Engineering of RNase P ribozyme for gene-targeting applications

Gene 313 (2003) 59–69 www.elsevier.com/locate/gene

Review

Engineering of RNase P ribozyme for gene-targeting applications Stephen M.L. Raj, Fenyong Liu* Division of Infectious Diseases, School of Public Health, 140 Warren Hall, University of California, Berkeley, CA 94720, USA Received 25 February 2003; received in revised form 2 April 2003; accepted 14 April 2003 Received by A.J. van Wijnen

Abstract Ribonuclease P (RNase P) is a ubiquitous ribonucleoprotein complex responsible for the biosynthesis of tRNA. This enzyme from Escherichia coli contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). M1 ribozyme cleaves an RNA helix that resembles the acceptor stem and T-stem structure of its natural tRNA substrate. When covalently linked with a guide sequence, M1 RNA can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which can cleave any target RNA sequences that base pair with the guide sequence. Recent studies indicate that M1GS ribozymes efficiently cleave the mRNAs of herpes simplex virus 1, human cytomegalovirus, and cancer causing BCR-ABL proteins in vitro and effectively inhibit the expression of these mRNAs in cultured cells. Moreover, RNase P ribozyme variants that are more active than the wild type M1 RNA can be generated using in vitro selection procedures and the selected variants are also more effective in inhibiting gene expression in cultured cells. These results demonstrate that engineered RNase P ribozymes represent a novel class of promising gene-targeting agents for applications in both basic research and clinical therapy. This review discusses the principle underlying M1GS-mediated gene inactivation and methodologies involved in effective M1GS construction, expression in vivo and emerging prospects of this technology for gene therapy. q 2003 Elsevier Science B.V. All rights reserved. Keywords: Ribozyme; RNase P; Herpes simplex virus; Cytomegalovirus; Gene therapy; Gene targeting

1. Introduction The observations of duplex formation between two independent, complementary oligonucleotides by means of the conventional Watson-Crick base pairing and the subsequent degradation of these double-stranded oligomers by endonucleases in a sequence-specific manner lead to the idea of exploiting this intermolecular phenomenon to manipulate gene expression in vivo (Zamecnik and Stephenson, 1978; Stein and Cheng, 1993). The target mRNA that hybridizes with a complementary antisense oligonucleotide is presumably cleaved by intracellular endonucleases (e.g. RNase H) before transmitting the message for protein production. The efficacy of the antisense oligomer can be further improved if the antisense molecule is tethered to a sequence or module that contributes catalytic activity as exemplified with ribozymes Abbreviations: RNase, P, ribonuclease P; pre-tRNA, precursor tRNA; HSV, herpes simplex virus; HCMV, human cytomegalovirus; DMS, dimethyl sulphate; TK, thymidine kinase. * Corresponding author. Tel.: þ 1-510-643-2436; fax: þ1-510-643-9955. E-mail address: [email protected] (F. Liu).

(Rossi, 1999; Sullenger and Gilboa, 2002) and DNA enzymes (Santoro and Joyce, 1997; Breaker, 2000). The discovery of RNA catalysts (Altman and Kirsebom, 1999; Doudna and Cech, 2002) has opened up the possibility of using such RNA enzymes or ribozymes for modulating the targeted gene expression in vivo. The highly efficient activity of these enzymes to cleave target RNAs in vitro suggests ribozymes as a novel class of agents for useful therapeutic application. For example, ribozymes derived from the hammerhead and hairpin motifs have been shown to inhibit viral replication in cells infected with human viruses, while a ribozyme derived from group I intron has been used to repair mutated mRNAs (Sarver et al., 1990; Yu et al., 1993; Lan et al., 1998). Moreover, ribonuclease P (RNase P), an essential cellular enzyme, is being explored to down-regulate the expression of specific genes in vivo (Altman, 1995; Li et al., 1992; Guerrier-Takada et al., 1997). Over the past few years, RNase P ribozyme, such as M1 RNA, the catalytic RNA subunit of E. coli RNase P, was engineered with the internal guide sequence and expressed in human cells to silence specific viral and cellular oncogenic messages (Liu and Altman, 1995; Cobaleda

0378-1119/03/$ - see front matter q 2003 Elsevier Science B.V. All rights reserved. doi:10.1016/S0378-1119(03)00677-2

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and Sanchez-Garcia, 2000; Trang et al., 2000a,b, 2001, 2002), whereas in other cases, the endogenous cellular human RNase P was recruited to cleave target mRNAs (Yuan et al., 1992; Kawa et al., 1998; Plehn-Dujowich and Altman, 1998; Dunn et al., 2001; Kraus et al., 2002). Exploiting endogenous RNase P for specific gene inactivation has been successful in E. coli (Li et al., 1992; Guerrier-Takada et al., 1997) and human cells (Yuan et al., 1992; Kawa et al., 1998; Plehn-Dujowich and Altman, 1998; Dunn et al., 2001; Kraus et al., 2002) by expressing sequence specific RNA called external guide sequence (EGS) that anneals with its cognate target mRNA and directs cellular RNase P for target degradation. The principle and methodologies governing the EGS-based technology in directing endogenous RNase P for cleavage are described elsewhere (Guerrier-Takada and Altman, 2000). This review will focus on the gene knock-down strategies based on RNase P ribozyme and discuss its possible use in gene therapy.

2. RNase P: molecular organization and catalytic mechanism RNase P is a ubiquitous ribonucleoprotein complex responsible for the 50 maturation of tRNAs and several other RNAs (e.g. 4.5S RNA) (Frank and Pace, 1998; Altman and Kirsebom, 1999). In eubacteria, RNase P consists of a catalytic RNA subunit (M1 RNA in E. coli) and a protein subunit (C5 protein in E. coli). M1 RNA acts as a catalyst and cleaves precursor tRNAs (pre-tRNAs) at certain ionic strength buffer conditions (e.g. . 50 mM MgCl2) in vitro even in the absence of C5 protein (Guerrier-Takada et al., 1983). The dramatic increase of catalytic activity of M1 RNA in the presence of C5 protein under physiological buffer condition is consistent with the notion that both the M1 and C5 subunits are essential for the tRNA processing activity in vivo (Frank and Pace, 1998; Altman and Kirsebom, 1999). Moreover, these observations suggest that M1 RNA may be stimulated by cellular proteins, including those from human cells. Meanwhile, the archael and eucaryotic RNase P enzymes are more complex with a single RNA and several protein subunits (Chamberlain et al., 1998; Hall and Brown, 2001; Jarrous and Altman, 2001). The description on structure, function and substrate recognition properties of eucaryotic RNase Ps, including those from human and yeast, is outside the purview of this article, and can be referred to several recently published reviews (Jarrous and Altman, 2001; Xiao et al., 2002). The three dimensional structures of both the holoenzyme and the catalytic M1 RNA subunit of E. coli RNase P remain unknown. However, phylogenetic comparative analysis on the RNase P RNA subunits from diverse organisms and mutational analysis on the consensus sequence of these RNAs provide significant insights into the potential tertiary interactions within the RNA subunit (Haas et al., 1994;

Chen et al., 1998; Massire et al., 1998). Sequence alignments of M1 RNA and the RNA subunits from various eubacteria reveal that maximal sequence conservation falls within the region that makes up the P4 helix (Fig. 1). Studies using sulfur substitutions in the phosphate oxygen have suggested a central role of the P4 helix in the catalysis for pre-tRNA cleavage (Christian et al., 2002). Similarly, several regions of M1 RNA that are also highly conserved among eubacterial RNase P RNAs are found to play important roles in binding the substrate. For example, the GGU residues present in the region linking P15 and P16 of M1 RNA are believed to base pair with the 30 CCA sequence of a pre-tRNA (Kirsebom and Svard, 1994). Moreover, studies have been carried out to understand how M1 RNA interacts with C5 to achieve efficient cleavage of a pretRNA substrate (Barrera et al., 2002; Tsai et al., 2003). Based on extensive biochemical and phylogenetic analyses, models of the secondary and three-dimensional structures of RNase P catalytic subunits including M1 RNA have been proposed and used to define the catalytic active site, regions of C5 docking, substrate binding and magnesium coordination in the ribozymes (Haas et al., 1994; Chen et al., 1998; Massire et al., 1998) (Fig. 1). The RNase P-mediated cleavage is a hydrolysis reaction generating 30 hydroxyl and 50 phosphate groups in the pretRNA cleavage site. Interaction with divalent metal ions is essential for maintaining the highly structured RNA subunit and the catalytic activity of the ribozyme. There are preliminary evidences supporting a hydroxyl attack on the scissile phosphate mediated by magnesium in coordination with water and a phosphate at the pre-tRNA cleavage site with possible interaction of oxygens from RNase P RNA (Rasmussen and Nolan, 2002). In addition, recent studies also suggest that C5 cofactor plays a role in magnesium coordination by enhancing the affinities of metal ions in order to stabilize tRNA binding with M1 RNA (Kurz and Fierke, 2002).

3. Principle underlying RNase P-mediated cleavage The principles governing the substrate specificity of E. coli RNase P have been extensively studied in order to exploit this enzyme for specific gene inactivation. Cleavage assays performed on various deletion derivatives of tRNA revealed that E. coli RNase P and M1 RNA cleave a simple stem-loop structure resembling the T stem stacked on the acceptor stem containing a 30 RCCA (Fig. 2A). Moreover, when the sequence of such a minimal substrate equivalent to the T-loop of a tRNA is nicked to derive a bipartite molecule, M1 RNA is still able to cleave at the canonical þ 1 position efficiently (McClain et al., 1987; Forster and Altman, 1990). The 30 proximal sequence of such a bipartite structure can be regarded as an external guide sequence (EGS) (Fig. 2A). These results lead to the development of a gene-targeting approach, in which M1 RNA or RNase P can

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Fig. 1. Proposed secondary structure (A) (Haas et al., 1994) and three-dimensional structure (B and C) (Massire et al., 1998) of the RNA subunit (M1 RNA) of RNase P from E. coli. (B) A complex of M1 RNA and a ptRNA substrate (in purple) (courtesy from Massire et al., 1998). (C) A complex of M1 RNA (in purple and red) and a substrate consisting of a GS and a mRNA targeting sequence (in white). The red regions represents those sequences that were found in close proximity to a mRNA substrate by UV crosslinking and nuclease footprint analysis (Kilani and Liu, 1999; Trang et al., 1999; Hsu et al., 2000). (C) is generated with a SGI-O2 Workstation using the two current three-dimensional models of M1 RNA from Dr. Eric Westhof’s and Dr. Norman Pace’s laboratories (Chen et al., 1998; Massire et al., 1998).

be directed to cleave a target mRNA if a custom-designed EGS is constructed to hybridize to the mRNA (Altman, 1995; Li et al., 1992; Guerrier-Takada et al., 1997). Moreover, M1 RNA can be converted into a sequencespecific endonuclease, M1GS RNA, by covalently attaching the ribozyme to a guide sequence that can complement with a target mRNA (Fig. 2B) (Frank et al., 1994; Liu and Altman, 1995). Thus, M1GS RNA binds to the mRNA through Watson-Crick base pairing interactions between the guide sequence and the target mRNA sequence (Altman, 1995). The RNase P ribozyme-based gene-targeting approach appears to be highly effective in down-regulating the expression of both cellular and viral genes in cultured cells. Inhibition of the expression of specific mRNAs by M1GS RNA in human and mouse cell lines has been reported from our laboratory as well as in other laboratories (Liu and Altman, 1995; Cobaleda and Sanchez-Garcia, 2000; Trang et al., 2000a,b, 2001, 2002). The following sections of this article will describe the ground rules for engineering RNase P ribozymes and summarize recent

progress on the biochemical characterization of M1GS RNAs and the investigation of these ribozymes for applications in inhibiting gene expression in cell culture.

4. Construction and in vitro characterization of RNase P ribozymes for specific inactivation of gene expression 4.1. Selection of a target for RNase P ribozyme-mediated cleavage Ribozyme approaches are generally suitable for targeting viral and other disease-associated RNAs that are distinct from normal cellular messages. Table 1 lists the various mRNA targets used for the M1GS RNA-based gene inactivation. The messages encoding viral essential functions, including structural proteins (e.g. capsid proteins), DNA and RNA polymerases, and the gene products playing regulatory roles in the expression of viral essential genes, are considered ideal targets for antiviral applications. For example, the mRNAs coding for immediate early gene

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Fig. 2. (A) Representation of the natural substrates (ptRNA and 4.5S RNA) and a small model substrate (EGS:mRNA) for RNase P and M1 RNA. The site of cleavage is marked with a filled arrow. (B) Representation of an M1GS RNA construct to which a target RNA has hybridized.

products IE1 and IE2 of human cytomegalovirus (HCMV), which are essential for the transcription of viral early and late genes (Mocarski and Courcelle, 2001), are suitable targets for RNase P ribozyme targeting. M1GS-mediated cleavage of the overlapping regions of IE1 and IE2 mRNAs leads to an overall inhibition of HCMV gene expression and a reduction of more than 150-fold in viral growth (Trang Table 1 List of various target mRNAs used for M1GS-mediated gene inactivation Gene target Viral target Thymidine kinase human herpes simplex virus 1 Major transcription activator ICP 4 of human herpes simplex virus 1 Major transcription activators IE1 and IE2 of human cytomegalovirus Oncogenic message BCR-ABL

Reference

Liu and Altman, 1995; Kilani et al., 2000 Trang et al., 2000a, 2001

Trang et al., 2000b, 2002

Cobaleda and Sanchez-Garcia, 2000

et al., 2000b). For RNA viruses, including HIV and many other human and animal viruses, virus-encoded enzymes like reverse transcriptase and RNA dependent RNA polymerase (replicase) are considered ideal targets (Rossi, 1999). Alternatively, the host-encoded proteins that help viruses establish the infection could also be served as the targets of interest. In fact, it would be possible to have more than one ribozymes designed against two different targets in order to completely abolish viral replication and subsequent disease progression. 4.2. Choosing target sites and designing effective guide sequences The ribozyme may not get access to the target RNA if the potential target sites are buried within the organized and folded RNA structure. Therefore, a major pre-requisite for M1GS design is to map the regions of the target mRNA that may be accessible to M1GS binding. Three different approaches have been used to determine the regions of a target mRNA that may be exposed to ribozyme binding. First, computer-generated structure predictions are used to

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identify the targets that are likely to have accessible conformations (Zuker and Jacobson, 1995). However, an accurate secondary structure prediction may not be possible using computer algorithms since it is almost impossible to simulate in vivo thermodynamic parameters for folding ‘in silico’. Second, the secondary structure of the target mRNA can be probed with chemicals or enzymes that have different specificity in interacting with an RNA molecule (Brunel and Romby, 2000). For example, the end-labeled mRNA sequence is digested with RNases such as RNase T1, nuclease S1, and RNase V1 separately and the cleavage products are resolved on a denaturing gel. Unlike RNase T1 and nuclease S1, which recognize the single-stranded regions, RNase V1 only cleaves the regions of the RNA that are either base-paired or involved in tertiary interactions. The third approach is to map the accessible regions of a target RNA in cellular environment in which RNA folding is influenced by cellular proteins that may interact with the mRNA. Mapping of the accessible regions of an mRNA is possible using an in vivo mapping method based on nucleotide modifications by dimethyl sulphate (DMS) (Ares and Igel, 1990; Liu and Altman, 1995; Zaug and Cech, 1995). When cells are incubated in medium containing DMS, the chemical compounds enter the cells and modify the nucleotides in the accessible regions of a mRNA. The regions of the mRNAs that are exposed and modified by DMS can be determined by primer extension analysis using reverse transcriptase. It is preferable to avoid both the 50 and 30 untranslated regions as they may engage with regulatory proteins and translational apparatus. In addition to the fact that the target region should be accessible, its flanking sequences should also exhibit several sequence features that need to be present in order to interact with an M1GS ribozyme to achieve efficient cleavage. These features include the requirement for a guanosine and a pyrimidine to be the nucleotide 30 and 50 adjacent to the site of cleavage, respectively (Liu and Altman, 1996). The interactions of these sequence elements with the M1GS ribozyme are critical for recognition and cleavage by the enzyme. The guide sequence often includes 13 –18 nucleotides complementary to the potential target sites of the mRNA to achieve sufficient base pairing and target specificity. Guide sequences with an optimal length are desirable for efficient cleavage, high targeting specificity, and rapid dissociation rates. An unpaired 30 terminal ACCA sequence, which is important for M1GS recognition and cleavage efficiency, should also be included in the guide sequence (Liu and Altman, 1996). 4.3. Interactions between M1GS RNA and an mRNA in the absence and presence of a protein factor While extensive studies have been carried out to understand how M1 RNA interacts with a tRNA molecule, little is known about the mRNA binding site of a M1GS ribozyme and how a M1GS RNA interacts with a model

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mRNA substrate. Recent studies have been carried out to address these issues. The regions of the ribozyme that are in close contact with a model mRNA substrate were mapped by both UV crosslinking and nuclease footprint analysis (Kilani and Liu, 1999; Trang et al., 1999). The results from these two complementary mapping approaches suggest that (1) the cleavage site of the mRNA substrate is positioned at the same regions of the ribozyme which bind to the cleavage site of a ptRNA, (2) the target sequence of a model mRNA substrate interacts with the same regions of the ribozyme that are in close proximity to the acceptor stem of a ptRNA, and (3) the 50 leader and 30 tail sequence of a model mRNA substrate (Fig. 2B) interact with some of the ribozyme regions (e.g. P12, P13, J11/14, and P14) which are not believed to interact with a ptRNA substrate (Fig. 1C). Identification of the binding sites of the ribozyme to different regions of the mRNA substrate serves as a starting point to investigate how a M1GS ribozyme recognizes its mRNA substrates and achieves sequence specificity. By mutating the binding site and manipulating the interactions between the binding site and the substrate, we may be able to construct ribozymes that achieve optimal substrate binding and cleavage efficiency. The effects of C5 on the interactions between a M1GS ribozyme and a model mRNA substrate are also studied in order to understand how a M1GS RNA catalyzes the cleavage of a mRNA substrate in the presence of cellular proteins (Hsu et al., 2000). In order to develop RNase P ribozymes that are highly effective in vivo, it is essential to understand how cellular proteins interact with a M1GS ribozyme and affect its activity in cells. Studies of the effects of C5 protein on the interactions between a M1GS ribozyme and an mRNA substrate provide a model system to understand how the ribozyme functions to cleave an mRNA substrate in vivo, in the presence of cellular proteins. Kinetic analyses under single-turnover conditions indicated that the presence of C5 protein increases the activity (kcat/Km) of a M1GS ribozyme to cleave an mRNA substrate by at least 100-fold (Hsu et al., 2000). The effects of C5 on the interactions between the M1GS ribozyme and substrate tk46, which contains a sequence of the mRNA (TK mRNA) encoding the thymidine kinase (TK) of herpes simplex virus 1 (HSV-1), were investigated by UV crosslink mapping. A set of mRNA model substrates containing photoactive 4-thio-uridines at different positions were crosslinked to the ribozyme both in the absence and presence of C5. The ribozyme regions that are in close proximity to different parts of the substrates were mapped by primer extension analyses of the crosslinked conjugates. These results suggest that C5 protein does not significantly affect the interaction of the ribozyme to the targeting sequence of the substrate adjacent to the cleavage site (Hsu et al., 2000). However, differences in the interactions of the ribozyme to the 50 leader and 30 tail sequence were found in the presence of the protein cofactor. For example, the presence of C5 favors the interactions between J11/14 and the 30 tail

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sequence of the substrate. Meanwhile, some of the ribozyme regions (e.g. J11/12), which were crosslinked to the leader sequence 50 immediately to the cleavage site in the presence of C5, were different from those regions (e.g. P3) found in the absence of the protein cofactor (Hsu et al., 2000). Understanding the effects of C5 protein on the interactions between a M1GS ribozyme and an mRNA model substrate may provide insight into developing effective RNase P ribozymes that recognize their mRNA substrates efficiently in vivo in the presence of cellular proteins. It is reasonable to suggest that the differences in ribozymemRNA substrate interactions in the presence of C5 may account for the better substrate recognition and the increased cleavage efficiency observed in the presence of the protein cofactor. If this is the case, a ribozyme mutant, which, in the absence of C5, interacts with an mRNA substrate under low salt conditions in a similar way as the wild type ribozyme-C5 complexes, is expected to exhibit optimal catalytic activity to cleave an mRNA substrate under physiological conditions in the absence of the protein cofactor. Indeed, a ribozyme variant with base substitution mutations at positions 224 and 225 of the J11/14 region exhibits at least a 10-fold higher efficiency to cleave a mRNA substrate in the absence of C5 (see below) (Kilani et al., 2000). Alternatively, ribozyme variants, which, upon binding a cellular protein, exhibit similar interactions to an mRNA substrate as those observed in the presence of C5, may function effectively in vivo. Further studies to generate these ribozyme variants and to investigate the mechanism of how they interact with a mRNA substrate will facilitate the construction of ribozymes that exhibit optimal substrate binding and high sequence specificity in vivo (Kilani and Liu, 1999; Trang et al., 1999; Hsu et al., 2000).

5. Delivery, expression, and activity of M1GS ribozymes in cultured cells 5.1. Delivery and expression of ribozymes The efficacies of M1GS ribozymes are investigated in vivo by either stably or transiently expressing them in cellular environment. Virus-based gene therapy vectors, including those derived from adenovirus, adeno-associated virus (AAV), and lentivirus as well as other retroviruses, can be used to deliver the sequences encoding ribozyme into numerous types of cells and tissues (Wang et al., 1999; Mautino, 2002; Horster et al., 1999; Rossi, 1999; Yu et al., 1993). Ribozymes expressed from these vectors are effective in inhibiting the expression of the target. Further studies on constructing better expression and delivery vectors will facilitate the development of the ribozymebased technology as a powerful gene-targeting approach that can be used in both basic research and clinical gene therapy applications. We have been employing retroviral vectors for the stable expression of M1GS ribozyme in both

human and murine cells. These vectors are desirable for ribozyme delivery because of their broad host range, efficient integration into the host genome, and abundant expression of ribozyme in cell lines and animal models. In our studies, the retroviral expression vector LXSN has been used as the viral shuttle vector (Miller and Rosman, 1989). M1GS RNAs are placed under the control of the U6 promoter and termination signal, which has been shown to express M1GS RNA steadily in cells (Bertrand et al., 1997; Good et al., 1997; Kawa et al., 1998; Kilani et al., 2000; Liu and Altman, 1995). The reasons for choosing this promoter are that (i) U6 snRNA is a very strong RNA polymerase III promoter which supports the synthesis of over 106 U6 snRNAs in every mammalian cell, (ii) RNA transcripts synthesized from this promoter remain primarily in the nucleus where RNase P is localized, and (iii) this promoter has been successfully used to express M1GS RNAs and other functional RNAs in the nucleus at a high level (Yuan et al., 1992; Bertrand et al., 1997; Good et al., 1997; Kawa et al., 1998). The U6 promoter with the 50 sequence that is required for capping is also used for the expression of the M1GSs in order to increase their stability and steady-state expression levels (Bertrand et al., 1997; Good et al., 1997). When the ribozyme needs to be delivered to target an mRNA in the cytoplasm, tRNA sequences may be linked with ribozymes and transcribed by native tRNA promoters in order to colocalize the target mRNA and ribozyme into the cytoplasm (Warashina et al., 2001). There are reports using promoters for the RNA polymerase II to express ribozymes in vivo (Sarver et al., 1990). As the RNA polymerase II-expressed messages usually contain 50 terminal cap and 30 poly A sequences, these additional structural elements might prevent proper folding of the active conformation of M1 RNA in vivo. Thus, the utility of different promoters for ribozyme expression in vivo varies upon the localization of target mRNA within the subcellular region or/and specific tissue type. Ex vivo delivery of ribozymes into human cells is gaining ground due to the selective advantage of this technique over the vector-based gene delivery approach. The ribozymes could be chemically synthesized and the nucleoside residues could be modified appropriately so that the synthetic RNA molecules are not susceptible for cellular nuclease degradation. The oligonucleotides with 20 hydroxyl modification and/or phosphorothioates are highly resistant to cellular endonucleases (Verma and Eckstein, 1998). The delivery of such modified oligonucleotides could be accomplished by encapsulating them in liposomes or other biodegradable polymeric matrices (Jackson et al., 2002). The efficacy of this method in vivo is being explored for antisense, ribozyme, and EGS molecules (Ma et al., 2000; Dunn et al., 2001). However, chemical synthesis of a functional active M1GS ribozyme is at present technically difficult and economically impractical due to its large size (, 400 nucleotides). Thus, endogenous and stable expression of a transgene coding for the RNase P ribozyme currently

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appears to be the superb choice for M1GS expression and delivery. 5.2. Kinetics of M1GS action in cells While little is known about the rate-limiting step of M1GS RNA cleavage reaction in cultured cells, studies on hammerhead and hairpin ribozymes suggest that binding of the ribozyme to its target RNA appears to be rate-limiting in vivo (Sullenger and Cech, 1993; Lee et al., 1999; zu Putlitz et al., 1999). Extensive studies have been carried out to develop strategies to express these ribozymes to colocalize with the substrates within a particular cellular compartment, and to design ribozymes to target the mRNA regions that are accessible to binding (Sullenger and Cech, 1993; Yu et al., 1993; Bertrand et al., 1997; Lee et al., 1999; zu Putlitz et al., 1999). These studies lead to significant improvements in the efficacies of hammerhead and hairpin ribozymes in cellular environments. In our studies, M1GS RNAs are constructed to target the mRNA regions that are accessible to modification by dimethyl sulphate in cell culture and also accessible to ribozyme binding (Kilani et al., 2000; Trang et al., 2000a,b). Moreover, the ribozymes are expressed primarily in the nuclei by using the promoter of small nuclear U6 RNA. This design would increase the probability for the constructed RNase P ribozyme to locate and bind to its target mRNA sequence. Under such conditions, it has been demonstrated that the rate of the RNase P ribozyme cleavage in culture cells is dictated by the overall cleavage rate (kcat/Km) of the ribozyme (Kilani et al., 2000; Trang et al., 2001, 2002). For example, R6 and R29, which are M1GS variants generated by an in vitro selection procedure, are at least 10 and 20-fold more active (kcat/Km) in cleaving HSV-1 TK mRNA in vitro than the ribozyme (i.e. M1-TK) derived from the wild type M1 sequence, respectively. A reduction of 95 and 99% in TK mRNA and protein expression was observed in cells expressing R6 and R29, respectively, while a reduction of 70% in TK expression was found in the M1-TK-expressing cells (Kilani et al., 2000). These results suggest that increasing the catalytic efficiency of the ribozyme should lead to more effective inhibition of the target mRNA expression in cultured cells.

6. Engineering of M1GS ribozymes by in vitro selection 6.1. In vitro selection of catalytically efficient M1GS ribozyme The catalytic efficiency of M1 RNA can be enhanced by using in vitro selection procedures, which have been successful in generating either new RNA catalysts or evolving more efficient variants from known ribozyme molecules (Szostak, 1992; Gold et al., 1993; Joyce, 2000). Recently, we have developed a selection system to generate

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RNase P ribozyme variants that efficiently cleave the HSV-1 TK mRNA sequence in vitro. Randomized mutations were introduced into several conserved regions of M1 RNA that are important for catalysis and substrate binding, and those M1GS RNA variants that efficiently cleaved the mRNA substrate in vitro were selected by our in vitro selection procedure. In this procedure (Fig. 3), the ribozyme molecules are annealed to 50 -biotinylated RNA substrate tk46, and the annealed complexes are allowed to bind to a streptavidin column in the absence of divalent ions such as Mg2þ, which is essential for RNase P ribozyme catalysis (Guerrier-Takada et al., 1983) (Fig. 3). All unbound ribozymes are washed away during this step. The cleavage buffer containing Mg2þ is then added to the column to allow the cleavage reaction to occur. The ribozymes that cleave their substrates are released from the column and loaded on a denaturing gel, and these active ribozymes are then recovered from the gel. The cDNA copies of these RNA molecules are synthesized and amplified by RT-PCR to generate DNA templates for the synthesis of ribozyme molecules for the next round of selection. The selection procedure can be repeated until no apparent enhancement of cleavage rate is observed (Fig. 3). The ribozyme variants identified by their higher catalytic efficiency are cloned and sequenced. The sequence information can be used to identify the mutations that are responsible for the increased catalytic efficiency of the selected variants (Kilani et al., 2000). 6.2. In vitro biochemical characterization of the selected ribozymes Biochemical characterization, including kinetic and structural analysis, can be carried out to study how the selected variants achieve higher activity than the ribozyme derived from the wild type M1 RNA sequence. Studies of some of these variants (Kilani et al., 2000; Trang et al., 2001, 2002) have provided significant insights into the catalytic mechanism of how RNase P ribozymes cleave an mRNA substrate. These results indicate that engineered RNase P ribozymes can increase their activities (the values of kcat/Km) in cleaving an mRNA in vitro by enhancing both the substrate binding and the rate of chemical cleavage. Specifically, these findings can be summarized as follows. (a) Variants with mutations (G224G225 ! A224A225) exhibit at least 10-fold higher cleavage efficiencies in cleaving HSV-1 TK mRNA than M1-TK (i.e. the ribozyme derived from the wild type M1 sequence). Moreover, the binding affinities (i.e. the value of Kd) of these selected ribozymes to TK mRNA are at least 50 times better than that of M1-TK (Kilani et al., 2000). UV crosslinking studies suggest that the mutated nucleotides are in close proximity to the 30 tail sequence of the substrate (Kilani and Liu, 1999; Kilani et al., 2000). Perhaps these mutated nucleotides strengthen the interactions between the ribozymes and the substrate by directly interacting with the substrate.

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Fig. 3. In vitro selection of M1GS ribozymes that efficiently cleave a mRNA.

(b) A point mutation at nucleotide 86 of M1 RNA (A86 ! C86) increases the rate of chemical cleavage while another mutation at nucleotide 205 (G205 ! C205) enhances substrate binding of the ribozyme (Trang et al., 2002). Moreover, ribozyme variant V71, which contains both these two mutations (A86 ! C86, G205 ! C205) is at least 25 times more efficient in cleaving the target mRNA in vitro than the ribozyme derived from the wild type M1 RNA sequence (Trang et al., 2002).

cleavage activities (kcat/Km) of RNase P ribozymes and their efficacies in cultured cells and further suggest that improvement of their in vitro catalytic efficiencies should lead to increased efficacies in tissue culture. Thus, in vitro selection may be used as a general approach to generate M1GS ribozymes that efficiently cleave a target mRNA and are effective in inhibiting its expression in cell culture.

7. Therapeutic use of M1GS ribozyme 6.3. Enhanced efficacy of the selected ribozymes in cultured cells The selected ribozyme variants are also more effective in down-regulating gene expression in cultured cells than the ribozyme derived from the wild type M1 RNA sequence. For example, a reduction of up to 99% in the expression of HSV-1 TK was observed in cells expressing one of the selected ribozymes (R29) while a reduction of about 70% was found in the cells that expressed M1-TK (Kilani et al., 2000). The selected ribozymes also exhibit higher efficacy in inhibiting the expression of other mRNAs, including HSV-1 ICP4 mRNA and HCMV IE1/IE2 mRNAs (Trang et al., 2001, 2002). In summary, novel RNase P ribozyme variants can be selected to cleave an mRNA substrate efficiently in vitro. Moreover, the selected ribozymes are also more effective in inhibiting the expression of the target mRNA in cultured cells than the ribozyme derived from the wild type M1 RNA. These studies provide the first direct evidence that RNase P ribozyme variants isolated by the selection procedure can be used for the construction of gene-targeting ribozymes that are highly effective in tissue culture. These results also demonstrate the correlation between the in vitro

7.1. Ribozyme-mediated inhibition of the replication of herpes viruses Human herpes simplex virus 1 (HSV-1) (Roizman and Knipe, 2001) and cytomegalovirus (HCMV) (Mocarski and Courcelle, 2001) are ubiquitous herpes viruses that cause mild or subclinical diseases in immunocompetent adults but may lead to severe morbidity or mortality in neonates and immunocompromised individuals. In particular, HSV-1 is the causative agent for cold sores and encephalitis in newborns. HCMV is one of the leading causes of birth defects in newborns and causes one of the most important opportunistic infections encountered in patients with AIDS. While there are several drugs currently available for anti-herpes treatment, the toxicity of these compounds and the emergence of the drug-resistant strains post the need to develop novel approaches and strategies for treatment and prevention of these viral infections. When M1GS ribozymes targeting the overlapping region of IE1 and IE2 mRNAs were expressed in HCMV-infected cells, a reduction of up to 98% in IE1 and IE2 expression and an inhibition of up to 3,000-fold in HCMV growth were observed (Trang et al., 2000b, 2002). Furthermore, there

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was a direct correlation between the reduction of IE1 and IE2 expression and inhibition of viral growth in the M1GSexpressing cells. The downregulation of IE1 and IE2 by M1GS RNA expression led to a significant reduction of the expression of other early and late gene products such as US2, UL44, gB, and gH. On the other hand, the expression of other viral immediate-early transcripts (e.g. 5 kb RNA and UL36 mRNA), which are not regulated by IE1 or IE2, was unaltered in the ribozyme-expressing cells (Trang et al., 2000b, 2002). Meanwhile, M1GS expression does not exhibit significant cytotoxicity as the M1GS-containing cells were indistinguishable from the parental cells in terms of cell growth and viability for up to two months. These results strongly suggest that M1GS is highly specific in cleaving its target mRNA. In another study, M1GS ribozyme was used to target the mRNA encoding the major transcription activator ICP4 of HSV-1 (Trang et al., 2000a). ICP4 is essential for the expression of most of the viral early and late genes. The ribozyme specifically cleaved the ICP4 mRNA in vitro and its expression in HSV-1 infected cells resulted in a reduction of about 80% in the expression of ICP4 and a 1000-fold decrease in viral growth. Expressions of other HSV-1 immediate-early genes (e.g. a47 and ICP27) that are not regulated by ICP4 were not affected in the M1GSexpressing cells, suggesting that the ribozyme specifically targets the ICP4 mRNA (Trang et al., 2000a). These results demonstrate the feasibility of using RNase P ribozymes for treating and preventing infections caused by these human viruses and furthermore, provide a foundation for future studies on evaluating the efficacy of M1GS in animal models. 7.2. Cleavage of cancer-causing message in vivo Because of the specific cleavage of targets, ribozymes are advocated as a logical remedy for effective treatment of autosomal dominant diseases ranging from cancer to other genetic disorders (Cobaleda and Sanchez-Garcia, 2000; Lewin and Hauswirth, 2001). While employing ribozymes for gene inactivation, it is important to distinguish the pathogenic message from the normal transcripts for cleavage. The chimeric transcripts that contain additional exons due to chromosomal translocation in the cancerous cells are considered ideal therapeutic targets because cells deprived of these messages rapidly undergo apoptotic cell death which in turn results into tumor suppression. The random chromosomal translocation leads to the generation of abundant genetic messages, and the chimeric fusion proteins encoded by these mRNAs, which may induce indiscriminate cell proliferation. For example, translocation between chromosome 9 and 22 fuses the genes of BCR and c-ABL to produce BCR-ABL (Shtivelman et al., 1985). M1GS-mediated cleavage on the BCR-ABL fusion transcripts could specifically destroy these malignant messages without affecting normal BCR or ABL transcripts that are transcribed

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from another copy of the chromosome. Cobaleda and Sanchez-Garcia (2000) have elegantly shown that M1GS RNA can effectively inhibit the expression of the BCR-ABL fusion transcripts found in the cancerous cells of leukemia patients. In their study, the guide sequence complementary to the fusion junction of BCR-ABL was attached to the M1GS ribozyme to specifically cleave the oncogenic BCR-ABL message in cultured cells. These experiments have great implication in utilizing M1GS RNA as a promising agent for cancer therapy. 8. Outlook Recent studies have clearly demonstrated that RNase P ribozymes are efficient and specific in cleaving an mRNA sequence in vitro, and are effective in down-regulating the expression of both cellular and viral genes, and blocking viral infection in cultured cells. Research progress on biochemical characterization of M1GS RNA has provided significant insights into the molecular mechanism of how RNase P ribozyme achieves high efficiency and sequence specificity in cleaving an mRNA substrate. Moreover, ribozyme variants that are more active and effective in inhibiting gene expression can be generated using in vitro selection procedures. These results should generate guidelines for constructing highly effective M1GS RNAs for gene-targeting applications. In order to further develop M1GS RNA for gene therapy applications, the efficacy of this ribozyme needs to be evaluated in animal models and ultimately, in human clinical trials. Several issues like ribozyme delivery, stability, colocalization and a sustained effect by ribozymes on their targets may also need to be addressed in order to achieve successful ribozyme-based gene therapy against viral infections and other human diseases. Meanwhile, the ribozyme approach represents a promising gene-targeting strategy for studying gene functions. Combined with the genomic information that is available, this approach can be used to identify genetic messages involved in pathogenic mechanisms and to define potential drug targets. As the sequence of the target is known, ribozymes can be readily designed to shut down the expression of the desired gene. Moreover, ribozymes could also be employed to elucidate the roles of an array of genetic messages (e.g. expressed sequence tags) that have been accumulated in the genomic databases and that have no known function. Further studies on the biochemistry of RNase P ribozyme in vitro and on its activity in cultured cells and in animal models should significantly facilitate the development of M1GS RNAs as a novel class of gene-targeting agents for applications in both basic research and clinical therapy settings. Acknowledgements We thank Manfred Lee, Phong Trang, and Kihoon Kim for critical reading and comments on the manuscript. F. L. is

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a Pew Scholar in Biomedical Sciences and a Scholar of Leukemia and Lymphoma Society, and a recipient of American Heart Association Established Investigator Award. This research was, in part, supported by State of California Universitywide AIDS Research Program, March of Dimes National Birth Defects Foundation, and NIH.

References Altman, S., 1995. RNase P in research and therapy. Biotechnology 13, 327– 329. Altman, S., Kirsebom, L., 1999. Ribonuclease P. In: Gesteland, R.F., Cech, T.R., Atkins, J.F. (Eds.), The RNA World, 2, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 351–380. Ares, M. Jr., Igel, A.H., 1990. Mutations define essential and nonessential U2 RNA structures. Mol. Biol. Rep. 14, 131–132. Barrera, A., Fang, X., Jacob, J., Casey, E., Thiyagarajan, P., Pan, T., 2002. Dimeric and monomeric Bacillus subtilis RNase P holoenzyme in the absence and presence of pre-tRNA substrates. Biochemistry 41, 12986–12994. Bertrand, E., Castanotto, D., Zhou, C., et al., 1997. The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3, 75–88. Breaker, R.R., 2000. Tech. Sight. Molecular biology. Making catalytic DNAs. Science 290, 2095–2096. Brunel, C., Romby, P., 2000. Probing RNA structure and RNA-ligand complexes with chemical probes. Methods Enzymol. 318, 3– 21. Chamberlain, J.R., Lee, Y., Lane, W.S., Engelke, D.R., 1998. Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP. Genes Dev. 12, 1678–1690. Chen, J.L., Nolan, J.M., Harris, M.E., Pace, N.R., 1998. Comparative photocross-linking analysis of the tertiary structures of Escherichia coli and Bacillus subtilis RNase P RNAs. EMBO J. 17, 1515– 1525. Christian, E.L., Kaye, N.M., Harris, M.E., 2002. Evidence for a polynuclear metal ion binding site in the catalytic domain of ribonuclease P RNA. EMBO J. 21, 2253–2262. Cobaleda, C., Sanchez-Garcia, I., 2000. In vivo inhibition by a site-specific catalytic RNA subunit of RNase P designed against the BCR-ABL oncogenic products: a novel approach for cancer treatment. Blood 95, 731– 737. Doudna, J.A., Cech, T.R., 2002. The chemical repertoire of natural ribozymes. Nature 418, 222– 228. Dunn, W., Trang, P., Khan, U., Zhu, J., Liu, F., 2001. RNase P-mediated inhibition of cytomegalovirus protease expression and viral DNA encapsidation by oligonucleotide external guide sequences. Proc. Natl. Acad. Sci. USA 98, 14831–14836. Forster, A.C., Altman, S., 1990. External guide sequences for an RNA enzyme. Science 249, 783– 786. Frank, D.N., Harris, M.E., Pace, N.R., 1994. Rational design of selfcleaving pre-tRNA-ribonuclease P RNA conjugates. Biochemistry 33, 10800–10808. Frank, D.N., Pace, N.R., 1998. Ribonuclease P: unity and diversity in a tRNA processing ribozyme. Annu. Rev. Biochem. 67, 153 –180. Gold, L., Allen, P., Binkley, J., Brown, D., Schneided, D., Eddy, S.R., Turek, C., Green, L.M., Tasset, S., et al., 1993. RNA: the shape of things to come. In: Gesteland, R.F., Atkins, J.F., et al. (Eds.), The RNA World, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, pp. 497 –509. Good, P.D., Krikos, A.J., Li, S.X., et al., 1997. Expression of small, therapeutic RNAs in human cell nuclei. Gene Ther. 4, 45–54. Guerrier-Takada, C., Gardiner, K., Marsh, T., Pace, N., Altman, S., 1983. The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849– 857.

Guerrier-Takada, C., Salavati, R., Altman, S., 1997. Phenotypic conversion of drug-resistant bacteria to drug sensitivity. Proc. Natl. Acad. Sci. USA 94, 8468–8472. Guerrier-Takada, C., Altman, S., 2000. Inactivation of gene expression using ribonuclease P and external guide sequences. Methods Enzymol. 313, 442–456. Haas, E.S., Brown, J.W., Pitulle, C., Pace, N.R., 1994. Further perspective on the catalytic core and secondary structure of ribonuclease P RNA. Proc. Natl. Acad. Sci. USA 91, 2527–2531. Hall, T.A., Brown, J.W., 2001. The ribonuclease P family. Methods Enzymol. 341, 56– 77. Horster, A., Teichmann, B., Hormes, R., Grimm, D., Kleinschmidt, J., Sczakiel, G., 1999. Recombinant AAV-2 harboring gfp-antisense/ ribozyme fusion sequences monitor transduction, gene expression, and show anti-HIV-1 efficacy. Gene Ther. 6, 1231–1238. Hsu, A.W., Kilani, A.F., Liou, K., Lee, J., Liu, F., 2000. Differential effects of the protein cofactor on the interactions between an RNase P ribozyme and its target mRNA substrate. Nucleic Acids Res. 28, 3105–3116. Jackson, J.K., Liang, L.S., Hunter, W.L., Reynolds, M., Sandberg, J.A., Springate, C., Burt, H.M., 2002. The encapsulation of ribozymes in biodegradable polymeric matrices. Int. J. Pharm. 243, 43–55. Jarrous, N., Altman, S., 2001. Human ribonuclease P. Methods Enzymol. 342, 93–100. Joyce, G.F., 2000. RNA structure. Ribozyme evolution at the crossroads. Science 289, 401– 402. Kawa, D., Wang, J., Yuan, Y., Liu, F., 1998. Inhibition of viral gene expression by human ribonuclease P. RNA 4, 1397–1406. Kilani, A.F., Liu, F., 1999. UV cross-link mapping of the substrate-binding site of an RNase P ribozyme to a target mRNA sequence. RNA 5, 1235–1247. Kilani, A.F., Trang, P., Jo, S., Hsu, A., Kim, J., Nepomuceno, E., Liou, K., Liu, F., 2000. RNase P ribozymes selected in vitro to cleave a viral mRNA effectively inhibit its expression in cell culture. J. Biol. Chem. 275, 10611–10622. Kirsebom, L.A., Svard, S.G., 1994. Base pairing between Escherichia coli RNase P RNA and its substrate. EMBO J. 13, 4870–4876. Kraus, G., Geffin, R., Spruill, G., Young, A.K., Seivright, R., Cardona, D., Burzawa, J., Hnatyszyn, H.J., 2002. Cross-clade inhibition of HIV-1 replication and cytopathology by using RNase P-associated external guide sequences. Proc. Natl. Acad. Sci. USA 99, 3406–3411. Kurz, J.C., Fierke, C.A., 2002. The affinity of magnesium binding sites in the Bacillus subtilis RNase P x pre-tRNA complex is enhanced by the protein subunit. Biochemistry 41, 9545– 9558. Lan, N., Howrey, R.P., Lee, S.W., Smith, C.A., Sullenger, B.A., 1998. Ribozyme-mediated repair of sickle beta-globin mRNAs in erythrocyte precursors. Science 280, 1593–1596. Lee, N.S., Bertrand, E., Rossi, J., 1999. mRNA localization signals can enhance the intracellular effectiveness of hammerhead ribozymes. RNA 5, 1200–1209. Lewin, A.S., Hauswirth, W.W., 2001. Ribozyme gene therapy: applications for molecular medicine. Trends Mol. Med. 7, 221 –228. Li, Y., Guerrier-Takada, C., Altman, S., 1992. Targeted cleavage of mRNA in vitro by RNase P from Escherichia coli. Proc. Natl. Acad. Sci. USA 89, 3185–3189. Liu, F., Altman, S., 1995. Inhibition of viral gene expression by the catalytic RNA subunit of RNase P from Escherichia coli. Genes Dev. 9, 471 –480. Liu, F., Altman, S., 1996. Requirements for cleavage by a modified RNase P of a small model substrate. Nucleic Acids Res. 24, 2690–2696. Ma, M., Benimetskaya, L., Lebedeva, I., Dignam, J., Takle, G., Stein, C.A., 2000. Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. Nat. Biotechnol. 18, 58–61. Massire, C., Jaeger, L., Westhof, E., 1998. Derivation of the threedimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis. J. Mol. Biol. 279, 773–793.

S.M.L. Raj, F. Liu / Gene 313 (2003) 59–69 Mautino, M.R., 2002. Lentiviral vectors for gene therapy of HIV-1 infection. Curr. Gene Ther. 2, 23–43. McClain, W.H., Guerrier-Takada, C., Altman, S., 1987. Model substrates for an RNA enzyme. Science 238, 527–530. Miller, A.D., Rosman, G.J., 1989. Improved retroviral vectors for gene transfer and expression. Biotechniques 7, 980–990.pp. 980-982, 984986, 989-990. Mocarski, E.S., Courcelle, T.C., 2001. Cytomegaloviruses and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Virology, 4, Virology, 2. Lippincott, Williams, and Wilkins, pp. 2629–2673. Plehn-Dujowich, D., Altman, S., 1998. Effective inhibition of influenza virus production in cultured cells by external guide sequences and ribonuclease P. Proc. Natl. Acad. Sci. USA 95, 7327–7332. Rasmussen, T., Nolan, J., 2002. G350 of Escherichia coli RNase P RNA contributes to Mg(2 þ ) binding near the active site of the enzyme. Gene 294, 177. Roizman, B., Knipe, D.M., 2001. Herpes simplex viruses and their replication. In: Knipe, D.M., Howley, P.M. (Eds.), Virology, 4, Virology, 2. Lippincott, Williams, and Wilkins, Philadelphia, PA, pp. 2629–2673. Rossi, J.J., 1999. Ribozymes, genomics and therapeutics. Chem. Biol. 6, R33–R37. Santoro, S.W., Joyce, G.F., 1997. A general purpose RNA-cleaving DNA enzyme. Proc. Natl. Acad. Sci. USA 94, 4262–4266. Sarver, N., Cantin, E.M., Chang, P.S., Zaia, J.A., Ladne, P.A., Stephens, D.A., Rossi, J.J., 1990. Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222– 1225. Shtivelman, E., Lifshitz, B., Gale, R.P., Canaani, E., 1985. Fused transcript of ABL and BCR genes in chronic myelogenous leukaemia. Nature 315, 550–554. Stein, C.A., Cheng, Y.C., 1993. Antisense oligonucleotides as therapeutic agents – is the bullet really magical? Science 261, 1004–1012. Sullenger, B.A., Cech, T.R., 1993. Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science 262, 1566–1569. Sullenger, B.A., Gilboa, E., 2002. Emerging clinical applications of RNA. Nature 418, 252– 258. Szostak, J.W., 1992. In vitro genetics. Trends Biochem. Sci. 17, 89–93. Trang, P., Hsu, A., Zhou, T., Lee, J., Kilani, A.F., Nepomuceno, E., Liu, F., 2002. Engineered RNase P ribozymes inhibit gene expression and growth of cytomegalovirus by increasing rate of cleavage and substrate binding. J. Mol. Biol. 315, 573–586. Trang, P., Hsu, A.W., Liu, F., 1999. Nuclease footprint analyses of the interactions between RNase P ribozyme and a model mRNA substrate. Nucleic Acids Res. 27, 4590–4597.

69

Trang, P., Kilani, A., Kim, J., Liu, F., 2000a. A ribozyme derived from the catalytic subunit of RNase P from Escherichia coli is highly effective in inhibiting replication of herpes simplex virus 1. J. Mol. Biol. 301, 817– 826. Trang, P., Lee, M., Nepomuceno, E., Kim, J., Zhu, H., Liu, F., 2000b. Effective inhibition of human cytomegalovirus gene expression and replication by a ribozyme derived from the catalytic RNA subunit of RNase P from Escherichia coli. Proc. Natl. Acad. Sci. USA 97, 5812–5817. Trang, P., Lee, J., Kilani, A.F., Kim, J., Liu, F., 2001. Effective inhibition of herpes simplex virus 1 gene expression and growth by engineered RNase P ribozyme. Nucleic Acids Res. 29, 5071–5078. Tsai, H.Y., Masquida, B., Biswas, R., Westhof, E., Gopalan, V., 2003. Molecular modeling of the three-dimensional structure of the bacterial RNase P holoenzyme. J. Mol. Biol. 325, 661–675. Verma, S., Eckstein, F., 1998. Modified oligonucleotides: synthesis and strategy for users. Annu. Rev. Biochem. 67, 99–134. Wang, J.P., Enjoji, M., Tiebel, M., Ochsner, S., Chan, L., Teng, B.B., 1999. Hammerhead ribozyme cleavage of apolipoprotein B mRNA generates a truncated protein. J. Biol. Chem. 274, 24161–24170. Warashina, M., Kuwabara, T., Kato, Y., Sano, M., Taira, K., 2001. RNAprotein hybrid ribozymes that efficiently cleave any mRNA independently of the structure of the target RNA. Proc. Natl. Acad. Sci. USA 98, 5572–5577. Xiao, S., Scott, F., Fierke, C.A., Engelke, D.R., 2002. Eukaryotic ribonuclease P: a plurality of ribonucleoprotein enzymes. Annu. Rev. Biochem. 71, 165–189. Yu, M., Ojwang, J., Yamada, O., Hampel, A., Rapapport, J., Looney, D., Wong-Staal, F., 1993. A hairpin ribozyme inhibits expression of diverse strains of human immunodeficiency virus type 1. Proc. Natl. Acad. Sci. USA 90, 6340–6344. Yuan, Y., Hwang, E.S., Altman, S., 1992. Targeted cleavage of mRNA by human RNase P. Proc. Natl. Acad. Sci. USA 89, 8006–8010. Zamecnik, P.C., Stephenson, M.L., 1978. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc. Natl. Acad. Sci. USA 75, 280–284. Zaug, A.J., Cech, T.R., 1995. Analysis of the structure of Tetrahymena nuclear RNAs in vivo: telomerase RNA, the self-splicing rRNA intron, and U2 snRNA. RNA 1, 363– 374. zu Putlitz, J., Yu, Q., Burke, J.M., Wands, J.R., 1999. Combinatorial screening and intracellular antiviral activity of hairpin ribozymes directed against hepatitis B virus. J. Virol. 73, 5381–5387. Zuker, M., Jacobson, A.B., 1995. “Well-determined” regions in RNA secondary structure prediction: analysis of small subunit ribosomal RNA. Nucleic Acids Res. 23, 2791– 2798.