RNA enzymes as tools for gene ablation

William James and Aymen Al-Shamkhani

University of Oxford, Oxford, UK

Ribozymes have the potential to ablate the expression of any gene in

a sequence-specific manner and, therefore, may be useful as therapeutic

molecules or as tools for the analysis of gene function. Although a number

of reports have described ribozymes that are effective in inhibiting gene

expression, few studies have attempted, systematically, to analyze the features

of ribozymes that affect their potency within cells. Experimental observations

suggest that emerging rules governing ribozyme potency in cells can be

understood in terms of the competitive interactions between RNA-binding

proteins, complementary RNAs and their internal secondary structure.

Current Opinion in Biotechnology 1995, 6:44-49

Introduction

In principle, truns-acting ribozymes enable one to de- stroy mRNAs of choice with sequence selectivity Con- sequently, they are potentially valuable tools for the inhibition of virus replication, modulation of tumour progression and analysis of cellular gene function. A ri- bozyme can be thought of as a chimaeric RNA molecule consisting of two stretches of antisense RNA flanking a nucleolytic motif. The antisense RNA component, re- ferred to below as the flanking complementary regions (FCRs), provide the target selectivity. Two generic types of ribozyme have been exploited: the ‘hammerhead’ ri- bozyme [l] and the ‘hairpin’ ribozyme [2]. Both can cleave GUC sites, but the hammerhead can also cleave at GUA, GUU, CUC and UUC with comparable effi- ciency [3] and sometimes at AUC [4,5].

Ribozymes can be delivered into cells as synthetic oligonucleotides or be synthesized within the cell from engineered genes. This review considers the latter sit- uation only, as long-term therapeutic or phenotypic studies require a continued source of inhibitory agent. In the following sections, we highlight recent develop- ments in the understanding and exploitation of ribozyme action in the living cell. First, we take a brief look at the theoretical considerations that might be expected to de- termine whether a given ribozyme will destroy its target RNA. In addition, we consider the evidence from cell- free systems on the behaviour of a ribozyme in an ideal environment. We then briefly review experiments that investigate the use of ribozymes in oivo and the extent to which data from these experiments can be inter- preted. For a summary of the state of the field up to 1991, the reader is referred to a previous review [6].

Some theoretical considerations for ribozyme action

Two possible benefits may accrue from the addition of a ribozyme motif to an ordinary antisense RNA. In the first place, unlike an antisense RNA, a ribozyme does not rely on the host cell’s machinery to inactivate the target RNA, but does so itself As the antisense ef- fect usually appears to result from the entrapment and degradation of long stable RNA duplexes in the nucleus, which may be an inefficient process, a ribozyme might be expected to inactivate a target RNA with much greater efficiency. In the second place, the ribozyme might be able to cleave more than one copy of target RNA if it could dissociate from its cleaved products and were in a position to engage a second target RNA. Although work has demonstrated that ribozyme motifs can enhance the inhibitory potency of antisense RNAs under certain circumstances [4,7-10,11*,12], but not all [13], it is not clear whether the presence of the ribozyme resulted in a greater destructive efficiency (without true catalysis) or enabled the cleavage of multiple copies of target RNA.

The relative contribution of the above two components of ribozyme activity is dependent on the kinetics of quite distinct steps in the RNA degradation reaction (see Fig. 1). That is, if true catalysis cannot be attained in the cell, then maximizing the annealing step (a function of the rate constant k+l and the concentration/localization of the two RNAs) will optimize ribozyme function in viva; in contrast, optimization of the dissociation step (essentially a function of k+3) will not be of help. Conversely, if the rate-limiting step in vim were the re- lease of ribozyme from cleaved products, then improving

44

Abbreviations FCR-flanking complementary region; hnRNP-heterogeneous nuclear ribonucleoprotein; snRNA-small nuclear RNA.

0 Current Biology Ltd ISSN 0958-1669

RNA enzymes as tools for gene ablation James and Al-Shamkhani 45

-

ie_,-ate Riboqm ColiqJlr;i

k+‘l 1 3

f

k-1

k-2 VV k+2

k+3

c I

A+

\

Ribozyn._ . VI 4. Products

819% Current Opinion in8iihdogy

Ribozyme - Products complex

the abundance and co-localization of ribozymes or their annealing properties would not be advantageous. Con- sequently, it would be helpful if we could get clues about which mode of action is important in viva. So, the im- portant questions are the following: does a high k,,, cor- relate with high potency; does a high k+l (established under physiologically relevant conditions) correlate with potency; what are the effective concentrations required for ribozyme action in vivo; and where and in what ef- fective volume do these interactions take place? For a discussion of these issues, please refer to a recent ar- ticle by Sczakiel and Goody [14]. These authors [14] demonstrate that under normal in vitro conditions for ribozyme reactions, the ribozyme effect can only pre- dominate over the antisense effect at low concentrations of reactants and that the kinetics of cleavage in vitro are so slow that the observed effect in vivo must result from the special circumstances of the intracellular environment.

Experimental use of ribozymes

Inhibition of viral replication

Table 1 summarizes some key papers describing the utility of ribozymes in the inhibition of virus replica- tion in cultured cells [7-10,15-181. Ribozymes have been shown to range in potency, horn molecules that are less inhibitory than their parental antisense RNAs to those that produce several logs of inhibition over a prolonged period. It is hard to find clear correlations be- tween ribozyme or target RNA properties and potency. For some target sites, long FCRs are necessary, whereas for others, short FCRs are sufficient. This suggests that the optimal FCR length is specific to the target site. The

Fig. 1. A scheme summarizing the typi-

cal steps of a ribozyme reaction. The two single-stranded RNAs, ribozyme and

substrate, may contain internal secondary

structure. If conditions are favourable, the

two RNAs hybridize over their comple-

mentary regions, forming a complex in

which the ribozyme motif takes up its

active conformation. The substrate RNA is then cleaved in a Mg2+dependent

protein-independent reaction. The disso- ciation of ribozyme from the products de-

pends on environmental conditions and the extent of base pairing. Only if dissoci- ation occurs can the ribozyme engage in

further cycles of cleavage, becoming truly catalytic.

degree of inhibition does not correlate precisely with the level of ribozyme expression [8,15]. In spite of efforts to release ribozymes horn non-complementary flanking se- quences using &acting trimming ribozymes [19], most of the effective approaches have used ribozymes embed- ded in long stretches of irrelevant RNA. In one study, this has explicitly been shown to have no deleterious ef- fect [20].

Inhibition of cellular gene expression

The use of ribozymes against cellular genes has met with mixed success. For example, an anti-U7 snRNA ribozyme has been shown to be less inhibitory than its parental antisense RNA [21]. Potter et al. [22] found that expression of methyl guanine-DNA methyl transferase was inhibited by 90% in one of 16 clones transfected with a 12:lO ribozyme (where the colon delineates the length in nucleotides of ribozyme helix I and helix III) expressed from an Rous sarcoma virus promoter in HeLa cells; however, 14 of the remaining clones showed no in- hibition, and no point mutant to control for antisense effects was employed. Efiat et al. [23**] have found a 45% inhibition of glucokinase mRNA levels in murine insulinoma TC cells stably transfected with a 12:12 ri- bozyme expressed f?om the insulin promoter, although again, no ribozyme control was used. Using vaccinia- infected cells, Huillier et al. [ 121 have observed up to 80% inhibition of alpha lactalbumin expression by a 12:12 ribozyme expressed from a T7 promoter in vaccinia- infected cells. In this case, the effects were clearly a result of ribozyme-mediated cleavage, because a G22+U mu- tation in the ribozyme motif abolished the inhibitory ef- fect. In this study, no clear correlation was seen between k cat? determined in vitro, and potency in the cell.

46 Analytical biotechnology

Tabk 1. Experiments using ribozymes to inhibit viral replication.

Target Cell

LCMV NItl3T3

HIV 5’ UTR Hela

Promoter

MMTV

pactin

FCR

length’

8:9

Hairpin

a:4

Delivery Inhibition of Controls Reference

viral replication

Stable transfection -97% Mismatch virus I1 51

Transient transfection 75-85% Disabled riborynw II01

HIV 5’ UTR Hela MLV LTR

and tRNA

cassette

Hairpin

a:4

Retroviral transduction 60-9096 NO”e [I61

HIV 5’ UTR HeLa MLV LTR

and tRNA

cassette

Hairpin

8:4

Transient transfection 70-95% None 1161

HIV faf Jurkat MLV LTR 1918 Stable transfection 90% (briefly) Parental antisense

RNA (more potent)

[I71

HIV 5’ UTR SW480 Pre-synthesized 12tk263 RNA ribozyme/DNA 9&95% Antisense RNA and [91

target co-transfection mismatched ribozyme

HIV tat Jurkat MLV LTR From 99 to Retroviral transduction rx99.996 Antisense RNA, sense [SI

45:564 and mutant ribozyme

ELV fax Bat lung RSV 8:8 Stable 61% Antisense RNA and I71 and rex fibroblasts mismatched ribozyme

HIV 5’ UTR SW480 Pre-synthesized From 01203 to RNA ribozyme/DNA O-86% As [91 I181 5:283 target co-transfection

‘The length in nucleotides of helix I and helix Ill is delineated by a colon. Abbreviations include: EILV, bat lung virus; HIV, human immunodeficiency virus; LCMV, lymphqtic

chorionwngitis virus; LTR, long terminal repeat; MLV, murine leukaemia virus; MMTV, mouse mammary tumcwr virus; RSV, Rous sarcoma virus; UTR, untranslated region.

Genes predisposing to a malignant phenotype have re- ceived special attention recently. For example, the BCR- ABL oncogene mRNA, which is found only in cells having the Philadelphia chromosomal translocation and is associated with chronic myelogenous leukemia, has been targeted by ribozymes. In one study, the trans- ient transfection of pre-formed ribozyme led to a five- fold reduction in BCR-ABL mRNA in K562 cells. A similar study using a DNA-RNA hybrid ribozyme also led to reduced expression of BCR-ABL product (p210) and diminished cell growth in vitro [25]. Expression of ribozymes targeted at the junction between BCR and ABL, mediated by stable retrovirus-mediated transduc- tion, eliminates detectable mRNA expression and ~210 kinase activity [26]. The multi-drug resistance pheno- type of many malignant tumours is a serious clinical problem and is conferred by P-glycoprotein, which is encoded by the MDR-1 gene. Two studies have shown that levels of MDR-1 mRNA can be substantially re- duced by endogenously expressed ribozymes and that the resulting cells have a markedly increased sensitivity to drugs such as trimetrexate and daunorubicin [27,28]. In a recent study, Kobayashi et al. [27] used a ribozyme mutant to demonstrate that the effect is ribozyme spe- cific. In the case of US, ribozyme studies have been used to substantiate the role of this oncogene in the malignant phenotype. For example, a ribozyme to H-ras expressed in EJ cells, reduced ras expression, altered cellular mor- phology and reduced the tendency cells to form tumours

and metastases in nude mice [29]. This observation has been confirmed recently using H-US transformed 3T3 cells and ribozyme motif mutant controls [30].

Ribozymes in transgenic animals

The first demonstration of ribozyme action in a trans- genie animal was in Drosophila [l 1.1. In this study, a heat-inducible promoter was used to drive expression of a ribozyme targeted against the jkhi turuzu @z) gene, and a Ftz-like phenotype was generated in heat-shocked embryos. This technique was used to confirm the role of ftz in central nervous system development and in- dicated that ribozymes might prove powerful tools for dissection of gene function in viva. In an attempt to produce an animal model of maturity-onset diabetes of the young, Efiat et al. [23**] have introduced into transgenic mice a 12:12 ribozyme targeted against pan- creatic beta cell glucokinase under the control of the insulin promoter. Although an 80% inhibition of glu- cokinase activity was demonstrated, no change in blood glucose or insulin levels was observed. It is hoped that these ribozyme transgenics may exhibit a predisposition to diabetes. In a third study, three anti-p2 microglobulin ribozymes have all proved effective in the inhibition of gene expression in cell lines and, when introduced into transgenic mice, produced variable, but significant levels

RNA enzymes as tools for gene ablation James and Al-Shamkhani 47

of inhibition [31]. Unfortunately, no ribozyme mutant control was used in either this or the glucokinase study, leaving open the possibility that the effect resulted from antisense or non-specific effects.

The effect of the cellular environment on ribozyme function

From the above studies, it is clear that ribozymes are becoming increasingly useful tools for gene ablation in living cells and that their effects are usually the re- sult of ribozyme-mediated direct cleavage of the target RNA (rather than antisense activity). The kinetics of ri- bozyme action in vitro, however, do not concur with the levels of ribozyme activity observed in viva. It is clear that the cellular environment must facilitate rates of ribozyme-target RNA annealing much higher than those achieved in vitro [19]. We have reached a similar conclusion concerning the activity of antisense RNA on the basis of observations on the optimal lengths of inhibitory molecules. We suggested that the single- stranded RNA-binding proteins of the nucleus (includ- ing the heterogeneous nuclear ribonucleoproteins [hn- RNPs]) reduce the free energy of the unwound com- plementary RNAs and allow much greater rates of anti- sense RNA-target RNA hybridization than would be observed in protein-free conditions; accordingly, these considerations might also apply to ribozymes [6,8,32,33]. Recent experiments have provided direct support for this notion. In one study, the hnRNP protein, Al, and the HIV nucleocapsid protein, P7, promoted both the an- nealing of ribozyme and target RNA and the disso- ciation of ribozyme from product, markedly increas- ing catalytic efficiency [34*]. In another report, these proteins have been shown to catalyze the dissociation step for ribozyme with short FCRs and the annealing step when the substrate is a very long RNA [35*]. A third group have also suggested that the enhancement of ribozyme-product dissociation by P7 increases catalytic efficiency [36].

The fundamental mechanism by which these ubiqui- tous RNA-binding proteins increase the kinetics of hy- bridization involves a reduction of the energetic barri- ers that would otherwise hinder the attainment of the most thermodynamically favourable state of comple- mentary RNAs. However, if the ribozyme-target RNA hybrid is of higher free energy than the single-stranded RNAs, even hnRNPs will not cause it to form. Con- sequently, if the effective concentrations of the reactants are known, one should be able to predict the likelihood of a ribozyme annealing effectively to its target by an appropriate use of thermodynamic principles.

One indication that favourable thermodynamics might be crucial in hybrid formation stems from the observa-

tion that the addition of extended FCR arms to one or more sides of an ineffective ribozyme motif can render it inhibitory in cells [8,9,18]. Although this is certainly not necessary at all target sites, it is possible that an extended region of antisense RNA adjacent to the ribozyme can facilitate intermolecular hybridization by targeting a re- gion that has a particularly low degree of secondary structure; however, intramolecular base-pairing can also occlude somewhat the cleavage site itself [37,38].

Finally, the issue of the intracellular site of ribozyme-tar- get RNA interaction is only just beginning to be ad- dressed. The cell is a highly structured body, in which most macromolecules are restrained by higher order structures. It is probable that increasing the effective concentration of a ribozyme will be better achieved by precise intracellular targeting than by simply in- creasing the strength of the expression cassette. In one study, linking a murine retrovirus packaging signal to both ribozyme and target RNA afforded sufficient co- localization to produce a high degree of inhibition of gene expression [39]. A particularly attractive idea is to use the signals found in small nuclear RNAs (snRNAs) that direct the interaction with other components of the splicing apparatus as a way of helping ribozymes to scan all mRNA precursors. One expression system based on the U6 snRNA gene has been proposed as a ribozyme expression vehicle [40].

Conclusions

Although examples of the successful use of ribozymes for gene ablation are still overshadowed in the literature by a much larger number of failed experiments, it is becom- ing increasingly clear that ribozymes are useful genetic tools. We know that the cellular environment has a pro- found effect on the kinetics of ribozyme-target interac- tions; thus, in vitro assays need to be interpreted with cau- tion. Even so, evidence does suggest that the intelligent use of appropriate in vitro and theoretical information is leading to the development of effective ribozymes. This trend will be enhanced by an ever increasing systematic and collaborative approach to the problem. It is hoped that these approaches, together with more precisely tar- geted expression systems, will make ribozymes a routine tool for genetic investigation in the coming years.

Acknowledgements

Work in the authors’ laboratory is supported by the Medical

Research Council of Great Britain and the Leukaemia Research Fund. We thank Stephanie Thompson and Georg Sczakiel for help&l comments and discussion of unpublished work.

48 Analytical biotechnology

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RNA enzymes as tools for gene ablation James and Al-Shamkhani 49

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W James and A Al-Shamkhani, Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK.