Ribozyme: A clinical tool

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Invited critical review

Ribozyme: A clinical tool

Asad U. Khan *

Interdisciplinary Biotechnology unit, Aligarh Muslim University, Aligarh 202002, India Received 19 October 2005; received in revised form 18 November 2005; accepted 22 November 2005

Available online 19 January 2006

Abstract

Catalytic RNAs (ribozymes) are capable of specifically cleaving RNA molecules, a property that enables them to act as potential antiviral and anti-cancer agents, as well as powerful tools for functional genomic studies. Recently, ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo. Phase I clinical trials using ribozyme gene therapy to treat AIDS patients have been conducted. Despite initial success, there are many areas that require further investigation. These include stability of ribozymes in cells and designing highly active ribozymes in vivo, identification of target sequence sites and co-localization of ribozymes and substrates, and their delivery to specific tissues and maintenance of its stable long-term expression. This review gives a brief introduction to ribozyme structure, catalysis and its potential applications in biological systems as therapeutic agents.

Keywords: Ribozyme; Clinical applications; Hammerhead; RNAse P

1. Introduction

A ribozyme, or RNA enzyme, is an RNA molecule that can catalyze a chemical reaction. Many natural ribozymes catalyze either their own cleavage or the cleavage of other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome[1]. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions.

Seven naturally occurring classes of ribozyme have been identified to date, all of which catalyze cleavage or ligation of the RNA backbone by transesterification or hydrolysis of phosphate groups. The hammerhead, hepatitis delta virus (HDV), hairpin and Neurospora Varkud satellite (VS) ribozymes are small RNAs of 50 – 150 nucleotides that perform site-specific self-cleavage [2 – 9]. Found in viral, virusoid or satellite RNA genomes, they process the products of rolling circle replication into genome-length

strands[10]. The general mechanism of these ribozymes is similar to that of many protein ribonucleases in which a 2’

oxygen nucleophile attacks the adjacent phosphate in the RNA backbone, resulting in cleavage products with 2V,3V- cyclic phosphate and 5V hydroxyl termini (Fig. 1). Unlike protein ribonucleases, however, ribozymes cleave only at a specific location, using base-pairing and tertiary interactions to help align the cleavage site within the catalytic core. The evolutionary maintenance of these sequences may be due to their site specificity and to the simplicity and efficiency of genome self-cleavage.

Groups I and II introns and ribonuclease P (RNAse P) belong to the larger, more structurally complex ribozymes of several hundred nucleotides in length [11 – 13]. RNAse P cleaves precursor RNA substrates at specific sites to generate functional 5V termini [14], and groups I and II introns catalyze two-step self-splicing reactions[15 – 19]. In these large ribozymes, the nucleophile and the labile phosphate are located on different molecules or are greatly separated in sequence. Thus, the complex folds of these RNAs serve to orient the nucleophile and phosphate to ensure accurate cleavage or splicing.

Recently, ribozymes catalytic centers have been incorporated into antisense RNA and shown to specifically

doi:10.1016/j.cca.2005.11.023

* Tel.: +91 571 2723088 (O), +91 989 7188786 (M); fax: +91 571 27217176.

E-mail address: [email protected].

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cleave a target RNA substrate. The enzymatic activity of the ribozyme catalytic center resulted in the cleavage and destruction of the target RNA. Ribozyme/substrate pairing needs to be of sufficient duration in order for the ribozyme to cleave the target RNA, causing a functional inactivation of the target [9]. Once the target is cleaved, the ribozyme can then dissociate and recycle itself (binding, cleavage and dissociation). This unique characteristic provides an added advantage over standard antisense RNA that only inacti- vates the target RNA without degrading it. As such, ribozyme technology has made considerable gains in applied bio-sciences and has a vast implications for application in molecular medicine[15,16,20,21].

Recently, a new system has been designed that allows the regulation of gene expression in response to an externally administered drug. This system is based on allosteric ribozymes, which can specifically cleave their own RNA in the absence of the drug or be inactivated in its presence [22]. Thus, if ribozymes were suitably inserted within mRNA transcripts, they would lead to its quick degradation unless the drug was present. Such catalytic RNAs can be rationally designed using known ribozymes such as hammerhead ribozyme and regulatory sequences called aptamers [23,24]. A cyclic nucleotide monophosphate compound was found to activate allosteric ribozymes at a concentration of 100 AM. Interestingly, catalytic rates of these ribozymes increased up to 5000-fold and reached activities similar to the wild-type hammerhead ribozyme [25]. An in vitro selection scheme has also been applied to obtain allosteric ribozymes that respond to the antibiotic doxycycline[26]. Inhibition was approximately 50-fold and the response could be achieved at very low concentration with inhibition constants as low as 20 nM.

The development of therapeutic agents that specifically target the viral genome is complicated by the fact that viral replication occurs intracellularly. Despite this limitation, genetic medicine targeting the viral genome has been promising with respect to selective toxicity for viral infection. Strategies that involve antisense DNA, antisense

RNA, ribozyme, aptamer, triplex and the gene itself have been enthusiastically studied over the past decades. Despite the slow start of genetic medicine research and early skepticism, the first antisense DNA technology is now on the market in the USA and Europe. The mechanism of antisense oligodeoxynucleoside technology is not, however, without controversy. It has been proposed that the observed therapeutic effects might result from the presence of CpG and thus reflect immune stimulation in some cell types[27].

Hopefully, the design of functional genetic medicine therapeutics to combat viral infection will eventually lead to mainstream development of antiviral agents. Despite early advances, a highly efficient drug delivery system is required in order to deliver antiviral agents, i.e., engineered antisense ribozymes, to their site of action.

Different molecular and cellular studies have highlighted the important role of some gene products in the cause and/or perpetuation of human pathology including cancer and autoimmune disease. The identification of such gene products has led to the development of new candidate ribozyme-based therapies. The discovery of catalytic nucleic acid enzymes has also provided researchers with a potentially important tool to block the expression of abnormal genes, provided their sequences are known. The cleavage specificity of these compounds is determined by their hybridizing antisense arms, which anneal with the target mRNA in a complementary fashion. Catalytic RNA can be delivered to cells either endogenously as gene encoding RNA enzymes (ribozymes) or exogenously as in vitro prepared agents. Given the progress reported over the last few years, a wide range of molecular designs and chemical modifications can be introduced into these compounds, in particular the hammerhead type ribozyme[28].

Here we review the background, structure and functional features of RNAse P and hammerhead ribozyme and their therapeutic applications. In addition, relevant gene targets and applications in molecular medicine with special reference to down regulation of viral and oncogene expression are illustrated.

2. RNAse P

Gene interference strategies using antisense oligonucleotides, ribozymes, DNAzymes and RNA interference (RNAi) represent powerful research tools as well as potential therapeutic agents for human disease [29]. Each of these approaches has its own merits and limitations with respect to targeting efficacy, sequence specificity, toxicity and delivery efficiency in vivo. Although antisense DNA and RNA have traditionally been used as basic research tools (studying gene function during development), they are now being investigated for potential clinical application[30,31].

Recently, RNA enzymes derived from hammerhead and hairpin ribozymes have been shown to be promising gene- targeting reagents to specifically cleave RNA sequences of Acid-base catalysis Two metal-ion catalysis

A B

Fig. 1. RNA cleavage by acid – base catalysis and two-metal ion catalysis.

:B is a general base and H – A is a general acid. Thick dotted lines show direct inner-sphere coordination between divalent cations MAand MBand oxygen atoms. N represents a purine or pyrimidine base and R denotes chain continuation.

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choice [32 – 36]. These ribozymes contain both a catalytic RNA domain that cleaves the target mRNA and a substrate- binding domain with a sequence antisense to the target mRNA sequence. Therefore, these gene-targeting ribozymes bind to the mRNA sequence through Watson – Crick interactions between the target sequence and the antisense sequence in the substrate binding domain of the ribozyme.

RNAse P is a ribonucleoprotein complex responsible for the 5V maturation of tRNAs [37,38]. RNAse P catalyses a hydrolysis reaction to remove a 5V leader sequence from tRNA precursors (ptRNA) and several small RNAs. In Escherichia coli, RNAse P consists of a catalytic RNA subunit (M1 RNA) of 377 nucleotides and a protein subunit (C5 protein) of 119 amino acids[37,38]. In vitro, M1 RNA can cleave its ptRNA substrate at high divalent ion concentration in the absence of C5 protein [39]. The addition of C5 protein dramatically increased the rate of cleavage by M1 RNA under low Mg²⁺ concentration in vitro and is required for RNAse P activity and cell viability in vivo [37,38]. It has been proposed that C5 protein, an extremely basic protein, functions to stabilize the conformation of M1 RNA and enhance the interaction between the enzyme and the ptRNA substrate[40,41].

Studies on substrate recognition by M1 RNA and RNAse P have led to the development of a general strategy in which M1 RNA and RNAse P can be used as gene targeting tools to cleave specific mRNA sequences [42,43]. The natural substrates for RNAse P in E. coli include ptRNAs, the precursor to 4.5S RNA and several small RNAs [44 – 46].

All these substrates can fold into a structure equivalent to the upper portion of a ptRNA molecule (i.e. a 5V leader sequence, an acceptor-stem like structure and a 3V CCA sequence) (Fig. 2). Deletion analyses of a tRNA substrate has revealed that a small model substrate containing a structure equivalent to the acceptor stem and T stem, the 3VCCA sequence and the 5V leader sequence of a ptRNA molecule can be cleaved efficiently by M1 RNA (Fig. 2) [42,47]. In this small model substrate, the 5V proximal

sequence (the 5V leader and 5V proximal acceptor stem sequence) base pairs to the 3V proximal sequence (the 3V proximal acceptor stem sequence). This 3V proximal sequence is called an external guide sequence (EGS) because of its ability to base pair with the targeted sequence and guide M1 RNA to cleave the substrate (Fig. 2)[42]. External guide sequences (EGS) are antisense oligoribonucleotides that have been used to diminish gene expression in bacteria [48,50] and mammalian cells[43,51] with the aid of either RNAse P or M1 RNA. One of the first studies using the EGS technology to target h-galactosidase and alkaline phospha- tase activity in E. coli showed a reduction of more than 50%

in the expression of these proteins in strains that harbored the appropriate EGSs. No reduction in their expression was observed in strains with non-specific EGSs[48]. Moreover, EGSs had also been used to target drug-resistant genes in E.

coli to convert the cellular phenotype from drug-resistant to drug-sensitive [49]. The EGS-based technology takes advantage of RNAse P or M1 RNA to cleave a targeted mRNA when the EGS hybridizes to the target RNA and forms a structure resembling a portion of the natural tRNA substrates of the enzymes[42,43].

3. Hammerhead ribozyme and its functional features The hammerhead ribozyme was first discovered as a self- cleaving domain in the RNA genome of different plant viroids and virusoids [52]. Replication of these pathogens occurs by a rolling circle mechanism [53], in which the circular RNA genome is copied into long, multimeric transcripts. These transcripts must then be cleaved into genome length strands. In several cases, however, this step did not involve protein enzymes, but resulted from a self- cleavage reaction catalyzed by a specific RNA motif, termed ‘‘hammerhead’’ [54]. Soon thereafter, it was demonstrated that the hammerhead motif could be incorporated into short synthetic oligonucleotides and transformed into a

Fig. 2. A modified diagram from Fig. 1 of Trang et al.[92]to represent the ptRNA and 4.5S RNA as natural substrates and a small model substrate (EGS:

mRNA) for RNAse P and M1 RNA.

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true, multiple turnover catalyst, suitable for cleaving in trans a variety of RNA targets [55,56]. All hammerhead motifs share a typical secondary structure consisting of three helical stems (I, II and III) that enclose a junction characterized by several invariant nucleotides (i.e., the Fcatalytic core_). In most trans-acting hammerhead ribozymes, helix II is formed intramolecularly by the catalyst, whereas helices I and III are formed by hybridization of the ribozyme with complementary sequences on the substrate (Fig. 3)[57 – 59].

The three-dimensional structure of the hammerhead was determined independently by two laboratories and described during the mid 1990s. In 1994, Pley et al. reported the structure of a hammerhead ribozyme construct com- plexed with a non-cleavable substrate analogue made entirely of DNA [60]. The core was composed of two structural domains: residues 3 – 6 (CUGA) formed a sharp turn identical to the uridine turn of transfer RNA, while residues 8, 9, 12 and 13 formed a non-standard duplex that extended helix II (Fig. 3). In 1995, Scott et al. confirmed this overall structure in a crystallographic study describing an all-RNA hammerhead construct with a single 2-O- methyl group at the site of cleavage[61]and, subsequent- ly, in a second study of the same all-RNA hammerhead construct which did not contain any chemical modification [60]. Although structures obtained in these crystallographic studies mirror the ribozyme conformation predomi- nant in solution at room temperature, substantial rearrangement of this structure is likely required to achieve catalysis [62 – 64].

4. Ribozyme as antiviral molecule

Following initial studies on the applications of catalytic nucleic acids, the use of these molecules for the treatment of viral diseases was recognized as particularly promising [65]. Although any viral infection might be treated with RNA enzymes designed to cleave essential mRNAs, viruses that carry an RNA genome not be discussed further. Two Fgene therapy_ clinical trials have been initiated in the USA. In these trials, retroviral vectors are used to produce CD4 lymphocytes or CD34 hematopoietic precursors that express ribozymes targeted against sequences in the human immunodeficiency virus-1 (HIV-1) RNA [66,67].

The catalytic activity of ribozymes is being exploited to inhibit HIV replication through an HIV-directed hammerhead ribozyme[68]. A clinical trial has been initiated using this ribozyme in patients suffering from acquired immune deficiency syndrome (AIDS). Peripheral blood T lymphocytes obtained from HIV-infected patients will be transduced with a retroviral vector construct coding from a specific ribozyme sequence. Transduced lymphocytes will then be re-injected into patients. Upon successful insertion of the ribozyme gene, it is expected that intracellular expression of ribozyme will abrogate the host genome HIV RNA expression in infected cells [69]. These experiments will likewise examine the potential resistance to HIV infection and survival capability of ribozyme-expressing T lymphocytes[70].

In addition, a hairpin ribozyme and a hammerhead ribozyme containing a 5VC(UUCG)G3V loop were designed to cleave the long terminal repeat of HIV-1[71]. Although hepatitis C virus (HCV) has been identified as the major infectious agent responsible for chronic hepatitis, no vaccine is currently available to protect against this RNA virus. In an investigation of new genetic approaches to the management of this infection, six hammerhead ribozymes directed against a conserved region of the plus and minus strands of the HCV genome were isolated from a ribozyme library, characterized and expressed using recombinant adenovirus vectors. The expressed ribozyme plus or minus strand HCV RNA were expressed in cell culture and primary human hepatocytes obtained from chronic HCV- infected patients [72]. To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic HCV infection, Macejak et al. [73] designed and synthe- sized hammerhead ribozymes targeting 15 conserved sites in the 5V untranslated region (UTR) of HCV RNA. This region forms an internal ribosome entry site that allows for efficient translation of the HCV polyprotein. The 15 synthetic ribozymes contained modified nucleotides and linkages that stabilize the molecules against nuclease degradation. All 15 ribozymes were tested for their ability to reduce expression in an HCV 5V-UTR/luciferase reporter system and for their ability to inhibit replication of an HCV-poliovirus (HCV-PV) chimera. Treatment with sever-

Fig. 3. Secondary structure of the hammerhead ribozyme, with bound substrate. The ribozyme is shown in bold and the substrate in thin letters. N represents any nucleotide and H represents any nucleotide except G. The hammerhead consists of three helices and 11 non-helical residues located in the highly conserved central region (here, these residues are numbered following the standard hammerhead nomenclature). Ribozymes commonly used for exogenous application are 35 – 40 nucleotides long and the intramolecular helix II is formed by four base pairs, as shown here.

However, some very active hammerhead variants have been described, where this helix is reduced to just two base pairs or even replaced with a loop.

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al ribozymes resulted in significant down-regulation of HCV 5V-UTR/luciferase reporter expression (range 40% to 80% inhibition, P < 0.05). Moreover, several ribozymes showed significant inhibition (> 90%, P < 0.001) of chimeric HCV-PV replication. It was also shown that the inhibitory activity of ribozymes targeting site 195 of HCV RNA exhibit a sequence-specific dose response, require an active catalytic ribozyme core, and is dependent on the presence of the HCV 5V-UTR. Treatment with synthetic stabilized anti-HCV ribozymes has the potential to aid patients who are infected with HCV by reducing the viral burden through specific targeting and cleavage of the viral genome[73 – 75].

Chronic hepatitis B virus (HBV) infection is frequently associated with liver cirrhosis and hepatocellular carcinoma and has thus become a major worldwide problem. Ham- merhead ribozymes have recently gained importance as potential tools to inhibit viral infection for which there are no effective therapies. The management of chronic infection of HBV may also benefit from ribozyme therapy[76]. As opposed to an ex vivo ribozyme gene transfer strategy in AIDS patients, HBV may be susceptible to the injection of synthetic ribozymes or to in vivo transfer of ribozyme expression vectors. Hsieh and Taylor[77], as well as Netter et al.[78], have exploited the natural tropism of the hepatitis delta virus to direct a biologically active RNA to the liver.

The approach leads to in vivo targeting of the liver and a hepatocyte-specific expression of ribozymes. Furthermore, expression of other therapeutically relevant genes in the liver could be enhanced and is being pursued in laboratories around the world.

A wide variety of sequences in the HIV genome have also been the target for hammerhead and hairpin ribozyme- mediated trans-interference gene expression. Some of the examples have demonstrated the ability of ribozymes to inhibit expression of reporter genes under control of HIV-1 genes or to suppress HIV[79,80]. These experiments were performed by endogenous delivery of ribozymes via plasmid or retroviral vectors containing the ribozyme sequence behind a suitable promoter. Exogenous delivery of synthetic ribozymes represents an alternative approach for inhibition of gene expression [81,82]. However, exogenous delivery has not yet been applied for the inhibition of the expression of HIV-1 gene with the exception of mainly a cellular-uptake study with a DNA – RNA ribozyme chimera. Bramlage et al. [28] have therefore tried to explore this mode of application and have chosen the HIV-1 LTR as a target because of its high degree of conservation among the known HIV-1 isolates and because of its presence in both early and late viral gene products.

It has been demonstrated that small inhibitory RNAs (siRNA) efficiently target and degrade HIV-1 RNA sequences and inhibit viral replication[83,84]. A recent report by Novina et al. has shown inhibition of HIV-1 infection by synthetic siRNA via impairment of CD4 expression[85].

5. Ribozyme in cancer therapy

Cancer is a disease of genes. Anti-oncogene and tumor suppressor gene therapies of cancer are the two strategies that aim at correcting genetic disorders of cancer. The potential effectiveness of these therapeutic approaches is, however, very much dependent on their precise targeting at the mechanisms of the disease [86].

A key difficulty in effective treatment of cancer is the ability to selectively distinguish tumor cells from normal cells. This problem contributes to the lack of effectiveness of current cancer treatments. There have been, however, remarkable advances in our understanding of the molecular biology of cancer that may provide alternative and more selective mechanisms for tumor destruction. The molecular characterization of tumor-specific chromosomal abnormalities has revealed that fusion proteins are involved in most types of cancer [87]. These fusion proteins result from chimeric genes created by the translocation and production of chimeric mRNA species that contain exons from each gene involved in the translocation. Chimeric molecules are ideal therapeutic targets because they are unique to the disease because they exist exclusively in tumor cells. Inhibition of chimeric gene expression by anti-tumor agents specifically kills leukemic cells without affecting normal cells.

Although zinc-finger proteins, antisense RNA and hammerhead-based ribozymes have potential as therapeutic agents, all have limitations. Zinc-finger proteins must act at the DNA level, interacting with the desired sequence and blocking transcription[88]. However, gene fusions at the DNA level occur within introns and this implies that a new zinc- finger has to be designed for every patient. Thus, directing the strategy at the mRNA level seems to be more practical.

Antisense molecules, either oligodeoxynucleotides or antisense RNA, act in a 1:1 stoichiometric relationship. This problem can be overcome with the use of ribozymes that, because of catalytic activity, process and destroy a higher number of target molecules, per molecule of ribozyme.

Although hammerhead ribozymes require the presence of specific nucleotide sequences in the target RNA to be cleaved, these requirements cannot always be fulfilled. These data imply that new therapeutic tools would be desirable to allow the inactivation of any chimeric fusion gene product[20,89].

M1 RNA is the catalytic RNA subunit of RNAse P from E. coli. RNAse P is a ribonucleoprotein complex that catalyzes the hydrolysis of a 5V leader sequence from tRNA precursors and several other small RNAs of similar structure. Studies on substrate recognition by the M1- RNA and RNAse P[90]have led to the development of a general strategy of gene targeting in which M1 RNA can be used as a tool to cleave any specific mRNA sequence simply by the 3V terminal addition to the ribozyme sequence of a so- called ‘‘guide sequence’’ (M1-GS) complementary to the target mRNA. This artificial strategy involves the formation of a base-pair match and leaves a 5V-ACCAC-3V unpaired stretch for the M1-GS RNA to recognize and cleave. Thus,

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M1-GS RNA, apart from some requirements to improve its cleavage efficiency, can be specifically directed to cut any mRNA sequence. Cobaleda and Sanchez-Garcia[91]have taken advantage of this property to destroy the tumor- specific fusion genes created as a result of chromosomal translocations. This approach has been well described by Khan and Lal[9].

An additional use of ribozymes could be envisioned to inhibit the aberrant expression of fusion proteins that result from chromosomal translocation. The presence of nearly 100 non-randomly occurring chromosomal translocations in cancers has been identified, most commonly in hematopoietic tissue. Neoproteins generated by the translation of fusion transcripts have been functionally implicated in tumor evolution and progression. In this respect, ribozymes could potentially be useful as tools for cancer therapy[20].

6. Conclusion

Although skeptically proposed a decade ago, the use of ribozyme therapy to combat viral diseases seems quite feasible now. Despite this progress, there is still a lot to be learned regarding intracellular mobility of RNA and the cellular factors that can impede ribozyme action in order to fully capitalize on the targeted RNA-inactivation property of ribozymes. The most effective approach to maximize ribozyme function in a complex intracellular environment is to more fully comprehend intracellular fate of the targeted RNA. It should be noted, however, that the substantial progress made to date has been due to advances in basic research techniques in cell biology.

Fundamental studies of ribozyme structure and mechanism of catalysis are flourishing at the academic and industrial level and many new developments can be expected in these area. Nevertheless, the next frontier for ribozyme application is to enhance our understanding of cell biology, trafficking and intracellular localization of RNA.

These are the fields in which it can be expected to show most research efforts in the immediate future. It is anticipated that research on ribozymes may well have an equally important impact on the diagnostic and therapeutic practices of human and veterinary medicine in the next decade. The emerging picture is one of great diversity. There is at this stage no general cell model or clearly preferable ribozyme structure. Each and every cell line (and tissue) may be unique and vary with respect to structural requirements for optimal uptake, activity and stability of ribozymes. Obviously, additional research is required on RNA- based enzymes and their roles in biology and medicine.

Acknowledgement

I thank Prof. M. Saleemuddin for providing working infrastructure. Mr. Shahper and Ms. Barira are also

acknowledged for the critical proofreading of the manu- script. Distribution Information Sub-Centre is also acknowledged for the computer facilities. This work was supported by the internal fund of Biotechnology Unit, AMU, Aligarh.

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