Antisense Catalytic RNAs as Therapeutic Agents

Antisense Catalytic RNAs as Therapeutic Agents Daniela CastanOtto,* John J. Rossi,* and Nava Sawed *Division of Biology Beckman Research Znstitute of the City of Hope Duarte, California 91010 ‘Developmental Therapeutics Branch Division of AZDS National Institute of Allergy and Infectious Diseases Bethesda, Maryland 20892

1. Introduction Over the last 50 years a large number of drugs that alter the biochemistry of the cell have been developed. Classical pharmacology is based on the principle of designing drugs to obstruct or modify a specific metabolic or biochemical pathway or receptor function by interacting with the active sites of the responsible protein. Therapeutic nucleic acids are emerging as a novel class of drugs applicable to diseases in disparate areas, ranging from agricultural to human viral infections and inborn metabolic errors. These new drugs could effect a dramatic increase in affinity and specificity compared to more traditional therapies, and their development represents one approach to rational drug design. Essentially, the role of nucleic acid therapeutics is to block the information transfer from gene to protein primarily by interfering with mRNA function. In addition to conventional nucleic acid therapies (e.g., antisense oligodeoxyribonucleotides),recent advances have been made in the application Advances in Pharmacology, Volume 25 Copyright 0 1% by Academic Press, Inc. AU rights of reproduction in any form reserved

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of ribozymes (catalytic RNAs) to the arsenal of nucleic acid drugs. Ribozymes can be designed to inhibit the expression of any gene by targeting its RNA: such inhibition may be able to reverse the pathogenesis of various diseases. Because of their high specificity, and the possibility of being endogenously synthesized within a cell, it is likely that ribozymes will be major players in the design and development of new drugs during the next several years. A highly promising strategy is the combined application of ribozymes and gene therapy technologies, and sufficient evidence has now been accumulated to raise some confidence that effective ribozymes-based gene therapy can be achieved. Despite the considerable progress that has been made, however. there are a number of problems that remain to be solved. I n uiuo delivery is perhaps the most serious issue. Other major hurdles are optimizing interaction of ribozymes with the target RNA and achieving reliable catalytic activity in an intracellular environment. Possible toxicity of ribozymes and their degradation products, should also be investigated. In addition to biogenetic studies, pharmacokinetic and pharmacodynamic analyses will play a key role in the development of this new technology. It is the purpose of this article to give a general overview of wellcharacterized ribozymes, with an emphasis on the problems that remain to be overcome before ribozyme therapy can be made a useful form of medical treatment.

II. Catalytic RNA Catalytic RNAs (ribozymes) were initially discovered in the group I intron of the Terrahymena pre-rRNA (Kruger et af., 1982) and in the RNase P enzyme purified from Escherichia coli (Guerrier-Takada et al., 1983).This revolutionary finding gave rise to innovative views on the origin of life, namely, that RNA could have been an early self-replicating molecule in the prebiotic world. Examples of catalytic RNAs have now been found in the plant and animal kingdoms. The continuously expanding list indicates a wide distribution throughout nature. To date, with the exception of RNase P, only self-cleaving RNA reactions have been identified, and in vitro modifications of the various RNA motifs are necessary to convert the self-cleaving (cis) reaction to a trans reaction before ribozymes can be used in an applied clinical mode. Nonetheless, it is possible that yet unidentified trans-cleavage RNA reactions exist and function in cellular processes. Catalytic RNAs have the potential to cleave multiple targets and can be directed against a wide variety of diseases of viral, bacterial, or parasitic origin, provided that the nucleotide sequence of the target RNA is known.

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A. Group I Introns Group I introns are found in fungal mitochondria, chloroplasts, rRNA genes of protists, T-even phages, and the genome of eubacteria. These introns are defined by complex, but phylogenetically conserved, sequence homologies and secondary structures (Michel and Dujon, 1983). Group I introns (Fig. 1) effect their own splicing in uitro (Zaug et al., 1983;Garriga

ti

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Fig. 1 Ribozyme secondary structures. The structure of the various ribozyme motifs is shown schematically; P1-p9 indicate the nine base-paired helical elements of group I introns. Stems I, 11, and 111 of the hammerhead ribozyme are shown. The 5' and 3' splice sites (SS) of the group I introns and the cleavage sites for the smaller ribozymes are indicated by arrows; the double arrow for RNase P represents a phylogenetically conserved pseudoknot structure. The location of the highly conserved internal guide sequence (IGS) of group I introns is also indicated. The IGS base-pairs with sequences adjacent to and including the 5' splice site.

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and Lambowitz, 1984; Van der Horst and Tabak, 1983, but many, if not all, require protein factors to splice efficiently in uiuo (Lambowitz and Perlman, 1990). The splicing mechanism involves two sequential transesterification reactions (Cech, 1988). First, the 3'-OH of a free guanosine cofactor attacks the 5' end of the intron. The 3'-OH of exon 1 then carries out a nucleophilic attack on the 3' splice junction. This results in ligation of exons 1 and 2 and release of the intron, intervening sequence, IVS). The cleaved products generated by the splicing reaction are a 3' hydroxyl and a 5' phosphate. A reversible cyclization reaction occurs after excision of the intron, generating a circular form of the IVS and a short oligoribonucleotide (Grabowski et al., 1981; Kruger et al., 1982; Zaug et al., 1983, 1985; Inoue et al., 1986; Been and Cech, 1987). The circular form retains many of the catalytic activities of the intact intron (Zaug and Cech, 1986). In addition to self-splicing activity, group I introns exhibit a degree of genetic mobility and can encode genes that enable them to insert into intronless sites of other genes (Dujon, 1989; Lambowitz, 1989; Perlman and Butow, 1989). In a cell-free system group I introns have been capable of self-inserting into different RNAs by reverse splicing (Woodson and Cech, 1989). It is the intrinsic structure of self-splicing introns that confers autocatalytic activity. Thus, identification of the stabilizing interactions and characterization of the folding pattern are crucial for understanding the mechanism of RNA catalysis. In general, group I introns exhibit a total of none base-paired helical elements (Pl-P9; Fig. 1). In addition, a 10th helix (P10) may exist in uiuo within most of these introns (Michel and Westhof, 1990; Partono and Lewin, 1990; Fernandez, 1992). For some of these elements, the primary sequence is critical, while for others only the secondary structure is important for function (Been et al., 1987). Located outside the core of group I introns are stem-loop structures, often containing open reading frames, which do not seem to be directly involved in the endowment of self-splicing activity (Price et ai., 1985; Doudna and Szostak. 1989). These structures may contribute to the catalytic activity by allowing the core to fold correctly and by stabilizing its active conformation (Doudna and Szostak, 1989; Beaudry and Joyce, 1990).Alternatively, noncore stem-loops may provide binding sites for proteins involved in the splicing reaction. The binding of metal ions (Mg2+,Mn2+, and, to lesser extent, Ca2+,S$+, and Ba2+)also contributes to the formation of the intron structure. These metal ions, in addition to promoting RNA folding, may be directly involved in the splicing chemistry and seem to bind mostly in the core and around the active sites (Sugimoto et al., 1988; Grosshans and Cech, 1989; Celander and Cech, 1991; Wang and Cech, 1992; Piccirilli et al., 1993). In addition to the extensive secondary struc-

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ture, several tertiary interactions have recently been proposed or identified (Tullius and Dombroski, 1985; Latham and Cech, 1989; Couture et al., 1990; Downs and Cech, 1990; Michel and Westhof, 1990; Celander and Cech, 1991; Heuer et al., 1991; Michel et al., 1992). Nonconserved (Young et al., 1991) or semiconserved (Pace and Szostak, 1991) sequence elements may contribute to the formation of these tertiary interactions. A highly conserved element, which ensures the specificity and efficiency of the group I reaction, is the internal guide sequence (IGS). This sequence is located near the 5’ end of the intron and plays a critical role in the 5’ cleavage and ligation reaction (Been and Cech, 1986; Waring et al., 1986). The IGS base-pairs with sequences adjacent to and including the 5’ splice site, which always coincides with a U : G base pair in the stem-loop (Fig. 1). The cis reaction of group I introns can be converted into the transsplicing reaction by separating the 5’ exon from the intron. In this case the IGS holds the 5’ exon in place for the attack on the phosphodiester bond at the 3’ splice site (Inoue et al., 1985; Garriga et al., 1986). In the absence of both 5’ and 3’ exons, the intron becomes a trans-acting catalyst (Fig. 2), which can cleave multiple RNA substrates complementary to the RNASE P

5 1 3 I I I I I I I

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Fig. 2 Strategies for directing the substrate specificity of group I introns and RNase P, hammerhead, and hairpin ribozymes. Sequences within the group I introns and hammerhead and hairpin ribozymes that can be altered to dictate the substrate specificity are indicated with Ns. S, Substrate; IGS, internal guide sequence of group I introns; IVS, Group I intron. In the case of RNase P, this sequence is not part of the ribozyme and is therefore called the external guide sequence (EGS). The cleavage sites of potential targets are indicated with arrows, and conserved nucleotide requirements are shown.

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IGS sequence (Zaug et al., 1986; Murphy and Cech, 1989). This catalytic reaction follows Michaelis-Menten kinetics, with K , and k,,, values of 42 ph4 and 1.7/min at 42°C. respectively (Zaug and Cech, 1986). While group I introns represent an excellent system from which to

elucidate the process of RNA catalysis. their application in biological systems to date has been limited owing to their lack of specificity, since as few as 2 bp with the IGS sequence can be sufficient for cleavage (Kay and Inoue, 1987; Murphy and Cech, 1989). However, with the development of genetic techniques for the selection of improved ribozymes (see Section VI), it may be possible to determine new interactions that will enable the engineering of highly specific group I ribozymes.

B. RNase P RNase P is a ubiquitous enzyme required for the maturation of tRNAs. Specifically, this enzyme is a site-specific endonuclease that cleaves the 5' leader segments from precursor tRNA molecules, making them competent for amino acid charging and translation (Robertson er al., 1972). As in group I introns. the products of the cleavage reaction are a 3' hydroxyl and a 5' phosphate (Guerrier-Takada er al., 1983). Another similarity is the requirement for divalent metal ions (Mg?+ or Mn'+ and, partially, Ca2+),believed to be directly involved in catalysis and in maintenance of the active structure (Kazakov and Altman, 1991; Smith ef ul., 1992). A comparison of the pH dependence of cleavage in the presence of Mg?+ and Mn? which have different p K , values, suggested that a metal hydroxide ion is the nucleophile that directly or indirectly attacks the phosphate bond at the cleavage site (Guerrier-Takada et al., 1986). The 2'-OH at the base upstream of and adjacent to the cleavage site may play only an indirect role in catalysis by interacting with water, Mg?', or enzyme components or by stabilizing the 3'-OH-leaving group (Forster and Altman, 1990). RNase P enzymes from diverse organisms, ranging from E. c d i to humans, contain an RNA component (called MI RNA in E. coli) and a protein component (Darr et al., 1992).While enzymatic activity, for typical precursor tRNA substrates [ K , 0.5 p M , k,,, l/min at 37°C (GuerrierTakada et nl., 1983)j is generally higher in the presence of the protein component, several of the RNA subunits of the holoenzyme remain active in the absence of the protein moiety, indicating that RNase P is a true ribozyme (Guerrier-Takada er ul., 1983; Gardiner ef nl., 1985). The highly conserved secondary structure (Fig. 11, in the presence of only a modest conservation in primary sequence, suggests an overall similarity in the three-dimensional conformation of ail RNase P RNAs (Darr er ul., 1992). +,

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RNase P is unique among naturally occurring ribozymes in that it binds and cleaves free substrate molecules in trans; all other naturally occurring ribozymes described to date function in cis. This natural transactivity makes RNase P an attractive candidate as a therapeutic agent. Another feature that distinguishes RNase P from all other ribozymes is the absence of Watson-Crick base-pairing between the catalytic RNA and the substrate. As is the case with most RNA-processing enzymes, the exact substrate requirements in terms of sequence and secondary structure are not well defined. Analysis of pre-tRNA deletion mutants showed that only the amino-acceptor stem and the T-loop and stem, which form a single coaxially stacked helix, are required for cleavage by RNase P (McClain et al., 1987). This suggests that any hairpin structure can be cleaved by this enzyme, provided that a single-stranded CCA trinucleotide is present at the 3' side of the hairpin. These results have been further substantiated by chemical interference, which demonstrated that the acceptor and Tstem-loops are the regions of the substrate primarily responsible for interactions with the RNase P RNA (Thurlow et al., 1991).Additional biochemical analyses have shown that the sequence of the amino-acceptor stem can influence both the kinetic characteristics (Kirsebom and Svard, 1992) and cleavage specificity (Holm and Krupp, 1992). The ability of RNase P to cleave a hairpin structure led directly to a test of whether a doublestranded RNA substrate, composed of two separate RNA molecules, was also a suitable cleavage substrate (Forster and Altman, 1990). Mixing two short complementary oligoribonucleotides containing a 3'-proximal NCCA sequence resulted in cleavage of the target RNA at the predicted site. It seems, then, that any RNA can be cleaved by endogenous bacterial RNase P, if an external guide sequence (EGS), containing a single-stranded NCCA at its 3' end, is provided which can hybridize with the chosen target (Fig. 2). The requirement for an EGS with the human RNase P is more complicated and requires a structure which closely resembles a tRNA structure (Yuan et al., 1992).

C. Viroids and Virusoids Viroids (Riesner and Gross, 1985;Diener, 1991)are small (- 300-nt) circular RNAs, which can infect and cause disease in several plant species. Both the RNA (Rigden and Rezaian, 1992) and cDNA (Cress et al., 1983) are infectious. Viroid RNAs possess rodlike structures as a consequence of extensive intramolecular base-pairing and encode no known proteins (Henco et al., 1979; Riesner et al., 1979). Virusoids (- 350-nt circular RNAs) are a subclass of plant satellite RNAs (Roossinck et al., 1992) with viroidlike characteristics. The major

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difference between virusoids and viroids is that the former require the assistance of a helper virus for replication. Viroids thus represent the smallest known independently replicating pathogens. A rolling-circle mode of replication has been proposed for viroids and virusoids, since concatenated forms, believed to represent replication-intermediate RNAs, are recovered from infected cells (Kiefer et al., 1982; Chu et al., 1983; Branch and Robertson. 1984). The self-cleaving regions of virusoid and several viroid RNAs can be folded into a phylogenetically conserved secondary structure called a “hammerhead” (Fig. I), which encompasses the cleavage site (Hutchins et a [ ., 1986; Forster and Symons, 1987). The hammerhead is the smallest known ribozyme motif. It consists of three stem-loops joined by an 11base single-stranded domain, 10 of which are highly conserved. The cleavage site is at a single unpaired nucleotide connecting two stem-loop structures. The 2 nt preceding the cleavage site are also conserved and form Watson-Crick base pairs in one of the stems (see Figs. 1 and 2). The three loops are dispensable, and a functional hammerhead structure can be generated in uitro using two (Uhlenbeck, 1987; Haseloff and Gerlach, 1988) or three (Koizumi et al., 1988) separate oligoribonucleotides. In these cases the oligoribonucleotides, which do not contain the cleavage site, act catalytically on substrate RNAs, such that multiple substrates are cleaved by a single transacting ribozyme. Thus, any RNA containing a 5‘-XUN-3’ (where X is A, C, G, or U and N is A, C, or U) can be cleaved by a transacting hammerhead ribozyme (Fig. 2). The cleavage products generated from the hammerhead cleavage reaction differ from those of group I and RNase P in that hammerhead cleavage generates 5‘hydroxyl groups and 2’,3’-cyclic phosphates instead of 5 ’ phosphates and 3’-hydroxyl groups. The hammerhead cleavage products are typical of simple base hydrolysis of RNA (Yang et af., 1992), in which the 2’-OH of ribose acts as a nucleophile. Support for this hydrolysis mechanism stems from the fact that the 2’-OH of the 5‘ nucleoside at the cleavage site is essential for cleavage (Perreault et al., 1990; Yang et al., 1992). The metal ion requirements of hammerhead cleavage are also different from those reported for group I introns and RNase P. Whereas Mg2+and MnZ+ are the principal metals that support cleavage of the former two ribozymes, cleavage by the hammerhead occurs in the presence of Co2+, Mg2+ , Mn2+ , Zn2+, Cd2+, and ST’+. However, Zn2+, Cd2+,and Sr2+ support cleavage only in the presence of spermidine (Dahm and Uhlenbeck, 1991). The phylogenetically conserved secondary structure of the hammerhead ribozyme has been supported by nuclear magnetic resonance data, but no tertiary interactions have yet been identified (Pease and Wemmer,

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1990; Heus and Pardi, 1991). In addition, site-specific mutagenesis experiments have verified the importance of the conserved single-stranded nucleotides (Koizumi et al., 1989; Sheldon and Symons, 1989; Ruffner et al., 1990; Pemman et al., 1992). Hammerhead ribozymes follow Michaelis-Menten enzyme kinetics (Uhlenbeck, 1987); mutations in the single-stranded conserved regions alter the kcat of the reaction (Ruffner et al., 1990), whereas mutations in the stem regions generally alter the K , (Fedor and Uhlenbeck, 1990). As in the case of hammerhead ribozyme, self-cleavage by the negative strand of tobacco ringspot virusoid produces 5’-hydroxyl and 2’,3’-cyclic phosphate termini (Feldstein et al., 1989; Hampel and Tritz, 1989; Haseloff and Gerlach, 1989). This molecule, however, lacks the ability to form a hammerhead structure (Buzayan et al., 1986),forming instead a “hairpin” configuration (Hampel et al., 1990) (Fig. 1). As with the hammerhead ribozyme, and 5’-GUC-3’ can be targeted by the hairpin ribozyme via base-pairing interactions within two short helices (Fig. 2). Cleavage by hairpin ribozyme is 5’ of the G residue, in contrast to 3’ of the C residue by hammerhead ribozyme. The G residue is absolutely required and cannot even be substituted with inosine, suggesting that the 2-amino group of the base coordinated the binding of a metal ion in the catalytic center (Chowrira et al., 1991). The hairpin reaction is reversible, and ligation serves to join the ends of linear monomers to form circular monomeric RNAs (Prody et al., 1986; van To1 et al., 1991). This reversibility should be addressed when designing hairpin ribozymes for inhibiting gene expression.

D. Other Ribozymes Self-splicing introns and RNase P, hammerhead, and hairpin ribozymes are not the only known catalytic RNAs. The hepatitis delta virus (HDV), consisting of a 1.7-kb circular single-stranded RNA genome, has extensive intramolecular base-pairing potential and is thought to replicate via a rolling-circle mechanism (Chen et al., 1986).A self-cleaving domain, which requires Mg2+ and produces a 2’,3‘-cyclic phosphate and a 5‘ hydroxyl (Wu et al., 1989), has been identified within this genome (Perrotta and Been, 1990). Denaturants that destabilize the rod-shaped genomic structure have been found to enhance the cleavage reaction (Wu and Lai, 1990). The secondary structure of HDV RNA has been difficult to determine due to limited phylogenetic data. However, a structure termed the “axehead” (Fig. l), proposed for both the genomic and antigenomic self-cleaving domains (Branch and Robertson, 1991), has been used to design transacting ribozymes (Perrotta and Been, 1992).

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Self-cleaving activity has also been seen with an RNA molecule that is encoded by an 881-nt mtDNA plasmid isolated from Neirrospora. These plasmids typically exist as head-to-tail concatemers and form multimeric RNAs, which self-cleave into monomeric units, similar to the viroids and virusoids (Saville and Collins, 1990). The secondary structure required for cleavage is presently unknown. The termini generated by this cleavage reaction, as well as the Mg" ion requirement, are the same as for the hammerhead (Saville and Collins, 1990). Two other metal-dependent cleavage activities have been discovered, whose biological significance is at present unknown. One is a Mn2+dependent cleavage of the 5' region of Tetrahymena rRNA (Dange et ul., 1990); the other is the lead-dependent self-cleavage reaction of yeast tKNAP1" (Werner et a / . , 1976). These reactions can be converted into trans reactions by separating the self-cleaving molecules into two parts. The kinetic parameters of the yeast tRNAPhelead-dependent reaction have been determined and a minimum structure has been defined (Deng and Termini. 1992).

111. Design of Trans-Acting Ribozymes As indicated above, substrate recognition by all ribozymes characterized to date, except for RNase P, involves simple Watson-Crick base-pairing, such that multiple trans-cleavage events can occur. While RNase P or a group I intron can target any site within cellular transcripts, cleavage activities at different sites can vary significantly (Li et al., 1992), which may be due to the different secondary structures of the target RNAs (Xing and Whitton, 1992). Little intracellular work has been attempted with group 1 intron, in part because their size is unfavorable (the smallest active derivative is about 180 nt long; Doudna and Szostak, 1989). These introns also lack specificity, due to the fact that as few as two base-pairing interactions with the IGS sequence are sufficient for cleavage (see Section I1I.A). lncreasing the complementarity to a target RNA, by increasing the number of base pairs in the IGS, can decrease target selectivity, because basepair mismatches are more tolerated over long sequences (Hershlag, 1991) and may result in nonspecific degradation of nontarget cellular RN As. It may be possible. however, to select more specific ribozymes, since mutations even in nonconserved regions can enhance activity and specificity (Wang and Cech, 1992). The hairpin and HDV are also promising candidates for in uiuo studies. However, as mentioned above, the considerable ligation activity of the hairpin may decrease its utility as an antimRNA agent.

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To date, the best candidate for in uivo experiments is the hammerhead ribozyme. This ribozyme has been well characterized and, due to its small size, has certain advantages over the larger catalytic motifs. The number of base pairs required for optimal ribozyme cleavage at different sites is variable and must be determined empirically for each specific site. However, 6-8 bp in each flanking arm encompassing the catalytic center is a good starting point, since fewer bases can result in poor binding and greater than 10 bp in each pairing arm can reduce turnover by slowing dissociation of ribozyme and cleavage products (Goodchild and Kohli, 1991; Bertrand et af., 1992; Heidenreich and Eckstein, 1992). In addition, the activity of the dsRNA unwinding/modifying enzyme, which requires only 15-20 bp of RNA (Nishikura et al., 1991), may inactivate the ribozyme. Nevertheless, the complexity of human RNA is about 100-fold lower than that for human DNA (Lewin, 1983), and specificity can be achieved with as few as 12-15 bp. The stability of the RNA-RNA duplex is affected by several factors, such as G-C content, temperature, pH, ionic concentration, and structure. Nearest-neighbor rules can provide a useful estimate of the stability of the duplex (Freier et af., 1992). Although the GUC trinucleotide is the most conserved and probably the most efficiently cleaved sequence, any XUN (see Section I1,C) can be selected (Ruffner et al., 1990; Koizumi et al., 1989); thus, each RNA target should contain several potential cleavage sites. However, some of these sites could be protected by RNA-associated proteins, and others might be inaccessible within secondary intramolecular structures. In addition to more simple secondary structures (stem-loops), higher-order pairing, such as pseudoknots or tertiary interactions, must be considered. Although the biophysical principles governing RNA folding are not well defined, computer RNA-folding programs are useful in making certain predictions of target accessibility. Computer-assisted RNA folding (Zuker, 1989), along with the computational analysis for three-dimensional modeling of RNA (Major er af., 1991), is certainly effective in guiding the choice of the cleavage sites. A definitive answer on target accessibility with current technology can only be obtained empirically by designing several different ribozymes to cleave at different sites. Because some of the cleavage sites behave differently in the context of different structures, it is not always possible to extrapolate results of cleavage at one target site to cleavage at others. Several chemical modifications of the ribozyme have been shown to increase stability. It is the 2’-hydroxyl group of ribose that renders RNA sensitive to ribonucleases. Synthesis of ribozymes containing 2’fluorocytidine and 2’-fluorouridine, or 2’-aminouridines, considerably increases their stability in serum without significantly decreasing their

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catalytic efficiencies (Pieken et al., 1991). Modification of the 2’-OH to 2‘-O-allylribonucleotides in all but 6 nt of the hammerhead’s conserved core gives similar results (Paolella et al., 1992). Any chemical modifications, however, must be compatible with the 2’-hydroxyl groups required for catalysis and cleavage activity of ribozymes (for a review on these studies, see Heidenreich et al., 1993). Replacing flanking sequences with DNA can increase intracellular stability 2- to 3-fold (Taylor et af., 1992). Phosphorothioate backbone substitutions can also render ribozymes more resistant to nucleases (Shibahara et al., 1989; Heidenreich and Eckstein, 1992). In addition to increased stability, these types of modificationscan be particularly informative for mechanistic, functional, and structural studies (Heidenreich et a / . , 1993).

IV. lntracellular Delivery One of the key tasks in the coming years will be to improve and expand the number of methods for in uiuo delivery of ribozymes. The intracellular transfer of ribozymes is continuously improving, but several problems remain. The efficiency of cellular uptake, targeting to specific tissue and organs, subcellular localization, and maintenance of ribozyme activity within the intracellular environment are some of the major issues to be addressed. Another question is how to time and regulate the activity of the ribozyme once inside the target cells. Many techniques have been developed to introduce functional naked DNA into mammalian cells in tissue culture studies, and some of these may also be applicable to the delivery of synthetic RNA molecules.

A. Exogenous Delivery The strategy for introducing catalytic RNA molecules into cells uses synthetic preformed RNA ribozyme, which is delivered exogenously (i.e., from without) into cells (Fig. 3). A major drawback of exogenous delivery is that the inhibitory effects of the nucleic acids so delivered are transient and require repeated administrations. From a therapeutic point of view, then, maintenance of an effective intracellular concentration might be costly, inconvenient, and impractical. However, the exogenous delivery can take full advantage of chemical modifications (see Section III), which can significantly increase the stability and efficacy of the ribozyme. In addition, chemically synthesized ribozymes can be free from extra nonbase-pairing flanking sequences, which would be present in an endogenous expression system, since in the latter case the ribozyme must be placed

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Fig. 3 A general strategy for ribozyme (Rz.)-dependent gene inhibition. hesynthesized ribozyme molecules and a plasmid for the endogenous expression of the ribozyme gene are shown. Examples of in uiuo delivery systems and potential RNA targets, which can be targeted by a ribozyme, are indicated.

within an intracellular transcriptional unit (see below). While these flanking sequences may protect the ribozyme from degradation, they may also alter its catalytic efficiency and intracellularlocalization (Taylor and Rossi, 1991). Further investigation is needed to establish whether the advantages of in uitro stabilization by chemical modifications will overcome the disadvantages inherent in exogenous delivery. One method of exogenous gene transfer is based on conjugating DNA to various compounds (e.g., polylysine) (for a review see Leonetti et al., 1990) or to lipophilic groups (Boutorin et al., 1989; Letsinger et al., 1989), which increase cellular uptake. The polylysine conjugate can also include receptor ligands for specific targeting and localization to defined types of cells (Wu et al., 1991). Additionally, conjugation of DNA with lipophilic derivatives can have higher antiviral activity, as shown in the case of human immunodeficiency virus 1 (HIV-1) (Letsinger et al., 1989; Abromova et al., 1991). The above procedures introduce plasmid DNA into the cytoplasm, but access to the nucleus is poor as a consequence of the physical barrier of the nuclear membrane, the rapid degradation of the DNA in the cytoplasm, or both. Some delivery techniques address this issue by complexing the DNA with nonhistone nuclear proteins (Kaneda et al., 1989) or by direct

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microinjection into the nucleus of the cloned genes (Capecchi, 1980). Phage particles (Ishiura ef al., 1982; Sugawa et al.. 1983, as well as nonhistone nuclear proteins, can mediate, to some extent, the migration of DNA to the nucleus. This effect may be achieved by facilitating the transfer through the nuclear membrane, protecting the DNA from degradation in the cytoplasm, or both. In the context of therapeutic gene delivery, promising in viuo techniques use liposomes and viral vectors. The former can be used for presynthesized RNA molecules as well as DNA genes, whereas the latter is used for transfer of DNA genes encoding the RNA (gene therapy).

B. Liposomes L,iposomes are composed of one or more concentric phospholipid bilayers (which can incorporate lipid-soluble substances) surrounding an aqueous compartment that can incorporate water-soluble substances. Size and lipid composition can vary, and different liposomes exhibit different characteristics as in uivo delivery systems. For example, negatively charged lipids can increase the efficiency of cellular uptake; saturated lipids and the presence of cholesterol can increase liposome stability. It is also possible to covalently attach the liposomes to antibody molecules, which results in specific binding to cellular antigens (for a review see Wright and Huang, 1989)and allows specific targeting to different types of cells. Other modifications include pH-sensitive liposomes whose membranes, once exposed to the low-pH environment of the endosomes, fuse with the endosome membranes. Immunoliposomes (pH-sensitive liposomes conjugated to monoclonal antibodies) were successfully targeted to cell surface receptors in uitro and in uivo (Ho et al., 1987). The primary mechanism of cellular uptake of liposomes seems to be endocytosis (Alving, 1988). Once in the cytoplasm, the liposomes are degraded, and the nucleic acids contained inside are released (or degraded), Intravenously administered liposomes can only leave the circulation if there is a discontinuous or damaged endothelium. They thus have a tendency to accumulate in inflamed areas, tumors, bone marrow, and especially in liver and spleen. This tendency, as well as leakage and lack of control in the rate of release of the liposomal contents once inside the cell, is among the major concerns for liposome delivery.

C. Endogenous Delivery Endogenous delivery involves the expression of a ribozyme from a DNA template permanently maintained within the cell. Expression of the ribo-

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zyme can be directed by polymerase (Pol) I1 (Cameron and Jennings, 1989; Sarver et al., 1990) or Pol I11 (Cotten et al., 1989) promoters. Pol I1 promoters used for ribozyme expression include promoters of viral origin, long terminal repeat (LTR) promoter sequences of retroviruses, and strong endogenous promoters (e.g., the actin promoter). Use of a vigorous promoter is advisable, but high levels of ribozyme expression may be difficult to obtain. Tandem repeats of the ribozyme gene, under the control of a single promoter, may help to alleviate this obstacle. In the event of strong ribozyme expression, other types of promoters could be utilized, such as inducible, repressible, or tissue-specific promoters. Such promoters would confer temporal and specific expression of ribozymes and may address other concerns, such as cellular toxicity generated by high levels of ribozyme within the cells. Endogenous expression from a Pol I1 promoter necessitates a polyadenylation signal, which allows transcription termination and the addition of a poly(A) tail. This, along with the 5’-m7 GpppG cap common to Pol I1 transcripts, may prolong the intracellular half-life of a ribozyme. Insertion of an intron could also be beneficial in increasing stability and efficacy of the ribozyme. Expressing ribozymes under the control of Pol I11 promoters affords additional advantages. First, Pol III-driven gene expression seems to occur at a high level in all tissues; second, the size of Pol III-transcribed genes is smaller, and thus, it may be easier to insert multiple copies of the ribozyme in the same transcriptional unit. Other modifications can be conceived. For example, an snRNA transcription unit (Guthrie and Patterson, 1988) which incorporates an antigenic protein (Sm) binding site within the ribozyme, could be used. This should result in the targeting of the ribozyme to the nucleus. Another option is to insert the ribozyme into the anticodon loop of a tRNA gene, which has resulted in higher expression and stability of the ribozyme (Cotten and Birnstiel, 1989). However, this tRNA expression system has been shown to alter post-transcriptional processing and cellular transport (Cotten and Birnstiel, 1989).

D. Viral Vectors The use of viral vectors is one of the most promising technologies for gene delivery and endogenous gene expression. Different viral vectors have the capacity to infect a variety of cell types as well as a significant number of the target cell population. Several classes of viral vectors are being exploited for delivery of genes in uiuo, including DNA [adenoviruses, herpes virus, and adenoassociated virus (AAV)] and RNA retroviruses. General concerns persist with the use of viral pathogens, such as residual infectivity, toxicity, and rescue of infectivity by recombi-

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nation. Additionally, each viral vector has its own set of advantages and disadvantages, which ultimately dictate its use in specific application. To date, the most extensively utilized viral vectors have been RNA retroviruses. This class of viruses can infect a variety of cell types and has the advantage of confemng stable long-term persistence due to integration into the host chromosome. However, the integration process requires cell replication, thereby restricting their usage to actively dividing cells. Other potential concerns are low vector titer, lack of specific integration sites, and the possibility of activation of protooncogenes and the potential for infectious helper virus rescue due to recombination. Nonetheless, retroviruses possess considerable potential for efficient and effective in uivo delivery systems and are, at the time of this writing, the method of choice. It is possible to engineer retroviral vectors with single or double genes which can be inserted in the same or the reverse orientation with respect to viral LTRs. The two genes can be under the control of two separate promoters or under the same promoter, in which case they could be regulated by alternative splicing or transcribed as a single unit. Alternatively, the genes can be under the control of promoter sequences contained in the viral LTRs. The choice of the internal promoter raises the possibility of an inducible or a tissue-specific expression system. Several retroviral vectors with deleted LTR promoter sequences have been developed (Linney et al., 1984; Yu et al., 1986; Hawley et al., 1987; Lim et al., 1987; Yee et al., 1987; Korczak et al., 1988). These vectors are less likely to activate cellular oncogenes although they often suffer from a low viral titer. In addition to the LTR promoter sequences, the construction of an expression system for a foreign gene delivery includes splice-site signals, a viral packaging signal, and usually a marker gene. A cell line for the packaging of the resultant plasmid, without the generation of wild-type retroviruses, must also be developed. This can be obtained by establishing a packaging cell line (such as NIH-3T3) which provides retroviral structural proteins, in trans from a plasmid encoding the retroviral structural genes (gag, pol, and env), but which cannot be encapsidated. The vector plasmid containing the transduced gene (e.g., ribozyme DNA template) is then transfected into the packaging cell line. Since only the vector plasmid contains the packaging signal ($), only this chimeric genome is packaged utilizing the structural proteins expressed from the resident defective retroviruses and is released from cells (Fig. 4). Because a single recombination event could, theoretically generate infectious helper virus, cell lines were developed where higher numbers of recombinationalevents are necessary to produce wild-type retroviruses (Miller and Buttimore, 1986; Bosselman et al., 1987; Danos and Mulligan, 1988; Markowitz et

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y”

Fig. 4 Schematic depiction of an expression and packaging system for a ribozyme (Rz.) gene incorporated into a retroviral vector. Above, a ribozyme gene is inserted downstream of an internal promoter (P) and upstream of the viral 3’ long terminal repeat (LTR) sequences. Alternatively, the gene can be under the control of the viral 5’ LTR promoter. The symbol 9 indicates a viral packaging signal. Below, a packaging strategy which can be used to produce retroviral vectors containing the ribozyme gene. Virus packaging lines are available which harbor a helper provirus. The helper provirus is capable of producing all of the viral proteins required for assembly and release, but is lacking the packaging signal. The helper virus RNA, therefore, cannot be packaged into mature virions. A ribozyme-retrovirus plasmid containing the signal, when transduced into the packaging cell line, uses the preexisting viral proteins for packaging, is assembled, and then released. The ribozyme-retrovirus vector is thus the only virion released free of helper (infectious) virions.

al., 1988), thereby reducing the likelihood of helper virus in the vector virus stocks. Owing to these features and improvements, retroviruses are currently being used in several clinical therapeutic protocols for cancer and inborn metabolic diseases. Larger genes, which cannot be accommodated within a retroviral genome, can be expressed using RNA viruses or herpes virus-derived vectors. Both are nonintegrating viruses, which can thus be used for nonrepli-

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cating or fully differentiated cells. Because of its autonomous replication mode, the use of adenovirus as a vector will require repeated administration during the course of the disease. Nonetheless, the propensity of adenovirus to lung tissue makes it a particularly attractive vehicle for the delivery of therapeutic genes in pulmonary diseases. Indeed, clinical protocols for the treatment of cystic fibrosis have recently been implemented at several clinical centers. Another promising vector system is derived from AAVs. The AAVs are nonpathogenic integrating viruses that require helper viruses for active replication of their genome. The AAV vector can exist autonomously at high copy number within a cell in the presence of helper virus (adenovirus or herpes virus) or can integrate into the host chromosome in the absence of helper virus. Integration with wild-type AAV is preferentially localized to a specific site within human chromosome 19 (Kotin et al., 1990) and does not lead to deleterious pathogenic consequences. As a final example, herpes simplex virus vectors are also being developed for specific targeting to the central nervous system, again because of the unique ability to these viruses to establish lifelong latent infection in postmitotic neural cells.

V. Applications inhibition of gene expression by trans-acting hammerhead ribozymes has been reported in E. coli (Sioud and Drlica, 19911, plant cells (Steinecke ef al., 1992). mammalian cells (Cameron and Jennings, 1989; Sarver et al., 1990; Scanlon et af., 1991), and Xenopus oocytes (Cotten et al., 1989; Saxena and Ackerman, 1990). Because ribozymes base-pair with their substrates, antisense effects may contribute to a decrease in steady-state levels of the RNA targets. Although this is advantageous from a practical point of view, the optimal design of ribozymes for therapeutic use will require the development of assays in which the antisense effects of ribozymes can be quantitated separately from the cleavage effects. One control for antisense effects is to create an RNA molecule identical to the ribozyme, except for one or two point mutations known to inactivate cleavage without altering the secondary structure. This is readily feasible for the hammerhead and RNase P by mutating single core nucleotides (Ruffner et ul., 1990) or the 3'-proximal CCA of the EGS (McClain er al., 1987), respectively. An inactivating mutation within the HDV ribozyme, which also could be used as a control for antisense activity. has recently been identified in one of the loops of the catalytic core (Kumar et al., 1992). It has been shown, in two separate sets of experiments, that wild-type hammerhead ribozymes can yield stronger inhibition than the mutants,

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demonstrating the biological effects of actual ribozyme cleavage intracellularly (Eckner et al., 1991; Steinecke et al., 1992). However, in another case, an antisence RNA proved to be more effective in nuclear extracts than a ribozyme (although the antisense molecule was not a mutant ribozyme) (Cotten et al., 1989). Cell-free substrate specificity and turnover can be assessed for group I introns (Murphy and Cech, 1989) and RNase P (Li et al., 1992), hammerhead (Haseloff and Gerlach, 1988), and hairpin ribozymes (Hampel and Tritz, 1989; Hampel et al., 1990; Kikuchi and Sasaki, 1991). Therefore, it should be possible to target any cellular transcript in v i m . As stated above, endogenous expression of ribozymes has been effectively demonstrated in several different areas, from the inhibition of chloramphenicol acetyltransferase (CAT) RNA in monkey cells (Cameron and Jennings, 1989), to the reduction of Xenopus 7-S snRNA (Cotten and Birnstiel, 1989), and to therapeutic applications against HIV-1 (Sarver et al., 1990) and cancer (Koizumi et al., 1989; Scanlon et al., 1991). This suggests that ribozymes might be used effectively against a wide spectrum of diseases, from viral infections (in which the ribozyme can be designed to cleave viral transcripts) to carcinogenesis (in which it can be designed to cleave oncogene-encoding mRNAs). Indeed, some oncogene transcripts can be specifically cleaved and discriminated from their respective protooncogenes as a consequence of activating point mutations, chromosomal rearrangements, aberrant initiation of transcription, and alternative splicing. For example, it is possible to target a ribozyme with high specificity to the c-H-ras mRNA, in which a mutation at codon 12 (from GGU to GUU) creates a suitable cleavage site not found in the wild-type transcript. Such ribozymes have no effect on a cellular ras protooncogene transcript that lacks this mutation (Koizumi et al., 1992). Mammalian cells transfected with plasmids expressing various ribozymes showed no impairment of growth, native gene expression, or longevity (Cameron and Jennings, 1989; Sarver et al., 1990); nonspecific cleavage with hammerheads (Steinecke et al., 1992) or RNase P (Li et al., 1992) does not seem to occur. Therefore, if ribozymes interfere with normal nontargeted cellular function, such interference is minimal. Ribozymes have also been used to probe certain aspects of RNA metabolism. Using a cis-cleaving ribozyme, Monforte et al. (1990) determined the number of nucleotides that must be synthesized before the nascent RNA transcript begins to fold. In another instance, it has been shown that 3'-end processing of histone mRNAs is linked to export of RNA transcripts from the nucleus (Eckner et at., 1991). Finally, a cis-acting HDV ribozyme has been used to define the 3' ends of viral transcripts, which was shown to be required for replication of genomic RNA in cul-

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tured cells (Patnalk et al., 1992). The application of ribozyme methodologies to these and other aspects of cellular RNA processing, such as RNA degradation, regulation, and cellular development should prove to be a valuable tool.

VI. Concluding Remarks Although comprehensive studies have been done on catalytic RNA, the use of ribozymes in uiuo is still at its roots. Basic problems, such as the ability of the ribozyme to find its target once within the cellular mileu and cleave it in a catalytic fashion, still must be addressed. To date, despite the potential for catalytic activity, great excesses of ribozyme over the substrate are often required to effect gene inhibition, and ribozymes may very well prove to be merely a slightly enhanced form of antisense RNA. However, ribozyme technology is continuously improving and in the nottoo-distant future the various ribozyme motifs will be brought to their full potential. Ribozymes hold the inherent advantages over conventional antisense molecules in not only neutralizing the target RNA, but also in cleaving it, ensuring its permanent inactivation. In addition, the effectiveness of an antisense RNA is unpredictable. Several reports indicate that, for unknown reasons, stable and abundant expression of antisense RNA sometimes has no detectable effect on expression of their target genes (Salmons et al., 1986; Gunning et al., 1987; Ken et al., 1988). Many techniques are currently being used to enhance ribozyme activity. Chemical modifications can be used to assess the sequence requirements for cleavage. In one case nucleotides that may naturally inhibit the cleavage reaction have been identified (Belinsky e? al., 1993). Another technique, developed originally by Tuerk and Gold (1990) to isolate RNAs that bind a specific RNA-binding protein, allows the cell-free selection of RNA molecules possessing desired functions from a heterogeneous population of chemically synthesized sequence variants. This approach (for a review see Burke and Berzal-Herranz, 1993) has been utilized to study sequence requirements of a loop region within the hairpin ribozyme (Berzal-Herranz et al., 1992). This “in uitro” evolution” has also been used to isolate mutant forms of a group I intron, which cleave DNA up to 100-foldmore efficiently than the wild-type intron (Robertson and Joyce, 1990; Beaudry and Joyce, 1992). Similarly, mutant ribozymes have been isolated that can function in the presence of Ca2+with no added Mg2+ and that still function as efficient catalysts using Mg2+or Mnz+(Lehman and Joyce, 1993). In addition to illustrating the remarkable power of testtube evolution, these results may have therapeutic implications. For example. the total Ca2+concentration in the human body is higher than the

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Mg2+concentration; in some instances the use of a Ca2+-dependentribozyme might be more appropriate than a Mg2+-dependentribozyme. Other characteristics that may confer high degree of specificity and function to the ribozyme could also be selected. These procedures may further clarify the role of RNA catalysis in molecular evolution. In uitro evolution takes advantage of the known ligation activities of the hairpin and group I ribozymes. Unfortunately, the hammerhead is less amenable to such in uitro selection because its ligation reaction is extremely inefficient. While no ligation reaction occurs, and therefore no in uitro selection procedure exists for increasing RNase P substrate specificity at this time, RNase P may ultimately prove to be the more effective therapeutic ribozyme because of its presence in all living organisms. In conclusion, ribozymes hold considerable promise for future therapeutic applications. We foresee that ribozymes will play a key role in the arsenal of pharmacotherapy .

Acknowledgments This work was supported by National Institutes of Health grants AI-25959 and AI-29329. We thank David Elkins and John Termini for their critical reading of the manuscript.

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