grassi et al. suggested that the cycling of the ribozyme from the wild-type binding site could be increased by introducing a mismatch between the target and one of the complementary arms of the ribozyme. The speci¢city of the ribozyme for the mutant allele achieved in the above study is encouraging for future use of ribozymes in OI and other dominant genetic disorders such as Marfans syndrome [94] and ¢broblastgrowth factor receptor (FGFR)-related disorders [95]. Another ribozyme-based strategy that has been developed for the treatment of genetic diseases is based on the cleavage and ligation capabilities of the T. thermophila group I intron which, during self-splicing, cleaves the phosphodiester bond that attaches its intron to its 5P exon, releases the intron, and then ligates its 3P exon onto the 5P exon [96]. This group I ribozyme can also transfer its 3P exon to a 5P exon that is present in trans [97,98]. Recently, an altered version of this trans-splicing ribozyme was shown to be able to repair truncated lacZ transcripts in E. coli [99] and in the cytoplasm of mammalian cells [100]. The reaction system used in the lacZ experiments has two RNA components: the target RNA, consisting of a truncated lacZ transcript with a ribozyme recognition site and a splice site, and a Tetrahymena ribozyme covalently linked to a 3P exon encoding the remainder of the lacZ sequence. During the trans-splicing reaction the ribozyme recognizes and binds to the 5P exon of the target lacZ RNA by means of an internal guide sequence (IGS), cleaves the RNA, releases the downstream sequence, and attaches the 3P exon onto the RNA. This ribozyme system was tested in both E. coli and mouse ¢broblasts where it was demonstrated that there was e¤cient trans-splicing of the lacZ 3P exon onto the truncated lacZ transcript, regenerating transcripts that could be translated and could produce L-gal activity. Trans-splicing ribozymes may become useful as therapeutic reagents for the repair of mutant RNAs in human cells. By repairing mutant mRNAs, such ribozymes should restore the regulated production of a wild type gene product while minimizing the production of any mutant product. Such an approach

has recently been described for the treatment of sickle cell anemia [101]. The genetic basis of sickle cell disease is an A to T transversion in the sixth codon of the L-globin gene, and individuals who are homozygous for the mutation accumulate long polymers of sickle hemoglobin (HbS) in their erythrocytes resulting in a chronic hemolytic anemia and cumulative tissue damage. A trans-splicing ribozyme was used to convert sickle L-globin transcripts into RNA encoding fetal Q-globin, which is itself thought to impede polymerization of HbS. In the splicing reaction, the ribozyme uses an IGS to recognize and base pair to the sickle L-globin (Ls -globin) transcript, cleave the Ls -globin RNA, release the mutation-containing cleavage product and splice on the revised Q-globin sequence. RNA repair may be a particularly appropriate method to treat sickle cell disease because the process should restore the regulated expression of anti-sickling versions of L-globin and simultaneously reduce the production of Ls -globin. In addition the e¤ciency of L-globin repair does not have to be 100% to bene¢t patients as sickle cell patients that express Q-globin at 10^20% the level of Ls -globin in most of their RBCs have greatly improved clinical status. Trans-splicing ribozymes might also be useful as therapeutic reagents for conditions such as Huntington's disease and myotonic dystrophy. These conditions, along with a number of others, are caused by trinucleotide repeat expansions (TREs) in, or close to, the disease-associated loci resulting in a change in the size of the genomic fragment and the resulting mRNA product. A potential therapeutic approach is to repair the expanded mRNA region using the trans-splicing ability of the group I ribozyme. Myotonic dystrophy (DM) has been used as a model to test this approach [102]. A trans-splicing ribozyme, designed to modify the 3P end of the human myotonic dystrophy protein kinase (DMPK) transcript, could replace 12 CUG repeat units of a synthetic DMPK mRNA with ¢ve CUG repeat units both in vitro and in mammalian cells. In addition, this ribozyme could successfully modify endogenous DMPK mRNA in human skin ¢broblasts. The use of trans-splicing ribozymes as therapeutic agents of genetic diseases has produced promising results so far. However, the general applicability of this novel technology will not be clear until the e¤-

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ciency with which the repair of mutant transcripts occurs is assessed in vivo. 6.5. Ribozymes as tools to study gene function In addition to their therapeutic potential, ribozymes can be used to study the function, regulation and expression of genes [8,9,103]. In the ¢rst application of ribozymes as tools to study animal development, Zhao and Pick [8] used ribozyme-mediated suppression of the fushia tarazu (ftz) gene in Drosophila to generate loss-of-function phenotypes. A heat inducible promoter was used to drive the expression of the anti-ftz ribozyme so that it could be experimentally activated/inactivated at various times during larval development. This allowed the role of the ftz protein to be de¢ned at di¡erent larval development stages. In particular, the ftz gene was known to be involved in development of the nervous system and timed induction of the ftz ribozyme allowed the characterization of the functional properties of the ftz product during two separate phases (¢rst in segmentation and later in neurogenesis). Based on the promising results described above, Xie et al. [9] used ribozyme-based experimental procedures to analyze gene function in a zebra¢sh developmental system. Such a system may be a good model for studying vertebrate development in general as there is evidence of a high level of nucleotide sequence conservation between analogous zebra¢sh and mammalian development genes. A hammerhead ribozyme targeted against the recessive dominant no tail (ntl) gene was expressed in developing zebra¢sh embryos. The ribozyme e¡ectively reduced ntl mRNA and protein levels and zebra¢sh with the expected phenotype (i.e. lacking a tail) were generated. In addition, no other developmental or structural abnormalities were observed thereby con¢rming the speci¢city of this ribozyme-mediated `knockdown' strategy. Future experiments using ribozyme-mediated `knockdown' of many other zebra¢sh genes should provide valuable information on their roles in development. In a recent study ribozymes were employed as tools to study the G protein signal transduction pathway [103]. G proteins are heterotrimeric proteins, consisting of K, L and Q subunits, that play a central role in signal transduction from cell surface

17

receptors to the cell nucleus. A large number of hormones, neurotransmitters and chemokines exert their e¡ects on cells by binding to G protein-coupled membrane receptors. Although G proteins play an important role in determining the speci¢city of the cellular response the exact mechanism whereby this is achieved is not clearly de¢ned. Wang et al. [103] used a ribozyme based system to directly test the hypothesis that the Q subunit contributes to the speci¢city of receptor signaling pathways in vivo. Using a transient transfection assay, a phosphorothioated DNARNA chimeric hammerhead ribozyme speci¢cally reduced both the mRNA and protein expression of the Q7 subunit in HEK 293 cells. There was a speci¢c reduction in expression of the L1 subunit coincident with the loss of the Q7 subunit. This suggests that the L1 and Q7 subunits are co-regulated and may interact to form a LQ dimer in vivo. In another recent study, regulated gene expression employing ribozymes was used to de¢ne the di¡erent roles for the co-activators p300 (a nuclear phosphoprotein) and CBP (cAMP-response element-binding protein) in retinoic acid-induced F9 cell di¡erentiation [104]. These two related proteins are transcriptional co-activators involved in growth control pathways, as has been established by their interaction with the tumor suppressor p53 and the viral oncogenes E1A and SV40 T antigen. The role of these proteins in retinoic acid-induced cell di¡erentiation, cell cycle exit and apoptosis was studied using hammerhead ribozymes directed against either p300 or CBP mRNAs. F9 cells expressing a p300-speci¢c ribozyme became resistant to retinoic acid-induced differentiation whereas cells expressing a CPB-speci¢c ribozyme were una¡ected. In contrast, both ribozymes blocked retinoic acid-induced apoptosis, indicating that both co-activators are required for this process. Thus ribozyme-mediated regulation of gene expression enabled the roles of p300 and CPB to be determined which, despite their similarities, have distinct biological functions. 7. Conclusion The discovery of catalytic RNA molecules almost two decades ago was a landmark in basic biological research. E¡orts to understand the structural require-

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ments governing their activity were accompanied by the realization that synthetic ribozymes would likely be valuable tools for manipulating gene expression in a range of experimental systems. Furthermore, their potential use as therapeutic agents to down-regulate or block the synthesis of pathogenic gene products was apparent. During the ensuing years much has been learned about the function of ribozymes, their rational design and e¤cient and e¡ective application in vitro and in vivo. This review has focused on some of the positive advances that have been made by leading investigators in the ¢eld who have established that ribozymes, indeed, constitute a novel technology with great potential. However, ribozyme research has not yet progressed to the stage where this technology can readily be adopted by the wider biomedical research community. The many failures experienced by those working with ribozymes attest to the fact that signi¢cant technical hurdles must be overcome and current research is directed to this end. Nevertheless, a small number of ribozymes have performed well in laboratory based experiments and are now undergoing clinical trials. Such successes indicate that the early promise of ribozymes as highly speci¢c biochemical modi¢ers and as a new class of therapeutic agents for the treatment of human disease should eventually be realized. References [1] T.R. Cech, A.J. Zaug, P.J. Grabowski, Cell 27 (1981) 487^ 496. [2] C. Guerrier-Takada, K. Gardiner, T. Marsh, N. Pace, S. Altman, Cell 35 (1983) 849^857. [3] H.N. Wu, Y.J. Lin, F.P. Lin, S. Makino, M.F. Chang, M.M.C. Lai, Proc. Natl. Acad. Sci. USA 86 (1989) 1831^ 1835. [4] B.J. Saville, R.H. Collins, Cell 61 (1990) 685^696. [5] P. Steinecke, T. Herget, P.H. Schreier, EMBO J. 11 (1992) 1525^1530. [6] R. Perriman, G. Bruening, E.S. Dennis, W.J. Peacock, Proc. Natl. Acad. Sci. USA 92 (1995) 6175^6179. [7] C.M. Flory, P.A. Pavco, T.C. Jarvis, M.E. Lesch, F.E. Wincott, L. Beigelman, S.W. Hunt, D.J. Schrier, Proc. Acad. Natl. Sci. USA 93 (1996) 754^758. [8] J.J. Zhao, L. Pick, Nature 365 (1993) 448^451. [9] Y. Xie, X. Chen, T. Wagner, Proc. Natl. Acad. Sci. USA 94 (1997) 13777^13781. [10] S.K. Saxena, E.J. Ackerman, J. Biol. Chem. 265 (1990) 17106^17109.

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