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Next Article 
Blood, Vol. 91 No. 2 (January 15), 1998:
pp. 371-382
REVIEW ARTICLE
The Therapeutic Potential of Ribozymes
By
Helen A. James and
Ian Gibson
From the School of Biological Sciences, University of East Anglia,
Norwich, Norfolk, UK.
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ABSTRACT |
Ribozymes are catalytic RNA molecules that recognize their target
RNA in a highly sequence-specific manner. They can therefore be used to
inhibit deleterious gene expression (by cleavage of the target mRNA) or
even repair mutant cellular RNAs. Targets such as the mRNAs of
oncogenes (resulting from base mutations or chromosome translocations,
eg, ras or bcr-abl) and viral genomes and transcripts
(human immunodeficiency virus-type 1 [HIV-1]) are ideal targets for
such sequence-specific agents. The aim of this review is therefore to
introduce the different classes of ribozymes, highlighting some of the
chemistry of the reactions they catalyze, to address the specific
inhibition of genes by ribozymes, the problems yet to be resolved, and
how new developments in the field give hope to the future for ribozymes
in the therapeutic field.
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INTRODUCTION |
RIBOZYMES ARE ribonucleic acid (RNA)
molecules with enzymatic activity that have a great potential as
therapeutic entities because of their ability to either cleave
deleterious RNAs or repair mutant cellular RNAs.1,2 They
form basepair-specific complexes and catalyze the hydrolysis of
specific phosphodiester bonds, causing RNA strand cleavage. Differences
exist between ribozymes in size and structure and, although most
naturally occurring ribozymes cleave intramolecularly in a cis
linkage, the RNA component of RNase-P, which is involved in the
processing of pre-t-RNA molecules, acts in trans, ie,
intermolecularly.3
Like its protein counterpart, a catalytic RNA or ribozyme greatly
accelerates the rate of a biochemical reaction and shows extraordinary
specificity with respect to the substrates it acts upon and the
products it produces. Ribozymes can cleave the normally unreactive
bonds of a phosphodiester linkage in an RNA molecule resulting in a
3 hydroxyl (3OH) and 5 phosphate (5OH) and 3 or 23-cyclic phosphate.4 There are
several different classes of ribozymes: the self-splicing group I and
group II introns; RNase P; and several distinct catalytic motifs found
in the small pathogenic RNAs (Fig 1).
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| Fig 1.
RNA catalyzed reactions. (1) The two-step self-splicing
reaction of group I introns. (2) The two-step self-splicing reaction of
group II introns. An internal hydroxyl initiates the attack. (3)
Cleavage of the 5 leader sequence from pre-tRNA by RNase P. (4)
Self-cleavage reaction of a number of small pathogenic RNAs and a few
other RNAs.
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SELF-SPLICING RNAs |
Many genes have their coding sequences (exons) interrupted by stretches
of noncoding DNA called introns. Transcripts of such genes must undergo
cleavage-ligation reactions to produce the mature functional RNA. The
splicing of most nuclear pre-mRNAs involves a two-step process,
generating an intron lariat and spliced exons, and has a requirement
for a number of small nuclear ribonucleoprotein particles (snRNPs) and
other proteins. Sequences in the RNA components of the snRNPs (U1, U2,
and U5 small nuclear RNAs) recognize the 5 splice site, branch
point, and 3 splice site, respectively, and, together with the
U4 and U6 snRNPs, create the spliceosome where the intron is excised
and the exons are ligated (Fig 2A; reviewed
in Maniatis and Reed5).
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| Fig 2.
Comparison of pre-mRNA nuclear splicing, self-splicing,
and trans-splicing. (A) Nuclear pre-mRNA splicing with
spliceosomes (small nuclear RNAs and proteins). (B) Self-splicing as
performed by the self-splicing group I and group II introns. (C)
Trans-splicing of a mutated mRNA with a modified group I
catalytic intron.
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Based on the nucleotide sequences and/or structures within and
adjacent to the introns, introns have been classified into four
classes: group I, group II, nuclear mRNA, and nuclear tRNA. Some
examples of group I and II introns are capable of self-splicing in
vitro in the absence of protein (Fig 2B).
Group I introns.
The self-splicing of group I introns, in the presence of a guanosine
cofactor and magnesium, was first observed for the intron of the
nuclear 26S rRNA gene in Tetrahymena
thermophila.6,7 Self-splicing proceeds by two
consecutive transesterification reactions, both initiated by
nucleophilic attack. The excised intron, with a small deletion, can be
converted into a true enzyme able to act in trans on specific
substrates.
The RNA cleavage and ligation activities are intrinsic to the structure
of the intron. By a number of basepaired regions, both the 3 and
5 splice sites are aligned for splicing by the internal guide
sequence, close to the guanosine binding site.8-11 The
catalytic domain of group I introns is formed by two structural domains, the crystal structures of which are now being
determined.12 The interactions between the two domains and
nucleotides important for cleavage activity are being
elucidated.13 These studies are leading to understanding
group I splicing: the mechanism of action and the roles played by the
guanosine and metal cofactors.14-16
Despite not understanding fully the group I intron self-splicing
mechanism of action these molecules are being manipulated to perform
trans-splicing, ie, the intentional modification of the
sequence of a targeted transcript in tissue culture
cells.2,17 It can be seen in Fig 2C that a mutation in the
RNA sequence can be corrected by replacing part of the mRNA with a new
sequence using a suitably modified ribozyme. Although currently not
very specific in choice of target RNA (the ribozyme recognizes and trans-splices several mRNAs), it should be possible to use this approach to develop safe therapeutic ribozymes that can repair mutant
RNAs associated with a variety of inherited diseases.
Group II introns.
Some group II introns are also able to undergo self-splicing. They
differ from group I introns by the structure of their catalytic core
and the products of the splicing: ligated exons and an excised intron-lariat18 (Fig 1). Again, the reaction consists of
two transesterifications. The first step can be initiated by
nucleophilic attack by an intronic 2hydroxyl group on the
phosphodiester linkage at the 5 splice site, leading to the
formation of the lariat structure. There is evidence for a second
pathway, that involving attack by H2O or OH.19
The second step is initiated by the 3OH of the 5 exon on
the 3 splice site. Basepairing interactions between sequences
known as the exon binding site and the intron binding site hold the
splice-sites in close proximity.20 This ability of group II
introns to specifically bind the 5 exon has been exploited to
encourage the intron to catalyze reactions on exogenous substrates.
Derivatives of group II introns reverse splicing by inserting
themselves between ligated exons.21 This insertion and
subsequent trans-splicing reactions can be used to shuffle
sequences22 and therefore link any RNA molecule to any
other RNA molecule, intentionally modifying the target sequence.
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RNase P |
Ribonuclease P (RNase P) is an ubiquitous endoribonuclease that
processes the 5 end of precursor tRNA molecules, producing 5 phosphate and 3OH termini.23 RNase P
consists of both protein and RNA components and it was shown that the
catalyst was the RNA moiety.24 As with the catalytic
introns, a divalent cation is required as cofactor.25 RNase
P can be directed to cleave any RNA when the target is in complex with
a short, complementary oligonucleotide called an external guide
sequence (EGS), thereby inactivating it.26-29
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SELF-CLEAVING RNAs: SMALL PATHOGENIC RNAs |
A number of small plant pathogenic RNAs (viroids, satellite RNAs, and
virusoids), an RNA transcript from Neurospora mitochondrial DNA, and an animal virus, HDV, all undergo a self-cleavage reaction in
vitro in the absence of protein (Table 1).
The reaction, which requires magnesium and produces
23-cyclic phosphate and 5OH termini, results from
several different catalytic motifs: the hammerhead, hairpin, and
axehead or pseudoknot (Fig 3). It is generally thought that the self-cleavage reaction is an integral part
of the small pathogenic RNAs' rolling circle method of
replication.53 Circular monomeric plus and minus RNAs act
as templates for the synthesis of longer-than-unit-length precursor
RNAs. The production of monomeric forms from these concatamers requires
specific cleavage thought to be performed by the RNA itself.
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| Fig 3.
Small catalytic RNAs. (A) Hammerhead ribozyme split into
substrate and catalyst,51 basepaired by means of two
flanking arms (in bold). Numbering is that of Hertel et
al.52 (B) Hairpin ribozyme split into substrate and
catalyst.42 (C) Pseudoknot motif of the HDV
ribozyme.45 Arrows show position of scissile bonds. IUB
codes used throughout: N = A, C, G, U; R = A, G; Y = C, U; B = C, G, U; D = A, U, G; H = A, C, U; V = A, C, G.
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The reaction is a simple nonhydrolytic cleavage whereby the scissile
bond undergoes a nucleophilic attack by the adjacent 2OH group
(Fig 1).54 The stereochemistry of cleavage and the role of
Mg2+ in catalysis has been greatly
studied.55,56 In the case of the hammerhead ribozyme, the
crystal structure57,58 and fluorescence resonance energy
transfer (FRET)59 measurements have shown the relative
orientation of the core helices and have allowed the hammerhead
catalytic core to be modelled.
Both of the crystallized hammerhead ribozymes contained modifications
to prevent self-cleavage: the first57 contained an all DNA
substrate analog; the second58, although all RNA, had the
active site 2 hydroxyl replaced with an inert 2 methoxy
group. As such, they may not represent the active structure. Scott et
al60 have used time-resolved crystallography to determine
the structure of an active hammerhead ribozyme in the absence of
divalent cation or with Mg2+ but at a pH unsuitable for
cleavage. A captured conformational intermediate showed that the most
significant conformational changes were limited to the active site of
the ribozyme.
It has been suggested that the function of the ribozyme catalytic core
is twofold. First, it would destabilize the substrate strand to allow
the scissile phosphodiester linkage to twist into a cleavable
conformation. Second, the core would assist in positioning the divalent
metal ion so as to facilitate catalysis.57 The more
information gained about the active structure of the hammerhead ribozyme, the more rational the design of specific ribozymes can be.
Once factors governing the specificity and mechanism of cleavage are
elucidated, it may be possible to design new catalysts that efficiently
cleave other sequences.
The hammerhead, hairpin, HDV, and Neurospora ribozymes have all
been converted from the naturally occurring cis-active
ribozymes to trans-active ribozymes by splitting the catalytic
core from the substrate sequence. The Neurospora VS ribozyme
has been converted once61 and is the least understood
motif. More work has been performed on the trans-active HDV
ribozyme. The development of this trans-active ribozyme is
hampered by the lack of firm knowledge of the secondary and tertiary
structure of the RNA and the alternative structures
proposed.45,62,63 Recent studies are providing more
detailed information.64,65 Despite this lack of
understanding of the structure, trans-active HDV ribozymes have
been developed.66,67
The development of the hammerhead and hairpin catalytic domains for
trans-cleavage are far more advanced. The structures and RNA
folding are fairly well understood and the models for the design of
trans-acting hammerhead and hairpin ribozymes are well tried
and tested.
The hammerhead (Fig 3A) consists of two flanking arms capable of
basepairing with the substrate to form two helices (I and III) and a
catalytic core with a helix (II) and several single-stranded regions.
The ribozyme recognizes sequences either side of a substrate NUH site
by means of the flanking arms. The catalytic core then cleaves the RNA
3 of the NUH triplet. The hairpin ribozyme (Fig 3B) has also
been split into substrate and catalytic core. Ribozymes are active in
vitro in cell-free systems and in living cells. This activity and their
sequence specificity makes them attractive as agents for the inhibition
of gene expression.
As described below, they have been used against oncogenes and viruses
and have helped in the understanding of the role of genes in
developmental processes. Many of the problems with ribozymes are
similar to those outlined for antisense oligodeoxy-nucleotides (ODNs).68 Efficient entry into cells, ribozyme stability,
and precise targeting to the substrate RNA sequences are receiving attention. Most, if not all, RNAs in vivo normally exist as
ribonucleoprotein (RNP) complexes and not as free molecules.
Interactions between ribozymes and substrates and these RNP proteins
could greatly influence activity. Ribozyme binding could be inhibited
by steric hindrance. Alternatively, proteins may enhance activity via
annealing and strand-exchange activities. The intracellular
environment, such as pH and the availability of divalent cations, must
also be considered. For example, the intracellular Mg2+
concentration is known to be approximately 0.8 mmol/L,69
much lower than is commonly used for in vitro reactions (10 to 20 mmol/L). A primary aim is to show that the ribozyme functions in vivo
in a catalytic manner producing two cleavage fragments.
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SPECIFIC GENE INHIBITION USING RIBOZYMES (TABLE
2) |
Specificity has, in many cases, been inferred from biological effects
without measuring target mRNA or protein levels or looking for ribozyme
cleavage products or for effects on unrelated mRNAs. Initial studies of
ribozyme activity in cultured cells have come across difficulties in
detecting the activity and the catalytic nature of the ribozyme.
Several groups have resorted to using a reporter gene, such as
chloramphenicol acetyltransferase (CAT),85 in which a
reduction in the CAT activity is taken as demonstrating ribozyme
activity, or neomycin phosphotransferase (npt).86 Ribozyme specificity has been demonstrated for the  APP mRNA when this was
shown to be reduced compared with control RNAs,  -actin, and G3PDH
mRNA.87 However, in this last study, a mutant ribozyme (catalytically inactive) was also effective at reducing levels, suggesting that the ribozyme-mediated degradation of APP mRNA in
COS-7 cells was not dependent on ribozyme cleavage.
Ribozyme cleavage has been demonstrated by an RNase protection assay,
where the cleavage products protected the probe.86 But care
has to be taken in interpreting such results. It was recently noted
that a hammerhead ribozyme cleaved its substrate during RNA preparation
and not while in the cell.82,88
Despite some of the problems already alluded to, many groups are
actively investigating ribozymes' therapeutic applications.
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OVERCOMING DRUG RESISTANCE |
The reversal of drug resistance is a popular goal for ribozyme
technology. Kobayashi et al70 showed they could reduce
MOLT-3 cells from approximately 700-fold resistant to vincristine to only 20- to 30-fold resistant when an anti-MDR-1 ribozyme was transfected in and stably expressed (under the -actin promoter). This increase in drug sensitivity, and reduction in MDR-1 mRNA levels,
was shown to be proportional to the amount of ribozyme expression. They
critically showed that, although they could not detect cleavage
products, a disabled ribozyme incapable of cleavage had no effects on
the drug resistance levels. They also tested their ribozyme in the
non-drug-resistant parental cell line and found no effect: the
ribozyme was not toxic. Kiehntopf et al71 found that
liposome-mediated transfection of drug-resistant mesothelioma cell
lines with either in vitro transcribed or chemically synthesized ribozymes significantly reduced expression of the MDR-1 gene. This
restored sensitivity towards chemotherapeutic drugs. They were also
able to demonstrate, using reverse transcription polymerase chain
reaction, the reduction in full-length MDR-1 mRNA levels.
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OVERCOMING THE TRANSFORMED PHENOTYPE |
The mRNAs from oncogenes have also been targeted by ribozyme
technology. H-ras is activated by a mutation at codon 12 (GGU  GUU), with the activated form being a
substrate for hammerhead ribozyme cleavage. When transformed NIH3T3
cells that displayed a neoplastic phenotype in vitro and were
tumorigenic in nude mice in vivo were transfected by a ribozyme
designed to cleave the activated H-ras mRNA, the transformed
phenotype was abrogated.72 A reduction in H-ras
mRNA was observed. A mutant ribozyme resulted in cells with an
intermediate phenotype, probably due to an antisense effect of the
ribozyme hybridizing arms. This group also addressed the question of
specificity by looking at the K-ras mRNA levels in control and
ribozyme-treated cells and showed no cross-reactivity. Ohta et
al73 demonstrated that a tissue-specific promoter for the
expression of an anti-H-ras ribozyme produced better results than using a viral promoter. This result suggests that, depending on
the target mRNA, the choice of viral promoter for ribozyme expression
is not always the best option. The transfected ribozyme appeared to
affect not only proliferation but also the differentiation process of
the melanoma cells in vitro.89
BCR-ABL in chronic myeloid leukemia.
Chromosome translocations and the resulting chimaeric genes are good
targets for sequence-specific strategies. The hybrid mRNA will only be
present in the cells with the translocation, and antisense ODNs or
ribozymes targeted to the hybrid's junction should be specific for the
hybrid and not affect the wild-type mRNA sequences. One such
translocation results in the Philadelphia chromosome (Ph+)
of chronic myeloid leukemia, and the bcr-abl oncogene. The
expression of the bcr-abl protein tyrosine kinase is thought to
be responsible for the malignant phenotype of Ph+ cells.
Both the wild-type abl and bcr proteins are thought to be important for normal cell proliferation. This makes Ph+
cells the ideal system to address the question of ribozyme sequence specificity. We have looked at the specificity of three different hammerhead ribozymes designed to cleave two splice variants of the
bcr-abl mRNA90 in an attempt to clear up some of
the contradictory results published.91-93 We showed the
specific nature of one ribozyme (cleaved only bcr-abl RNA) but
a lack of specificity of two others (cleaved both bcr-abl and
abl RNAs). In a cell line system, the question of specificity
has not really been addressed, but a decrease in cell
proliferation,74 a decrease in bcr-abl mRNA and
protein levels,75,76 and a decrease in bcr-abl
kinase activity93 have been observed
(Table 3). These studies all used slightly different hammerhead ribozymes: varying lengths of the flanking arms
and various modifications to improve nuclease resistance or expression
from vectors. Some groups observed quite substantial effects,75,76,93 whereas others have seen more
modest91 or shown very few effects (our unpublished
data). This is probably due to differences in the
ribozymes' structures. Until the structure-function relationship is
understood, such effects will not be predictable.
The hybrid gene AML1/MTG8 mRNA that results from a translocation
between chromosomes 8 and 21 (associated with acute myeloid leukemia)
is also being targeted by hammerhead ribozymes.94
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OVERCOMING VIRAL DISEASE |
Another clinical situation amenable to ribozyme targeting is viral
disease, such as that associated with the human immunodeficiency virus
(HIV). Two classes of ribozymes are being used to combat HIV:
hammerhead79,81,95,96 and hairpin.97
Weerasinghe et al95 showed that MT4 cells
transformed with vectors expressing an HIV-1 ribozyme were resistant to
varying degrees of HIV-1 infection. The choice of constitutive versus inducible promoters for the expression of the anti-HIV-1 ribozymes had
quite an influence on the degree of resistance to HIV-1 infection. A
similar study was performed by Lo et al96 with a hammerhead ribozyme designed to cleave the HIV-1 tat RNA. Interestingly, although the anti-tat ribozyme-producing cells inhibited the
replication of HIV-1, an antisense producing vector conferred a greater
resistance to HIV-1 replication.
A third group have shown that T lymphocytes expressing anti-HIV-1
ribozymes showed resistance to HIV-1 replication.79 Cells transformed with a mutant ribozyme showed little resistance to viral
replication. They argued that this demonstrated that the functional
ribozymes were cleaving the HIV-1 RNA and were specific for this RNA.
They have also looked at the effects of using different retroviral
vectors to express the ribozymes: despite different ribozyme levels,
there was a similar level of resistance to HIV-1 replication.98 This indicated that other factors had
determining roles in the effectiveness of the ribozyme.
Bauer et al81 have made an important step by treating
CD34(+) cells from individuals already infected with HIV-1.
They transfected the cells with a retroviral vector containing a double
tat-rev ribozyme and showed up to a 1,000-fold inhibition of
HIV-1 replication after further challenge with the virus.
A hairpin ribozyme has been shown to have similar effects to the
hammerhead ribozyme: cells expressing the ribozyme were resistant to
various strains of HIV-1. It was also demonstrated that the hairpin
ribozyme significantly reduced the efficiency of the incoming virus to
synthesize viral DNA.97 A hairpin ribozyme is in the preliminary stages of a phase I clinical trial against
HIV-1.99 CD4(+) lymphocytes from
HIV-1-infected donors were transduced with ribozyme or control vectors
and viral replication was delayed by 2 to 3 weeks.80 The
rapid progress of ribozymes to early clinical trials can be attributed
to their specificity and ability to be delivered by viral vectors to
the target cells. Another reason ribozymes could be successful against
retroviruses is that they can potentially target several stages in the
viral life cycle: the incoming genomic RNA, the mRNA transcribed from
the integrated genome, and the progeny RNA genomes.
HIV-1 is not the only virus being targeted. Beck and
Nassal82 are targeting a hammerhead ribozyme to the
encapsidation signal  of Hepatitis-B virus, the causative agent of
B-type hepatitis. Unfortunately, ribozyme cleavage has only been
observed in vitro and in cell extracts, but not in intact cells. Even
when the ribozyme was placed in cis with an artificial
substrate, little ribozyme activity was observed (most of the activity
could be attributed to antisense effects). The investigators speculated
that there was something inhibiting cleavage within the cell, because
efficient cleavage was observed upon RNA extraction. Hepatitis-C virus
RNA is also the subject of selective targeting and destruction by ribozymes,83,100,101 as is the mRNA for influenza A
virus.102
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RIBOZYMES IN ANIMAL MODEL SYSTEMS |
Some researchers are beginning to assay ribozyme effects in animal
model systems.77,84,103 We have been investigating the action of an ex vivo purging of a murine cell line containing the
bcr-abl oncogene with ribozymes against the bcr-abl
mRNA before injection into SCID mice. Effectiveness of the ribozyme is
demonstrated by the increased survival of the mice (when compared with
mice injected with control-treated bcr-abl-containing
cells).77
Flory et al84 have used a rabbit model of
interleukin-1-induced arthritis to assess the localization, stability,
and efficacy of exogenous anti-stromelysin hammerhead ribozymes. They
observed that exogenously delivered ribozymes were taken up by cells in the synovial lining and synovial interleukin-1-induced stromelysin mRNA levels were reduced. Catalytically inactive ribozymes were ineffective supporting a cleavage mechanism for ribozyme activity. Using nude mice, Czubayko et al78 have rather nicely
demonstrated the relationship between the secreted growth factor
pleiotrophin (PTN) from melanoma cells and the melanoma cells'
metastasis to the lungs. Introducing ribozymes against PTN into a cell
line, which reduced PTN mRNA and growth factor activity, concomitantly prevented the metastatic spread of the tumors. Larsson et
al103 looked at hammerhead ribozymes against the
2-microglobulin (2M) mRNA in both cell lines and transgenic mice.
They observed ribozyme expression in lung, kidney and spleen with
greatest reduction in the 2M mRNA levels in the lung. However, it
should be noted that ribozyme levels and reduction in 2M mRNA varied
widely between litter mates. Lieber and Kay104 have looked
at ribozymes (against human growth hormone) expressed from adenovirus
vectors after somatic gene transfer into transgenic mice. A reduction
of up to 96% in growth hormone was observed in correlation with the mRNA levels. Using transgenic mice is obviously quite different from
using a retroviral vector to express a ribozyme. Although inappropriate
for therapeutic administration, transgenic animals can provide
important information about ribozyme expression levels in different
tissues and ribozyme efficacy, as well as information about the role of
the targeted mRNA.
Very little has been done so far with respect to pharmacokinetic and
pharmacodynamic data for ribozymes in animal model systems. However,
one study of pharmacokinetic properties of synthetic, chemically
modified hammerhead ribozymes after intravenous
injections105 has shown the prolonged presence of a
cytochrome P-450 ribozyme in the plasma (up to 48 hours postinjection)
and perfusion into tissues other than the vascular system (kidney and
liver). This group has also injected a similarly modified ribozyme to
amelogenin mRNA, with no carrier to assist cellular uptake, and shown a
surprisingly successful knock-out of the targeted gene's
expression.106 The various works detailing transgenic
animals with a gene construct for an antisense RNA or ribozyme sequence
has recently been reviewed.107
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OTHER USES OF RIBOZYMES |
As well as their potential as therapeutic agents, ribozymes can be used
to generate loss-of-function phenotypes to elucidate the roles of
genes. This has been performed for the Fushi taragu gene in
Drosophila,108 for c-fos,109 and for
matrix metalloproteinase 2.110
Ribozymes have also been used in other systems to varying degrees of
success: in plant protoplasts111,112 and in yeast
(Saccharomyces cerevisiae113 and S
pombe114). Transgenic plants with ribozymes to the
lignin-forming peroxidase of tobacco have been created, with reduction
in the peroxidase mRNA and protein levels observed.115
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THE DESIGN OF RIBOZYMES AND PROBLEMS TO OVERCOME |
(For assistance in designing and applying ribozyme technology please
refer to two recently published methods books: Ribozyme Protocols116 and Antisense and Ribozyme
Methodology.117)
As detailed above, ribozymes have been successfully used to target
specific mRNAs. To use a ribozyme strategy, several parameters must be
decided. The first choice is the type of ribozyme to use. The
hammerhead ribozyme is the best-studied type. The target sequence requirements for the hammerhead are slightly less stringent than for
the hairpin, and, because the hammerhead is smaller than the hairpin,
it is cheaper to synthesize and higher yields can be obtained. The next
decision is the choice of target mRNA. It has been suggested that
targeting the mutated gene sequence may not always be the most
effective approach. Targeting an mRNA of a protein downstream of the
mutated gene product may result in a more effective inhibition.
However, this could lead to a decrease in specificity between normal
and affected cells. The position of the NUH target site within the mRNA
must then be selected. The efficiency of the ribozyme is dictated by
the sequence (different NUH sequences can vary by >100-fold in the
rates of their cleavage, and the sequence context of the site is also
an important factor118) and accessibility of target site
due to secondary and tertiary structures of the mRNA.119
Obviously, before cleavage can occur, the ribozyme must bind to its
substrate via the sequences either side of the target site.
Experimental analysis such as nuclease mapping or chemical probing can
be performed to monitor accessibility.120 Alternatively
computer RNA folding programs can be used to help determine accessible
sites121,122 with less expense but at a reduced
reliability.
Most computational algorithms for RNA structure prediction are based on
calculation of the minimum free energy ( G). For a stretch of bases
the program determines the most stable structure, the lowest G. The
computer then looks at the next stretch of bases (which may or may not
overlap with the previous stretch) and calculate the next G. In this
way, local maxima and minima G values are obtained that correspond
to relatively unstable or stable regions of the RNA, respectively. The
value of G is dependent on which algorithm is used to determine the
structure, and this is where the errors can be introduced. A minima in
G does not necessarily mean that a stable structure exists in vitro or in vivo. Current programs are also limited to the length of the
sequence to be analyzed; distant interactions in a long mRNA cannot be
predicted.
The length of the ribozyme's hybridizing arms (which basepair with the
substrate) must strike a balance between providing specificity for the
substrate while allowing product dissociation.123,124 It
has been suggested that asymmetric arms may be more effective than
symmetric arms.125 It is also important to realize that results suggesting an efficient arm length in a cell-free system may
not be ideal in a cellular system.126,127 An increasing
number of laboratories are applying in vitro selection, or in vitro
evolution, techniques (SELEX) to develop more efficient ribozymes and
ribozymes with new activities.128,129 The principal of the
technique is based on the screening by selection of a pool of RNA
molecules. Starting with either a totally or partially random pool of
RNA molecules, a cycling of binding or activity, partitioning, and amplification steps results in a novel ribozyme or ribozymes with the
desired binding or catalytic activity. Using this technique, and by
chance, both RNA and antisense DNA molecules have been developed that
are aptameric in activity, ie, they can interact directly
with the target protein in a sequence-specific
manner.130-132
Many groups are looking at ways to improve ribozyme catalytic activity.
One way to do this is to use multiunit ribozymes (ie, transcripts with
more than one catalytic domain) targeting different sites within the
same mRNA. Chen et al133 developed a nona-ribozyme against
HIV-1 and showed that this was more effective at cleaving substrate RNA
in vitro and inhibiting HIV-1 replication in cell lines than
mono-ribozymes. This multimeric ribozyme has a second advantage, ie,
HIV-1 escape mutants are less likely. A multiunit anti-BCR-ABL
ribozyme (3 catalytic domains) was shown to be more effective than the
three ribozyme domains added separately, both in vitro and in cell
lines, by reducing the BCR/ABL mRNA.134
Efforts to increase exogenously delivered ribozymes' stability and
hence activity have concentrated on chemical modifications to the
ribozyme structure. 2-fluoro or 2-amino135
and 2-O-allyl and 2-O-methyl106,136 are a few
of the modifications being investigated.
Ribozymes can be delivered to cells in two ways: as preformed ribozymes
(exogenous delivery) or as ribozyme genes, a method of endogenous
delivery. The means of transfection (eg, lipofection or
electroporation) is important for the former, whereas the choice of
promoter (pol II, pol III, viral, etc) is important for the latter.137 The latter could be termed ribozyme gene
therapy, whereby the introduced ribozymes downregulate the targeted
gene expression or repair mutant mRNAs.138
Endogenous delivery has been achieved by inserting ribozyme sequences
into the untranslated regions (UTRs) of genes transcribed by RNA
polymerase II (pol II), such as the SV40 early promoter85 or the actin gene.139 The RNA polymerase III (pol III)
promoters from the U6 small nuclear RNA140 or from certain
tRNAs have also been successfully used to express ribozymes and achieve
effects.141 Tissue-specific promoters, such as the
tyrosinase promoter, have also been investigated.73
Both retroviral-derived and more recently adeno-associated viral
(AAV)-derived vectors are commonly used. Ribozyme genes can be
expressed from the viral long terminal repeat (LTR) promoters or from
introduced pol II or pol III promoters. Retroviral vectors are
relatively efficient, safe, and capable of (randomly) integrating stably in the host genome of replicating cells. AAV is nonpathogenic and has the advantage of integrating into a defined region of the host
genome without requiring cell division. Zhou et al98 compared three types of promoters: Molony murine leukemia virus (MoMuLV) LTR, the human CMV promoter, and a human tRNAmet
cassette for the transcriptional control of a pair of anti-HIV-1 ribozymes. The LTR promoter produced the highest expression levels of
the ribozyme, but the ability of each to confer resistance to HIV-1
replication was very similar. The investigators concluded that other
factors (antisense effects, protein influence, and localization) than
the absolute levels of the ribozymes played a role in determining the
effectiveness of the ribozymes. The recent work of Bertrand et
al137 shows that colocalization is the most important
factor. In this study, promoters used for the expression cassettes were
derived from the human tRNAmeti (pol III), the human U1
snRNA (pol II), the human U6 snRNA (pol III), and the Rous sarcoma
virus (RSV) LTR (pol II). Each cassette was introduced into a cell line
with both AAV- and MoMuLV-based vectors. All of the cassettes produced
ribozymes when in context of the AAV vector, whereas the tRNA and U6
cassettes were inactive with the viral vector. Anti-HIV ribozymes
derived from the RSV cassette were expressed at the lowest levels and
were cytoplasmic (consistant with being capped and polyadenylated). The
other transcripts were predominantly nuclear and expressed at higher
levels. When the cells were challenged with HIV, surprisingly only
those containing the capped, poly-A RNAs that were cytoplasmically
localized were able to suppress HIV replication.
Sullenger and Cech142 have successfully looked at using a
viral packaging signal to direct their ribozymes to the same
subcellular locations as the viral targets. It has also been observed
that the 3UTRs of some mRNAs carry the signal responsible for
localization.143,144 These, and others yet to be
discovered, could be attached to ribozyme sequences to aid their
colocalization. In a hope to understand RNA trafficking in the cell, we
have been following ribozyme distribution using fluorescently tagged
ribozymes.145 Because the nucleus is thought by many to be
the site of action for ribozymes, several groups are looking at
ribozyme activity when expressed with a nuclear localization
signal140 or in isolated nuclei.146 It can be
concluded that the type of promoter and its context can determine the
intracellular compartmentalization of the ribozyme and that
colocalizing the ribozyme with the substrate is a primary determinant
of ribozyme efficacy in vivo.
Measurements for assaying ribozyme activity can be made at the mRNA
(RNase protection assays or reverse-ligation polymerase chain
reaction147) or protein levels (Western blotting and
antibody probing), function of the protein (eg, kinase activity) and
effects on cell differentiation, or the onset of apoptosis. In general, ribozyme cleavage activity in the cell system or in vivo has not been
empirically demonstrated, but instead has been inferred from the lack
of effects by a catalytically inactive ribozyme if included as a
control.
Stein and Krieg148 pointed out various considerations for
the interpretation of data derived from the use of antisense
ODNs. Charged ODNs are polyanions and as such can bind and
sequester growth factors to the basement membrane. Phosphorothioate
ODNs may exhibit nonantisense but sequence-dependent effects, eg, a G
quartet149 or a TAT triplet at the 3
end.150 These considerations, although not proven, are
probably just as valid for ribozymes. To show specific ribozyme
cleavage, the following controls are required: a catalytically inactive
ribozyme (to show antisense effects), a conventional antisense ODN
equivalent to the ribozyme arms (again, to show antisense effects), an
active but unrelated ribozyme (to show specificity for target mRNA), a
scrambled ODN, and finally the transfection agent or vector only
(nonspecific toxicity effects).
Ribozymes and antibiotics.
It has been demonstrated that certain classes of antibiotics can
interact with some ribozymes and, in some cases, influence their
cleavage activity. Aminoglycoside antibiotics inhibit group I intron
function but not group II introns.151 The aminoglycosides, in particular neomycin B, have been shown to inhibit hammerhead ribozyme activity,152 as have the
tetracyclins.153 The HDV ribozyme is also inhibited by both
aminoglycosides and tetracyclins.154 Contrary to this, the
Neurospora VS ribozyme cleavage activity is actually enhanced
by viomycin, a tuberactinomycin antibiotic.155 The
modulation of ribozyme activity by other agents, in particular antibiotics, should be taken into consideration when designing experiments with cell cultures (often grown in the presence of antibiotics) and possibly in a clinical setting.
| |
THE FUTURE FOR RIBOZYMES |
For ribozymes to become realistic therapeutic agents several obstacles
need first to be overcome. These obstacles are the efficient delivery
to a high percentage of the cell population, efficient expression of
the ribozyme from a vector or intracellular ribozyme concentration,
colocalization of the ribozyme with the target, specificity of ribozyme
for the desired mRNA, and an enhancement of ribozyme-mediated substrate
turnover.
Despite these reservations, results with ribozymes so far look
promising, particularly in the HIV-1 studies. As our knowledge of RNA
structure, secondary and tertiary, increases, we will be able to target
the RNA more rationally, which may help with the problems of
specificity. At the same time, the understanding of the physical
localization of RNA in cells and its tracking as it moves from the
nucleus to cytoplasm will also help in ensuring colocalization of the
ribozyme and target. Modifications of the ribozymes, eg, the 2
ribose with allyl group, increases the stability to nucleases quite
dramatically. Similarly, DNA sequences allied to the ribozymes in use
in our laboratory increase the stability. Entry into cells with
liposomes or via vectors are also looking hopeful. Catalytic activity
is maintained but research is still ongoing at tackling the problem of
increasing the number of ribozyme molecules in proportion to the
substrate molecules a requirement for success. These molecules must
retain their catalytic potential, must reach an accessible site in the
substrate, and eventually be synthesized from the appropriate vector
chosen for clinical trials. Work in the antisense DNA field would also
benefit from solutions to these problems.
| |
FOOTNOTES |
Submitted June 3, 1997;
accepted September 11, 1997.
Supported by Grant No. 9667 of the Leukaemia Research Fund, London, UK.
Address reprint requests to Helen A. James, PhD, School of
Biological Sciences, University of East Anglia, Norwich, Norfolk, NR4
7TJ UK.
| |
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Abstract
Introduction
Abstract