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Development of hammerhead ribozymes for HIV-1 gene therapy: principles and progress

Dr. , Sadhna Joshi et. al.

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Gene Therapy and Molecular Biology Vol 3, page 271

Gene Ther Mol Biol Vol 3, 271-280. August 1999.

Development of hammerhead ribozymes for HIV-1 gene therapy: principles and progress

Review Article

A. Ramezani and Sadhna Joshi

Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S
3E2, Canada

Corresponding Author: Sadhna Joshi, Ph.D., 150 College Street, #212, Department of Medical Genetics and Microbiology, Faculty of Medicine, University of Toronto, Toronto, Ontario M5S 3E2, Canada. Tel: (416) 978-2499; Fax: (416) 638-1459; E-mail: sadhna.joshi.sukhwal@utoronto.ca

Key words: Hammerhead ribozymes, HIV-1 replication, gene therapy, multimeric ribozymes, retroviral vectors, HIV-1 Env Abbreviations: HIV-1, human immunodeficiency virus type-1; FCRs, flanking complementary regions; nts, nucleotides; TAR, trans-activation response; LTR, long terminal repeat; , retroviral RNA packaging signal

Received: 18 December 1998; accepted: 22 December 1998

Summary

Hammerhead ribozymes are small RNA molecules that can be designed to specifically recognize and cleave a target RNA. A single ribozyme can theoretically act in a catalytic manner thus cleaving more than one molecule of its target RNA. Because of their sequence specificity, ribozymes are being developed as therapeutic agents to eliminate unwanted cellular and viral RNAs. Ribozymes are being used to inhibit human immunodeficiency virus type-1 (HIV-1) replication. Promising results have been obtained by several groups using ribozymes targeted against various sites within the HIV-1 genome. This article reviews some of the factors relevant to the design of hammerhead ribozymes with a particular focus on their application in HIV-1 gene therapy.

I. Introduction

Ribozymes are small RNA molecules with endoribonuclease activity. They can be designed to cleave RNAs in a sequence-specific manner. Ribozymes base-pair with nucleotides around the cleavage site of the target RNA and catalyze the hydrolysis of a specific phosphodiester bond. There are several different types of ribozymes: the self-splicing group I and II introns (Zaug et al., 1986), RNase P (Guerrier-Takada et al ., 1983), an RNA transcript from Neurospora mitochondrial DNA (Saville and Collins,
1990), the hepatitis delta virus (Wu et al., 1989), the hammerhead ribozyme (Foster and Symons, 1987), and the hairpin ribozyme (Haseloff and Gerlach, 1989).
The hammerhead ribozyme is the smallest of the above ribozymes. It was first discovered in small circular pathogenic RNAs such as the satellite RNA of the Tobacco ringspot virus (Buzayan et al., 1986) and the Avocado sunblotch viroid (Hutchins et al., 1986). In its natural setting, the ribozyme motif has evolved to mediate a single turnover reaction in cis. However, to have therapeutic
applications, the ribozyme must cleave its target RNA in trans and in a catalytic manner. The hammerhead ribozyme could be adopted to cleave in trans by separating the ribozyme and substrate motifs (Feder and Uhlenbeck, 1992; Hasseloff and Gerlach, 1988; Uhlenbeck, 1987). The trans- cleaving hammerhead ribozyme (Fig. 1 ) contains a catalytic domain consisting of 11 conserved nts and two flanking complementary regions (FCRs). The catalytic domain consists of 11 nts that are highly conserved. This region also contains a stem-loop. The sequence and length of this stem- loop can vary except for the innermost G-C base pair, which is conserved. Target RNA specificity is provided by the FCRs on either side of the catalytic domain. FCRs are designed to contain sequences that are complementary to those surrounding the cleavage site within the target RNA. These FCRs allow the ribozyme to recognize and hybridize with the target RNA. Cleavage of the target RNA occurs 3’ to the NUH sequence (where N is any nucleotide, and H is any nucleotide but G), although not all NUH sites are cleaved equally well (Foster and Symons, 1987).
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Fig. 1. Schematic representation of a trans- cleaving hammerhead ribozyme. The conserved nucleotides within the catalytic

domain are indicated in boldface. The cleavage site within the target RNA is italicized. N represents non-conserved nucleotides within the 5’/3’ FCRs. The arrow points to the site of cleavage.

Structural and functional characteristics have led to the development of hammerhead ribozymes that can cleave in trans. Factors which affect ribozyme-mediated cleavage of cellular or viral RNA have also been investigated. This review discusses factors affecting hammerhead ribozyme activity and provides the progress to date for ribozyme- mediated inhibition of HIV-1 replication.

II. Ribozyme design

A. Flanking complementary regions (FCRs)

In vitro studies suggest that the catalytic activity of hammerhead ribozymes with symmetrical FCRs is greatest if the 5’ and 3’ FCRs contain 6 to 8 nts (Fedor and Uhlenbeck,

1990; Goodchild and Kohli, 1991). However, short FCRs may not allow the ribozyme to specifically associate with the target RNA in vivo.
The effect of the length of the 5’ and 3’ FCRs on ribozyme activity have been studied and compared both in vitro and in vivo . Crisell et al. (1993) designed and tested a set of ribozymes targeted against the first coding exon of HIV-1 tat. These ribozymes were designed to contain either symmetrical 5’/3’ FCRs containing 9/9, 12/12, 15/15, 18/18,
21/21, 24/24, 27/27, 30/30, and 33/33 nts or asymmetrical
5’/3’ FCRs containing 45/70 and 45/564 nts. Optimum activity in vitro was observed with ribozymes containing 9/9 nt-long 5’/3’ FCRs, whereas the inhibition of HIV-1 replication was greatest with ribozymes containing 33/33 nt-long FCRs. Increasing the length of FCRs may have enhanced the ability of the ribozyme to “melt” the secondary structure of the target RNA in vivo. However, long FCRs

could decrease the rate of dissociation of the ribozyme from the cleaved target RNA and hence reduce the catalytic activity of the ribozyme.
Tabler et al. (1994) constructed ribozymes with asymmetric 5’/3’ FCRs against the leader-gag region of HIV-1. These ribozymes contained a long 3’ FCR (289 nts) and a short 5’ FCR which varied in length (0, 1, 2, 3, 5, 8, and 13 nts). In this study, as few as 3 nts in the 5’ FCR were found to be sufficient for effective cleavage of target RNA in vitro. Ribozymes with 3 and 5 nt-long 5’ FCRs were also shown to be effective at inhibiting HIV-1 replication in vivo. Although the 3’ cleavage product was shown to be released in vitro, the 5’ cleavage product could not be dissociated. Therefore, the design of asymetric ribozymes with long FCRs may decrease ribozyme catalytic activity.
The optimal length of the 3’ FCR to be used with ribozymes containing the 3 nt-long 5’ FCR has been investigated using a set of ribozymes against the HIV-1 tat coding region (Hormes et al., 1997). These ribozymes contained a 3 nt-long 5’ FCR and variable length 3’ FCRs (20, 25, 31, 35, 41, 45, 51, 60, and 195 nts). All ribozymes were shown to be active in vitro . A minimum 51 nt-long 3’ FCR was found to be necessary to inhibit virus replication in a microinjection experiment where HIV-1 provirus DNA and ribozyme RNA were injected into the nucleus of human cells.

B. The catalytic domain and the stem-loop region

Since most nucleotides in the single-stranded regions of the catalytic domain are highly conserved, efforts have been
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made to modify the stem-loop region and determine its effect on cleavage. While decreasing the length of this stem from the conventional 4 bps to 2 bps did not alter the cleavage activity, its further reduction significantly decreased the cleavage activity in vitro (Tuschl and Eckstein, 1993). Elongating the stem from 4 bps to 6, 10, 21 or 22 bps seemed to have no effect on in vitro cleavage activity of ribozymes, although this resulted in a reduction in their ability to inhibit HIV-1 inhibition (Homann et al.,
1994). Therefore, except for the innermost G-C bp that must be conserved, the length or the composition of remaining bps in the stem-loop region does not seem to be critical for ribozyme activity (Tuschl and Eckstein, 1993).

III. Target site selection

The choice of HIV-1 RNA that must be targeted and cleaved is critical as inactivation of not all HIV-1 RNAs would have the same impact on virus replication. Furthermore, within any given HIV-1 RNA only conserved and accessible regions should be targeted. Finally, although hammerhead ribozymes could be designed to cleave HIV-1
RNA at any NUH cleavage site, the most efficiently cleaved sites should be targeted.

A. Target RNA

HIV-1 provirus DNA transcription gives rise to over 20 distinct mRNA species in an infected host cell (Fig. 2). Although it has been reported that the unspliced HIV-1 RNA in the nucleus is the primary target of ribozymes (Paik et al.,
1997), ribozyme activity may not be exclusively limited to the nucleus and may also occur within the cytoplasm. Ribozymes were also shown to be very effective at cleaving RNA within the progeny virus (Sullenger and Cech, 1993; Westaway et al. , 1998). The choice of the target site within HIV-1 RNA only matters for cleavage of spliced mRNAs, as all sites are present within the unspliced HIV-1 RNA. Thus, to allow cleavage of all HIV-1 RNAs within the cell and within the progeny virus, ribozymes may be targeted against regions that are common to all HIV-1 RNAs (Joshi and Joshi, 1996).

B. Target site

The most important factor that must be considered while selecting a ribozyme target site within HIV-1 RNA is the genetic variability. Fortunately, there are numerous regions throughout the viral genome that are highly conserved in all HIV-1 isolates within a given subtype. Some of these sites are also conserved within various subtypes of HIV-1. The ribozyme target sites should be selected from these highly conserved regions. This would decrease the emergence of escape mutants since mutations within these conserved regions are likely to be genetically attenuated and/or
replication defective. However, escape mutants may still arise from mutations within non-conserved regions adjacent to the cleavage site. Such mutations may alter target RNA structure and thereby prevent its accessibility to the ribozyme.
To cleave target RNA, a ribozyme must also be able to properly associate with it. However, long RNAs contain complex secondary and tertiary structures involving significant intramolecular base pairing that may reduce the accessibility of a particular target site (Fedor and Uhlenbeck,
1990; Uhlenbeck et al., 1997). Single-stranded regions containing ribozyme target sites should therefore be mapped by enzymatic digestions or chemical modifications (Ehresmann et al., 1987). For example, HIV-1 RNA incubation with oligodeoxyribonucleotides containing potential ribozyme FCRs followed by RNase H digestion could locate target sites within the conserved regions that are likely to be accessible for ribozyme binding (Ho et al., 1996; Scherr and Rossi, 1998). It may be desirable to perform these analyses on the full-length target RNA similar to what is to be cleaved in vivo. Computer programs may also be used to predict the most probable RNA secondary structures with minimum free energy parameters (Jaeger et al., 1989), although the reliability of computer prediction for very large RNAs is still not high. Once the information on the target RNA secondary structure is available, target sites should be selected within the single-stranded regions that are accessible and highly conserved (Zhao and Lemake, 1998).

C. Cleavage site

With the exception of the AUA-cleaving satellite RNA of the barley yellow dwarf virus (Miller et al., 1991) and the GUA-cleaving lucerne transient streak virus (Foster and Symons, 1987), all naturally occurring hammerhead ribozymes cleave their target RNA at a GUC site (Bruening,
1990). Mutagenesis studies have been performed to determine cleavage sites that are best cleaved by the hammerhead ribozyme (Sheldon and Symons, 1989; Ruffner et al. , 1990; Perriman et al ., 1992). Although initial studies led to the development of the general NUH rule (Koizumi et al., 1989), detailed kinetic analyses of a target RNA with all possible mutations at the cleavage site (Zoumadakis and Tabler, 1995) identified GUC as the most efficiently cleaved site. The influence of bases surrounding the cleavage triplet was demonstrated by Clouet-d’Orval and Uhlenbeck (1997) who analyzed a hammerhead ribozyme with a 10-fold higher cleavage rate than what was previously reported for hammerhead ribozymes. Mutational analyses demonstrated that the increased cleavage rate was due to the presence of an AU immediately after the GUC cleavage site within the target RNA.
Many attempts have been made to compare and identify ribozyme target sites within HIV-1 RNA that are best cleaved in vitro or that inhibit HIV-1 replication most
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Fig. 2. Ribozyme interference sites within the HIV-1 life cycle. Following entry inside the cell, HIV-1 RNA reverse transcribes and integrates within the cellular genome. Upon transcription, the full length 9.3 kb viral RNA is produced, which is differentially spliced to give rise to various HIV-1 mRNAs. The 2 kb RNAs then give rise to Tat which enhances gene expression and Rev which allows export of 4-5 and 9.3 kb HIV-1 RNAs. Translation of these RNAs then gives rise to various structural and maturation proteins.

Lys

Virus assembly then takes place and recruits 2 copies of full length HIV RNA and cellular tRNA3

. Some of the steps taking place

during the subsequent round of infection are also shown. Ribozymes ( ) may cleave HIV-1 RNA in the nucleus, cytoplasm, or

progeny virus such that either no virus will be produced or virus produced will be non-infectious.

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Table 1 Summary of HIV-1 inhibition results obtained using monomeric hammerhead ribozymes.

Target site Target cell Expression HIV-1 replication Reference

(pool/clone) (compared to controls)

R

U5

HeLa CD4/pool

T-cell line/pool

Transient

Stable

Suppressed for 9 days

Delayed for 18 days

Dropulic and Jeang, 1994

Weerasinghe et al., 1991

T-cell line/pool

HeLa CD4/pool

Stable

Transient

Suppressed for 5-7 days

Suppressed for 10 days

Dropulic et al., 1992

Dropulic et al., 1992

T-cell line/clone

Stable

Suppressed for 12 days

Sun et al., 1994

RRE

HeLa CD4/pool

Transient

Suppressed for 6-9 days

Dropulic and Jeang, 1994

gag

HeLa CD4/pool

T-cell line/pool

Transient

Stable

Suppressed for 7 days

Delayed for 9 days

Sarver et al., 1990

Ramezani and Joshi, 1996

pro

T-cell line/pool

Stable

Delayed for 15 days

Ramezani and Joshi, 1996

RT

T-cell line/pool

Stable

Delayed for 9 days

Ramezani and Joshi, 1996

tat

T-cell line/clone

Stable

delayed for 8 days

Lo et al., 1992

T-cell line/pool

T-cell line/pool

Stable

Stable

Delayed for 6 days

Delayed for 6-8 days

Crisell et al., 1993

Zhou et al., 1994

T-cell line/pool

T-cell line/pool

Stable

Stable

Suppressed for 12 days

Suppressed for 9 days

Sun et al., 1995

Wang et al., 1998

rev

T-cell line/clone

T-cell line/pool

Stable

Stable

Suppressed for 18 days

Delayed for 6-8 days

Michienzi et al., 1998

Zhou et al., 1994

tat and rev

LTBMC/pool

T-cell line/pool

Stable

Stable

Suppressed

Delayed for 6-8 days

Bauer et al., 1997

Zhou et al., 1994

env

T-cell line/pool

Stable

Delayed for 18 days

Ramezani et al., 1996

nef

T-cell line/clone

Stable

Delayed up to 14 days

Larsson et al., 1996

LTBMC: long-term bone marrow culture.

efficiently in vivo ( Table 1 ). While targeting some sites has been found to be more effective at inhibiting virus replication than others, the in vitro cleavage results could not always be correlated with the results obtained in vivo (Dropulic and Jeang, 1994; Ramezani and Joshi, 1996). Even ribozymes with poor in vitro cleavage activities have been shown to significantly inhibit HIV-1 replication (Crisell et al., 1993; Ramezani and Joshi, 1996).

IV. Ribozyme delivery, expression, and localization

A. Ribozyme delivery and expression vectors

Retroviral vectors are commonly used for the delivery and expression of genes (Friedman, 1989). Since these
vectors are unable to transduce non-dividing cells such as hematopoietic stem cells, lentiviral vectors have recently been developed and successfully used to deliver genes into non-dividing cells (Naldini et al., 1996; Uchida et al., 1998). Among the cells transduced with HIV-1 based vectors are the human hematopoietic stem cells (Uchida et al., 1998), macrophages (Corbeau et al., 1998), and terminally differentiated neurons (Naldini et al., 1996). The restricted host range of HIV-1, which is limited to CD4+ cells, could be extended using the amphotropic envelope protein from the Moloney murine leukemia virus (MoMuLV) or the G protein from vesicular stomatitis virus (Naldini et al., 1996). The later envelope is also more stable and allows ultracentrifugal concentration of virions to high titers (Naldini et al., 1996; Reiser et al., 1996).
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Table 2 In vitro selection studies aimed at improving/altering the cleavage activity of various ribozymes.

Ribozyme # of nts mutated/ Results (approximate improvement Reference

mutation rate compared to unselected RNA)

Tetrahymena

group I intron

140/5% per position

100-fold improved DNA-cleaving activity

Beaudry and Joyce, 1992

Tetrahymena

group I intron

140/5% per position

170-fold improved catalytic activity utilizing an altered metal cation

Lehman and Joyce, 1993

RNase P

9/random

30-fold improved catalytic activity

Yuan and Altman, 1994

Hairpin

50/3 mutations per molecule

20-fold improved trans-cleavage activity

Joseph and Burket, 1993

Hammerhead

14/random

Consensus activity

Ishizaka et al., 1995

Hammerhead

4/random

Less efficient than consensus

Thomson et al., 1996

Hammerhead

10/random

Less efficient than consensus

Vaish et al., 1997

Ribozyme genes are expressed form the retroviral long terminal repeat (LTR) promoter and/or from internal promoters. However, internal promoters often function poorly when inserted downstream of LTR promoters due to transcriptional interference between promoters (Emerman and Temin, 1984). Optimum functional expression of ribozyme genes has been shown to occur when ribozymes are expressed as part of the long viral RNAs transcribed form the 5’ LTR promoter by RNA polymerase (pol) II, rather than as part of transcripts produced form internal pol II (CMV, U1 snRNA) or pol III (tRNA, U6 snRNA) promoters (Zhou et al., 1996; Bertrand et al., 1997). High
the ability of the ribozyme to better associate with the target RNA. Nuclear extracts have been shown to improve the hybridization of complementary RNAs (Portman and Dreyfuss, 1994) and ribozyme activity in vitro (Bertrand and Rossi, 1994; Heidenreich et al., 1995).
Alternatively, ribozymes could be designed so that they would be co-packaged with HIV-1 virion RNA. Cleavage of HIV-1 RNA within the progeny virus should prevent subsequent viral spread. Sullenger and Cech (1993) used the retroviral RNA packaging signal ( ) to develop a packagable ribozyme and demonstrated the feasibility of this approach in a MoMuLV-based system. A chimeric

Lys

level expression was obtained from pol III promoters when
tRNA3
-ribozyme was also developed and shown to be
cloned within the 3’ LTR (Ilves et al ., 1996). Upon reverse
transcription, this design also resulted in gene duplication within both the 5’ and 3’ LTRs.

B. Ribozyme localization

Proper co-localization of the ribozyme with its target RNA is essential for ribozyme activity. Pol III-driven human tRNA or U6 snRNA as well as pol II-driven U1 snRNA have been shown to allow nuclear localization of ribozymes, while pol II promoters such as the Rous sarcoma virus LTR promoter mainly localized the ribozyme to the cytoplasm (Bertrand et al., 1997).
Anti-HIV ribozymes may be localized to the cytoplasm to inactivate the incoming virion RNA before it is reverse- transcribed. However, hammerhead ribozymes expressed under the control of the pol II promoter were not found to inhibit the incoming HIV-1 RNA (Ramezani et al., 1997; Paik et al., 1997). In contrast, hammerhead ribozymes have been shown to cleave HIV-1 transcripts in the nucleus (Paik et al., 1997). Enhanced ribozyme activity in the nucleus may be due to certain RNA-protein interactions which increase
packaged by HIV-1 (Westaway et al., 1995; 1998). As
expected, infectivity of the progeny virus was shown to be significantly decreased.

V. In vitro selection of ribozymes

In vitro selection involves isolation of molecules with a desired phenotype (i.e. altered or improved catalytic activity) from a pool of partially or randomly mutated molecules. This strategy has been successfully applied (Table 2) to RNase P (Yuan and Altman, 1994), the hairpin ribozyme (Joseph and Burke, 1993), and the Tetrahymena group I ribozyme (Lehman and Joyce, 1993; Beaudry and Joyce,
1992; Tsang and Joyce, 1994). Larger ribozymes, such as the Tetrahymena group I intron, appear to have a very high potential for improvement. In contrast, it seems difficult to further improve upon the catalytic activity of smaller ribozymes, like the hammerhead ribozyme. Many unsuccessful attempts have been reported for selecting hammerhead ribozymes with improved catalytic activities (Nakamaye and Eckstein, 1994; Long and Uhlenbeck, 1994;
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Ishizaka et al., 1995; Vaish et al., 1997; Thomson et al.,
1996; Ramezani and Joshi, unpublished results).
Long and Uhlenbeck (1994) replaced the stem-loop region within the catalytic domain of a hammerhead ribozyme with 4 or 6 random nts and applied 3 rounds of in vitro selection to isolate active ribozymes. However, all selected ribozymes displayed poor catalytic activity. The most effective of these ribozymes contained 6 nts with a tetranucleotide loop and the consensus G-C base pair next to the single stranded regions of the catalytic domain.
Thomson et al. (1996) replaced the consensus GAA sequence within the catalytic domain with 4 random nts and used 2 rounds of in vitro selection to isolate ribozymes with an improved catalytic activity. However, the most active ribozymes that could be selected contained the sequence HGAA (where H is A, C, or U) instead of GAA and possessed a reduced catalytic activity.
Ishizaka et al. (1995) randomized all of the conserved nucleotides in the catalytic domain of a minizyme lacking the stem-loop region, and conducted in vitro selection to isolate active ribozymes. However, the ribozymes selected after 7 to 8 rounds of selection contained consensus sequences. Similar results were also reported by Vaish et al. (1997), suggesting that the naturally occurring ribozymes might have already evolved to optimum or near optimum levels.

VI. The efficacy of anti-HIV-1 ribozymes

A. Monomeric ribozymes

A variety of sites within HIV-1 RNA have been targeted by monomeric ribozymes (Table 1). These include coding regions such as gag (Sarver et al., 1990; Ramezani and Joshi, 1996), pol (Dropuic and Jeang, 1994), pro (Ramezani and Joshi, 1996), RT (Ramezani and Joshi, 1996), tat (Lo et al. 1992; Crisell et al., 1993; Zhou et al., 1994; Sun et al.,
1995; Ramezani and Joshi, 1996, Wang et al. 1998), tat/rev (Zhou et al., 1994), env (Ramezani and Joshi, 1996), and nef (Larsson et al., 1996), as well as non-coding regions such as the repeat (R) region (Dropulic and Jeang, 1994), the trans- activation response (TAR) element (Ventura et al., 1994), the unique 5’ (U5) region (Weeraninghe et al., 1991; Dropulic et al ., 1992, Westaway et al., 1995), the region (Sun et al., 1994), and the Rev response element (RRE) (Dropulic and Jeang, 1994).
While the above studies have all demonstrated varying degrees of virus inhibition, there has not yet been any report of complete inhibition of virus replication using monomeric ribozymes. Virus replication eventually resumed despite the use of improved expression vectors or co-localization strategies. Partial inhibition was observed even in an extremely exaggerated experimental setting where a ribozyme was expressed as part of the HIV-1 RNA (Dropulic et al., 1992; Dropuic and Jeang, 1994). Interestingly, the break-through of virus production was not
due to escape virus production, demonstrating the inability of monomeric ribozymes to completely inhibit virus replication.

B. Multimeric ribozymes

The limited success at inhibiting HIV-1 replication using the existing monomeric ribozymes calls for new strategies to further improve on the intracellular activity of ribozymes. The efficiency of the ribozyme is largely dictated by the accessibility of the target site. However, it is unlikely that any given site will be available for cleavage at all times as RNA constantly folds and unfolds and is often masked by various cellular or viral proteins. Targeting various sites within a given RNA would significantly increase the possibility of having at least one site cleaved, which is sufficient for inactivation of the target RNA. Targeting multiple sites within HIV-1 RNA should also reduce the chance of developing escape mutants in clinical settings.
Multimeric ribozymes may be designed to contain ribozymes targeted against various sites within the same or different HIV RNA molecules. Multitarget ribozymes could even be designed to target various subtypes of HIV-1 and/or HIV-2, which would potentially confer simultaneous resistance.
Multimeric ribozymes may be expressed in tandem as part of a single RNA molecule or could be flanked by cis- acting ribozymes such that individual ribozymes will be liberated (Chowrira et al., 1994; He et al., 1993; Ventura et al., 1993; Yuyama et al., 1992, 1994). Self-liberating multimeric ribozymes have been shown to be very effective at cleaving their target RNA in vitro, although the efficacy of these ribozymes is yet to be demonstrated in vivo (Ohkawa et al., 1993). These ribozymes may be more effective at inhibiting virus replication because of their smaller size and their ability to act simultaneously. However, issues related to the stability and compartmentalization of individual ribozymes remain to be addressed. Furthermore, the use of cis-acting ribozymes limits the application of this strategy as it can not make use of retroviral vectors for efficient gene delivery.
Chen et al. (1992) constructed several multimeric ribozymes (mono-, di-, tetra-, penta-, and nonameric ribozymes) targeted against various sites within the env coding region of HIV-1 RNA. In co-transfection experiments, these ribozymes were able to confer significant inhibition of virus replication. Paik et al. (1997) used a defective HIV-1 DNA to express the nonameric ribozyme in HeLa T4 cells. Compared to control cells, which were highly permissive for HIV-1 and HIV-2 replication, only HIV-1 replication was inhibited in multimeric ribozyme-expressing cells.
We developed retroviral vectors expressing mono- or nonameric ribozymes against conserved sites within the HIV-1 Env RNA and compared their anti-HIV-1 efficacy in
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a stably transduced T-cell line (Ramezani et al., 1997). The monomeric ribozymes could only delay virus replication, whereas almost no virus production could be detected from nonameric ribozyme-expressing cells for up to sixty days (the length of the experiment). We have also tested the mono- and nonameric ribozyme expression vectors in human peripheral blood lymphocytes (PBLs). When challenged with a primary, patient-derived isolate of HIV-1 (Ramezani and Joshi, 1998), the nonameric ribozyme-expressing PBLs were shown to delay virus replication more than the monomeric ribozyme-expressing PBLs.

VII. Prospects

The hammerhead ribozyme offers a potentially effective means of inhibiting HIV-1 replication. Although the catalytic activity of the hammerhead ribozyme might already be close to optimal, a number of other factors affecting ribozyme design and activity could improve ribozyme activity in vivo . Promising results have been obtained using multimeric ribozymes. Combination strategies could be used to achieve even better inhibition of HIV-1 replication.

Acknowledgments

This work is supported by a grant from the Medical
Research Council of Canada.

References

Bauer, G., Valdez, P., Kearns, K., Bahner, I., Wen, S. F., Zaia, J.

A., Kohn, D. B. (1997). Inhibition of HIV-1 replication after transduction of granulocyte colony-stimulating factor- mobilized CD34+ cells from HIV-1-infected donors using retroviral vectors containing anti-HIV-1 genes. Blood 89,

2259-2267.

Beaudry, A. A., and Joyce, G. F. (1992). Directed evolution of an

RNA enzyme. Science 257, 635-641.

Bertrand, E., and Rossi, J. J. (1994). Facilitation of hammerhead ribozyme catalysis by the nucleocapsid protein of HIV-1 and the heterogeneous nuclear ribonucleoprotein A1. EMBO J 13,

2904-2912.

Bertrand, E., Castanotto, D., Zhou, C., Carbonnelle, C., Lee, N. S., Good, P., Chatterjee, S., Grange, T., Pictet, R., Kohn, D., Engelke, D., and Rossi, J. J. (1997). The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization. RNA 3, 75-

88.

Bruening, G. (1990). Compilation of self-cleaving sequences from plant virus satellite RNAs and other sources. Methods Enzymol. 180, 546-558.

Buzayan, J. M., Gerlach, W. L., and Bruening, G. (1986). Non- enzymatic cleavage and ligation of RNAs complementary to a plant virus satellite RNA. Nature 326, 349-353.

Chen, C. J., Banerjee, A. C., Harmison, G. G., Haglund, K., and Schubert, M. (1992). Multitarget-ribozyme directed to cleave at up to nine highly conserved HIV-1 env RNA regions inhibits

HIV-1 replication – potential effectiveness against most presently sequenced HIV-1 isolates. Nucleic Acids Res 20,

4581-4589

Chowrira, B. M., Pavco, P. A., and McSwiggen, J. A. (1994). In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes. J Biol Chem 269, 25856-25864.

Clouet-d’Orval, B., and Uhlenbeck, O. C. (1997). Hammerhead ribozymes with a faster cleavage rate. Biochemistry 36, 9087-

9092.

Corbeau, P., Kraus, G., and Wong-Staal, F. ( 1998). Transduction of human macrophages using a stable HIV-1/HIV-2-derivedgene delivery system. Gene Ther 5, 99-104.

Crisell, P., Thompson, S., and James, W. (1993). Inhibition of HIV-

1 replication by ribozymes that show poor activity in vitro.

Nucleic Acids Res 21, 5251-5255.

Dropulic, B., and Jeang, K-T. (1994). Intracellular susceptibility to ribozymes in a tethered substrate-ribozyme provirus model is not predicted by secondary structure of HIV-1 RNAs in vitro . Antisense Nucleic Acid Drug Dev 4, 217-221.

Dropulic, B., Lin, N. H., Martin, M. A., and Jeang, K-T. (1992).

Functional characterization of a U5 ribozyme, Intracellular suppression of HIV-1 expression. J Virol 66, 1432-1441.

Ehresmann, C., Baudin, F., Mougel, M., Romby, P., Ebel, J. P., and Ehresmann, B. (1987). Probing the structure of RNAs in solution. Nucleic Acids Res 15, 9109-9128.

Emerman, M., and Temin, H. (1984). Genes with promoters in retrovirus vectors can be independently suppressed by an epigenetic mechanism. Cell 39, 459-467.

Fedor, M. J., and Uhlenbeck O. C. (1990). Substrate sequence effects on hammerhead RNA catalytic efficiency. Proc Natl Acad Sci USA 87, 1668-1672.

Fedor, M. J., and Uhlenbeck, O. C. (1992). Kinetics of intermolecular cleavage by hammerhead ribozymes. Biochemistry 31, 12042-12054.

Foster, A. C., and Symons, R. H. (1987). Self-cleavage of plus and minus RNAs of a virusoid and a structural model for the active sites. Cell 49, 211-220.

Friedman, T. (1989). Progress toward human gene therapy. Science

244, 1275-1292.

Goodchild, J., and Kohli, V. (1991). Ribozymes that cleave an RNA sequence form HIV, The effect of flanking sequence on rate. Arch Biochem Biophys 284, 386-391.

Guerrier-Takada, C., Gardiner, k., Marsh, T., Pace, N., and Altman, S. (1983). The RNA moiety of ribonuclease P is the catalytic subunit of the enzyme. Cell 35, 849-857.

Haseloff, J., and Gerlach, W. L. (1988). Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature

334, 585-591.

Haseloff, J., and Gerlach, W. L. (1989). Sequences required for self-catalyzed cleavage of the satellite RNA of tobacco ringspot virus. Gene 82, 43-51.

He, Y. K., Lu, C. D., and Qi, G. R. (1993). In vitro cleavage of HPV 16 E6 and E7 RNA fragments by synthetic ribozymes and transcribed ribozymes from RNA-trimming plasmids. FEBS Lett 322, 21-24.

278

Gene Therapy and Molecular Biology Vol 3, page 279

Heidenreich, O., Kang, S. H., Brown, D. A., Xu, X., Swiderski, P., Rossi, J. J., Eckstein, F., and Nerenberg, M. (1995). Ribozyme- mediated RNA degradation in nuclei suspension. Nucleic Acids Res 23, 2223-2228.

Ho, S. P., Britton, D. H., Stone, B. A., Behrens, D. L., Leffet, L.

M., Hobbs, F. W., Miller, J. A., and Trainor, G. L. (1996). Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNA- accessible sites with oligonuceotide libraries. Nucleic Acids Res 24, 1901-1907.

Homann, M., Tabler, M., Tzortzakaki, S., and Sczakiel, G. (1994).

Extension of helix II of an HIV-1-directed hammerhead ribozyme with ling antisense flanks does not alter kinetic parameters in vitro but causes loss of the inhibitory potential in living cells. Nucleic Acids Res 22, 3951-3957.

Hormes, R., Homann, M., Oelze, I., Marschall, P., Tabler, M., Eckstein, F., and Sczakiel, G. (1997). The subcellular localization and length of hammerhead ribozymes determine efficacy in human cells. Nucleic Acids Res 25, 769-775.

Hutchins, C. J., Rathjen, P. D., Forster, A. C., and Symons, R. H. (1986). Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res 14, 3627-3640

Ilves, H., Barske, C., Junker, U., Bohnlein, E., and Veres, G. (1996). Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts, a comparative study. Gene 171, 203-208.

Ishizaka, M., Ohshima, Y., and Tani, T. (1995). Isolation of active ribozymes from an RNA pool of random sequences using an anchored substrate RNA. Biochem Biophys Res Commun

214, 403-409.

Jaeger, J. A., Turner, D. H., and Zuker, M. (1989). Improved predictions of secondary structures of RNA. Proc Natl Acad Sci USA 86, 7706-7710.

Joseph, S., and Burke, J. M. (1993). Optimization of an anti-HIV

hairpin ribozyme by in vitro selection. J Biol Chem 268,

24515-24518.

Joshi, S., and Joshi, R. L. (1996). Molecular biology of HIV-1.

Transfus Sci 17, 351-378.

Koizumi, M., Hayase, Y., Iwai, S., Kamiya, H., Inoue, H., and Ohtsuka, E. (1989). Design of RNA enzymes distinguishing a single base mutation in RNA. Nucleic Acids Res 17, 7059-

7071.

Larsson, S., Hotchkiss, G., Su, J., Kebede, T., Andang, M., Nyholm, T., Johansson, B., Sonnerborg, A., Vahlne, A., Britton, S., and Ahrlund-Richter, L. (1996). A novel ribozyme target site located in the HIV-1 nef open reading frame. Virology 219, 161-169.

Lehman, N., and Joyce, G. F. (1993). Evolution in vitro of an RNA

enzyme with altered metal dependence. Nature 361, 182-185.

Lo, K. M. S., Biasolo, M. A., Dehni, G., Palu, G., and Haseltine, W. A. (1992). Inhibition of replication of HIV-1 by retroviral vectors expressing tat-antisense and anti-tat ribozyme RNA. Virology 190, 176-183.

Long, D. M., and Uhlenbeck, O. C. (1994). Kinetic characterization of intramolecular and intermolecular hammerhead RNAs with stem II deletions. Proc Natl Acad Sci USA 91, 6977-6981.

Michienzi, A., Conti, L., Varano, B., Prislei, S., Gessani, S., and

Bozzoni, I. (1998). Inhibition of HIV-1 replication by nuclear

chimeric anti-HIV ribozymes in a human T lymphoblastoid cell line. Hum Gene Ther 9, 621-628.

Miller, W.A., Hercus, T., Waterhouse, P. M., and Gerlach, W. L. (1991). A satellite RNA of barley yellow dwarf virus contains a novel hammerhead structure in the self-cleavage domain. Virology 183, 711-720.

Nakamaye, K. L., and Eckstein, F. (1994). AUA-cleaving hammerhead ribozymes: Attempted selection for improved cleavage. Biochemistry 33, 1271-1277.

Naldini, L., Blomer, U., Gallay, P., Ory, D., Mulligan, R., Gage, F.

H., Verma, I. M., and Trono, D. (1996). In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector. Science 272, 263-267.

Ohkawa, J., Yuyama, N., Takebe, Y., Nishikawa, S., and Taira, K. (1993). Importance of independence in ribozyme reactions: Kinetic behavior of trimmed and simply connected ribozymes with potential activity against HIV. Proc Natl Acad Sci USA

90, 11302-11306.

Paik, S-Y., Banerjea, A., Chen, C-J., Ye, Z., Harmison, G. G., and Schubert, M. (1997). Defective HIV-1 provirus encoding a multitarget-ribozyme inhibits accumulation of spliced and unspliced HIV-1 mRNAs, reduces infectivity of viral progeny, and protects the cells from pathogenesis. Hum Gene Ther 8,

1115-1124.

Perriman, R., Delves, A., and Gerlach, W. L. (1992). Extended target-site specificity for a hammerhead ribozyme. Gene 113,

157-163.

Portman, D. S., and Dreyfuss, G. (1994). RNA annealing activities in HeLa nuclei. EMBO J 13, 213-221.

Ramezani, A., and Joshi, S. (1998). Inhibition of HIV-1 replication in human peripheral blood lymphocytes by retroviral vectors expressing monomeric and multimeric hammerhead ribozymes.

12t h World AIDS Conference 435-439.

Ramezani, A., and Joshi, S. (1996). Comparative analysis of five highly conserved target sites within the HIV-1 RNA for their susceptibility to hammerhead ribozyme-mediated cleavage in vitro and in vivo. Antisense Nucleic Acid Drug Dev 6, 229-235.

Ramezani, A., Ding, S. F., and Joshi, S. (1997). Inhibition of HIV-1 replication by retroviral vectors expressing mono- and multimeric hammerhead ribozymes. Gene Ther 4, 861-867.

Reiser, J., Harmison, G., Kluepfel-Stahl, S., Brady, R. O., Karlsson, S., and Schubert, M. (1996). Transduction of non-dividing cells using pseudotyped defective high titre HIV-1 particles. Proc Natl Acad Sci USA 93, 15266-15271.

Ruffner, D. E., Stormo, G. D., and Uhlenbeck, O. C. (1990).

Sequence requirements of the hammerhead RNA self-cleavage reaction. Biochemistry 29, 10695-10702.

Sarver, N., Cantin, E. M., Chang, P. S., Zaia, J. A., Ladne, P. A., Stephens, D. A., and Rossi, J. J. (1990). Ribozymes as potential anti-HIV-1 therapeutic agents. Science 247, 1222-1225.

Saville, B. J., and Collins, R. A. (1990). A site-specific self- cleavage reaction performed by a novel RNA I Neurospora mitochondria. Cell 61, 685.

Scherr, M., and Rossi, J. J. (1998). Rapid determination and quantitation of the accessibility to native RNAs by antisense oligodeoxynucleotides in murine cell extracts. Nucleic Acids Res 26, 5079-5085.

279

Ramezani and Joshi: Hammerhead ribozymes for HIV-1 gene therapy

Sheldon, C. C., and Symons, R. H. (1989). Mutagenesis analysis of a self-cleaving RNA. Nucleic Acids Res 17, 5679-5685.

Sullenger, B. A., and Cech, T. R. (1993). Tethering ribozymes to a retroviral packaging signal for destruction of viral RNA. Science 262, 1566-1569.

Sun, L-Q., Wang, L., Gerlach, W. L., and Symonds, G. (1995).

Target sequence-specific inhibition of HIV-1 replication by ribozymes directed to tat RNA. Nucleic Acids Res 23, 2909-

2913.

Sun, L-Q., Warrilow, D., Wang, L., Witherington, C., and Macpherson, J. (1994). Ribozyme-mediated suppression of MoMuLV and HIV-1 replication in permissive cell lines. Proc Natl Acad Sci USA 91, 9715-9719.

Tabler, M., Homann, M., Tzortzakaki, S., and Sczakiel, G. (1994).

A three-nucleotide helix I is sufficient for full activity of a hammerhead ribozyme: advantages of an asymmetric design. Nucleic Acids Res 22, 3958-3965.

Thomson, J. B., Sigurdsson, S. T., Zeuch, A., and Eckstein, F. (1996). In vitro selection of hammerhead ribozymes containing a bulged nucleotide in stem II. Nucleic Acids Res 24, 4401-

4406.

Tsang, J. and Joyce, G. F. (1994). Evolutionary optimization of the catalytic properties of a DNA-cleaving ribozyme. Biochemistry 33, 5966-5973.

Tuschl, T., and Eckstein, F. (1993). Hammerhead ribozymes: importance of stem-loop II for activity. Proc Natl Acad Sci USA 90, 6991-6994.

Uchida, N., Sutton, R. E., Friera, A. M., He, D., Reitsma, M. J., Chang, W. C., Veres, G., Scollay, R., and Weissman, I. L. (1998). HIV, but not murine leukemia virus, vectors mediate high efficiency gene transfer into freshly isolated G0/G1 human hematopoietic stem cells. Proc Natl Acad Sci USA 95,

11939-11944.

Uhlenbeck, O. C. (1987). A small catalytic oligoribonucleotide.

Nature 328, 596-600.

Uhlenbeck, O. C., Pardi, A., and Feigon, J. (1997). RNA structure comes of age. Cell 90, 833-840.

Vaish, N. K., Heaton, P. A., and Eckstein, F. (1997). Isolation of hammerhead ribozymes with altered core sequences by in vitro selection. Biochemistry 36, 6495-6501.

Ventura, M., Wang, P., Franck, N., and Saragosti, S. (1994).

Ribozyme targeting of HIV-1 LTR. Biochem Biophys Res

Commun 203, 889-898.

Ventura, M., Wang, P., Ragot, T., Perricaudet, M., and Saragosti, S. (1993). Activation of HIV-specific ribozyme activity by self- cleavage. Nucleic Acids Res 21, 3249-3255.

Wang, L., Witherington, C., King, A., Gerlach, W. L., Carr, A., Penny, R., Cooper, D., Symonds, G., and Sun, L. Q. (1998). Preclinical characterization of an anti-tat ribozyme for therapeutic application. Hum Gene Ther 9, 1283-1291

Weerasinghe, M., Liem, S. E., Asad, S., Read, S. E., and Joshi, S. (1991). Resistance to HIV-1 infection in human CD4+ lymphocyte-derived cell lines using retroviral vectors expressing an HIV-1 RNA specific ribozyme. J Virol 65,

5531-5534.

Westaway, S. K., Larson, G. P., Li, S., Zaia, J. A., and Rossi, J. J. (1995). A chimeric tRNA Lys-ribozyme inhibits HIV replication following virion assembly. Nucleic Acids Symp Ser 33, 194-

199.

Westaway, S. K., Cagnon, L., Chang, Z., Li, S., Li, H., Larson, G.

P., Zaia, J. A., and Rossi, J. J. (1998). Virion encapsidation of tRNA Lys-ribozyme chimeric RNAs inhibits HIV infection. Antisense Nucleic Acid Drug Dev 8, 185-197.

Wu, H. N., Lin, Y. J., Lin, F. P., Makino, S., Chang, M. F., and Lai, M. M. (1989). Human hepatitis delta virus RNA subfragments contain an autocleavage activity. Proc Natl Acad Sci USA 86,

1831-1835.

Yuan, Y., and Altman, S. (1994). Selection of guide sequences that direct efficient cleavage of mRNA by human ribonuclease P. Science 263, 1269-1273.

Yuyama, N., Ohkawa, J., Koguma, T., Shirai, M., and Taira, K. (1994). A multifunctional expression vector for an anti-HIV-1 ribozyme that produces a 5’-and 3’-trimmed trans-acting ribozyme, targeted against HIV-1 RNA, and cis-acting ribozymes that are designed to bind to and thereby sequester trans-activator proteins such as Tat and Rev. Nucleic Acids Res 22, 5060-5067.

Yuyama, N., Ohkawa, J., Inokuchi, Y., Shirai, M., Sato, A., Nishikawa, S., and Taira, K. (1992). Construction of a tRNA- embedded-ribozyme trimming plasmid. Biochem Biophys Res Commun 186, 1271-1279.

Zaug, A. J., Been, M. D., and Cech, T. R. ( 1986). The Tetrahymena ribozyme acts like an RNA restriction endonuclease. Nature

324, 429-433.

Zhao, J. J., and Lemake, G. (1998). Rules for ribozymes. Mol Cell

Neurosc 11, 92-97.

Zhou, C., Bahner, I., Rossi, J. J., and Kohn, D. B. (1996).

Expression of hammerhead ribozymes by retroviral vectors to inhibit HIV-1 replication: Comparison of RNA levels and viral inhibition. Antisense Nucleic Acid Drug Dev 6, 17-24.

Zhou, C., Bahner, I. C., Larson, G. P., Zaia, J. A., Rossi, J. J., and Kohn, D. B. (1994). Inhibition of HIV-1 in human T- lymphocytes by retrovirally transduced anti-tat and rev hammerhead ribozymes. Gene 149, 33-39.

Zoumadakis, M., and Tabler, M. (1995). Comparative analysis of cleavage rates after systematic permutation of the NUX consensus target motif for hammerhead ribozymes. Nucleic Acids Res 23, 1192-1196.

280