REVIEW
Antisense strategies in cell and developmental biology
ALAN COLMAN
School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK
Introduction
The ability of specific nucleotides to base-pair, or
hybridise, is fundamental to the role of nucleic acids in
information transfer. The elucidation of the complementary nature of the two strands of the DNA helix was soon
followed by the in vitro demonstration of DNA: DNA
hybridisation, RNA:DNA hybridisation, and RNA:RNA
hybridisation. Initially hybridisation was employed analytically to monitor particular classes of RNA in cell
extracts, or to deduce the presence of repetitive DNA
sequences in the eucaryotic genome (see Lewin, 1974).
However, in the late 1970s, for the first time, in vitro
hybridisation was used to interfere with the translation of
specific mRNAs by a process known as hybrid arrest of
translation (Paterson et al. 1977). Subsequently, the
availability of cloned genes and synthetic oligonucleotides
has permitted many attempts to interfere with gene
expression in vivo by exploiting the tendency of complementary sequences to hybridise. The principle of the
antisense approach is to inhibit the expression of a specific
gene by the provision of complementary RNA or DNA
sequences that will hybridise to the transcripts of the
gene. In this review I will assess the rationale behind
antisense experiments, the antisense reagents and,
finally, attempt a critical analysis of the successes and
failures of this fashionable technique, of which it might be
fairly said that it has promised more than it has delivered.
I will confine most of my analysis to experiments that use
antisense strategies to understand the contribution of a
gene to cellular and developmental processes. I will not
review the burgeoning applications of antisense reagents
for therapeutic purposes in man or commercial purposes in
plants. For reviews that incorporate these developments
the reader is directed to Krol et al. (1988a), Cohen (1990)
and Rossi and Sarver (1990).
The rationale
The ultimate objective of most antisense strategies is to
disrupt either gene expression or, in certain cases (e.g.
virus infection), gene replication. As an approach it has
attracted particular attention in eucaryotic studies where
this objective cannot easily be satisfied by the provision of
appropriate mutants. In effect it provides a 'reverse
genetic' approach to understanding gene function and in
this context can be viewed against several other approaches such as ectopic expression (Kintner, 1988;
McMahon and Moon, 1989), homologous recombination
(Rossant and Joyner, 1989) and overexpression (Driever
and Nusslein-Volhard, 1988), the last of which shares with
Journal of Cell Science 97, 399-409 (1990)
Printed in Great Britain © The Company of Biologists Limited 1990
antisense the possibility of generating a dominantnegative control of gene expression. Of these techniques
there is no doubt that homologous recombination offers
the most precise and effective extinction of gene activity
(Thomas and Capecchi, 1990); however, it is still of
extremely restricted application. Antisense offers a more
broadly available approach and has the advantage over
homologous recombination that the timing of gene
inactivation can be manipulated to some extent by, for
example, the choice of promotor in the case of antisense
RNA produced in vivo, or the timing of injection or
external application in the case of synthetic RNA and
oligonucleotides.
The tools of the trade
Three different classes of antisense reagent are currently
in use: (1) antisense oligodeoxynucleotides are short
(usually less than 30) nucleotide sequences designed to be
complementary to a specific intracellular target RNA.
Since they are synthesised chemically, they are either
injected into cells or are applied externally. Consequently,
under most circumstances their effects will be transient.
(2) Antisense RNAs can be synthesised in vitro and
injected into cells, but the more common production route
is through the introduction of the encoding, antisense
DNA gene into cultured cells or germ lines. Depending on
the promotor chosen to drive the expression, antisense
RNA production can be transient or constitutive. (3)
Ribozymes are antisense RNAs that also have enzyme
activity and can cleave RNAs at preselected sites. In all
cases reagent specificity and efficacy will depend on
intracellular access to the target RNA, secondary structure of both reagent and target, uniqueness of target
sequence within the cell, strength of binding, mode of
inhibition, nuclease resistance and, in the case of
oligonucleotides, ability to enter the cell. Inevitably the
nucleotide sequence selected for the antisense reagent is
at best a compromise, that most satisfies those parameters
about which something is known.
Antisense oligodeoxynucleotides (ODNs)
The first successful demonstration of an antisense strategy
in vivo was the inhibition of Rous sarcoma virus
proliferation in cell culture by the addition of synthetic
ODNs to the culture medium (Zamecnik and Stephenson,
1978); these ODNs contained an unmodified phosphodiester backbone (Fig. 1). Subsequent studies showed that
cultured cells could be protected to varying extents from
infection from influenza virus (Zerial et al. 1987), vesicular
stomatitis virus (Agris et al. 1986), herpes simplex virus
399
-O
phosphodiester ( P - 0 )
- CH 3 methyl phosphonale ( P-CH3 )
- S~
phosphorolhioate (P-S)
x
— P
||
o
CH 2
I
Fig. 1. Oligodeoxynucleotides (ODNs) and common analogues.
(Smith et al. 1986) and human immunodeficiency virus
(Matsukura et al. 1987). One immediate problem, which
was recognised early into these studies, was that unmodified ODNs were often (but not always, see Holt et al. 1988)
degraded by nucleases present in the culture medium
(Wickstrom, 1986), thus necessitating their addition at
concentrations up to 100 jUM. This factor combined with a
prevalent belief that the negative charge on unmodified,
phosphodiester (P-O)-based ODNs compromised penetration through the plasma membrane, quickly led to a
search for effective derivatives that would be more stable
in vivo and more lipid-soluble. The first generation of such
derivatives include ODNs with methyl phosphonate
(P-CH 3 ) or phosphorothioate (P-S) backbones (Fig. 1).
Comparisons where the same sequence was represented by
all three types of ODN indicated that P-S ODNs were
effective at much lower concentrations than the others and
that 15-mers seemed the ideal size (Marcus-Sekura et al.
1987). Subsequent development has focussed on the
chemical modification of ODNs with a view to increasing
their reactivity (for detailed review, see Cohen, 1990).
Such modifications include the addition of the crosslinking moiety, psoralen (Kean et al. 1989), alkylating
moieties (Knorre et al. 1985), metal complexes that cleave
both RNA and DNA (Boutorin et al. 1984), acridine rings,
which increase the affinity of ODNs for RNA (Toulme et al.
1986), and various adducts designed to encourage membrane penetration (e.g. polylysine: Lemaitre et al. 1987;
cholesterol: Letsinger et al. 1989). By and large no clear
generalisations as to the design and use of ODNs have
emerged from cultured cell studies. To a large extent this
is due to shortcomings in this type of experimental system,
and from concern about the specificity of ODN hybridisation.
The effects of P - 0 ODNs on the activities of cultured
cells was unexpected because their entry into such cells
was not predicted. It is still not clear how cell entry occurs.
Loke et al. (1989) used several fluorescent, acridinelabeled ODN-dT n s to visualise and study the entry
characteristics of ODN entry into human HL60 myeloid
cells. They concluded that the compounds were taken up
by cells in a manner compatible with receptor-mediated
endocytosis and, using affinity chromatography to
ODN-dT cellulose, were able to identify a 80xl0 3 Mr
surface protein that may mediate transport. However,
they did not show that the acridine associated with the
cells was still covalently attached to the ODN, and a
400
A. Colman
diagnostic test for a receptor-mediated event, the removal
of ligand bound at 4°C using proteolytic enzymes, was not
reported. Similar criticism can be made of the data of
Yakubov et al. (1989), who performed binding and uptake
experiments on mouse fibroblasts with P—0 ODNs, some
of which contained alkylating groups and were used to
cross-link ODNs to putative receptor proteins. In fact
there are very few direct data attesting to the ability of an
externally applied ODN to find its target within the cell,
since diagnostic duplexes or reaction products (see below)
are rarely reported. The most convincing demonstration of
the intracellular targeting of an ODN is that of Holt et al.
(1988), who provided nuclease-protection evidence for the
presence of a duplex between c-myc RNA and a complementary ODN in HL60 cells. Notwithstanding our
ignorance of the entry route, the fact that the plasma
membrane presents a hurdle has complicated comparisons
of the relative efficacies of different ODNs. Most information concerning the possible modes of action of ODNs
comes from in vitro studies and in vivo microinjection of
Xenopus oocytes.
Paterson et al. (1977) were the first to use complementary polynucleotides, in this case cDNA, to interfere with
mRNA translation in the wheat germ cell-free system.
However the technique did not work well in the reticulocyte lysate. Minshull and Hunt (1987) conjectured and
subsequently showed that this was because the wheat
germ extract was particularly rich in an RNase H activity
that recognises DNA: RNA duplexes and digests the RNA
partner. It has been convincingly shown that with P - 0
ODNs, the principle effect of an ODN on translation both
in vitro and in Xenopus oocytes is a consequence of RNase
H-mediated cleavage of the RNA (Fig. 2A). Thus Walder
and Walder (1988) showed that in the presence of this
enzyme, ODNs directed at various parts of the coding and
upstream parts of mouse globin mRNA were equally
effective at inhibiting translation; however, when the
enzyme was inhibited by the addition of a competitor
RNA: DNA hybrid [poly(dT):(rA)], only ODNs directed to
the 5' cap sequence worked, presumably through steric
interference with ribosome recruitment. Likewise, in
oocytes several studies have shown that a variety of
mRNAs, both endogenous (calmodulin: Dash et al. 1987;
histone: Shuttleworth and Colman, 1988; heat shock:
Shuttleworth and Colman, 1988; cyclins: Colman, Hunt
and Minshull, unpublished; Vgl: Shuttleworth and Colman, 1988; Woolf et al. 1990) and exogenous (interleukin 2:
Kawasaki, 1985; globin: Cazenave et al. 1987a) are
specifically cleaved only by complementary ODNs,
although the degree of cleavage varies in an unpredictable
way from ODN to ODN even on the same mRNA
(Shuttleworth et al. 1988). Fortunately the efficacy of a
given ODN in an in vitro assay seems to be a good indicator
of its behaviour in oocytes (Baker et al. 1990). In most of
these studies extremely high molar ratios of ODN to
mRNA were required. This reflects the slow reaction
kinetics and rapid degradation of the ODNs in oocytes
(«j<20min, Cazenave et al. 19876; Woolf et al. 1990). It is
not clear whether the rate-limiting step in cleavage is the
formation of perfect hybrids or the RNase H reaction. It
seems that RNase H activity has a widespread distribution amongst eucaryotic cell types (Wagner and Nishikura, 1988), presumably due to its involvement in DNA
replication, so that this mode of RNA ablation may also
occur in cultured cells, although there is no direct evidence
for this. It certainly occurs in the unicellular trypanosomes, where Tschudi and Ullu (1990) have recently
shown that various small nuclear RNAs are cleaved in an
RNase H-like manner after the exposure of lysolecithinpermeabilised cells to specific P - 0 ODNs.
P-S ODNs can also mediate RNase H-like cleavage but
P-CH 3 ODNs cannot (Walder and Walder, 1988), so that
the observed specific effects of these latter ODNs on
mRNA translation (Murakami et al. 1985) and viral
growth (Agrawal et al. 1989) are attributable to other
causes such as steric interference with splice excision or
ribosome binding. Both these ODN modifications increase
their nuclease resistance and this is the main reason why
RNA cleavage in oocytes can be achieved with much lower
amounts of P-S ODNs (Cazenave et al. 1989; Baker et al.
1990; Woolf et al. 1990). Interestingly, mixed ODNs
containing phosphodiester and methyl phosphonate linkA. RNA cleavage by oligodeoxynucleotides
(ODNs)
1
ages can mediate RNase H cleavage and might combine
stability with efficacy (Agrawal et al. 1990).
A major consideration in the design and use of any
antisense reagent is the specificity of its action. This in
turn will be a function of the specificity of hybridisation.
This will be enhanced if the target sequence is unique
within the cell. Computations on the likelihood of a
random sequence of given length reoccurring more than
once in the RNA complement of a cell are necessarily
inaccurate but values of 11—15 (Helene, personal communication) or 12 (Miller and Tso, 1987) consecutive
nucleotides as defining a unique sequence have been
quoted, even though 'random' is an uncomfortable concept
in this area (Agrawal et al. 1990). Longer ODNs will form
more stable hybrids; however, increasing length brings
B. Inhibition by antisense RNA
mRNA/pre mRNA
ODN
o
n n
CXJ
"Anti-sense RNA
RIBOSOMES
PROTEIN
PROTEIN
C. Xenopus unwindase activity
D. RNA cleavage by ribozymes
mRNA
Antisense RNA
cleavage site
T
hybridisation
lj
RIBOZYME
covalent moditicalion (A—•-!)
unwinding
A
f
PROTEIN
PROTEIN
Fig. 2. Mechanisms of antisense regulation in eucaryotes. Possible ways by which the various antisense reagents cause inhibition
of gene expression are shown. The asterisks in C indicate the enzymic modification of adenines (A) to inosines (I).
Antisense strategies in cell and developmental biology
401
with it potential self-foldback problems and increasing
permutations of shorter stretches of consecutive nucleotides that might facilitate hybridisation to other RNAs,
especially as relatively short regions of duplex can serve as
RNase H templates (4-mer in vitro: Donis-Keller, 1979; 10mer in oocytes: Shuttleworth et al. 1988). Surprisingly,
cells can show remarkable discrimination between perfect
and mismatched hybrids. Wang et al. (1985) synthesised
64 ODNs (14-mers), based on a short tumour necrosis
factor (TNF) protein fragment, and coinjected pools of
these ODNs in Xenopus oocytes along with a poly(A)+
preparation that contained TNF mRNA. ODN pools
capable of inhibiting TNF translation were further
subdivided until one ODN, with the sequence GCTACAGGCTTGTC, was identified and used in the successful
screening for a TNF cDNA. In this process of ODN
elimination, GCTACAGGCTTGTC was clearly distinguished from GCCACAGGCTTGTC. Unfortunately,
very few details were given of the effects of the various
ODNs on the TNF assay so that the quantitative
consequences of mismatch recognition were not revealed.
In another study also based on a biological assay, Holt et
al. (1988) found that small degrees of mismatch could
completely compromise ODN action when the mismatched
base pairs were appropriately sited. They found that a twobase mismatch (at positions 6 and 10 in the ODN) between
a 15-mer ODN and c-myc mRNA was sufficient to prevent
an elongation of HL60 cell doubling times. Neither of
these studies rule out some inhibition due to regions of
partial homology. In fact, an indication that ODNs can
cleave mRNAs at secondary sites was reported by
Casenave et al. (1987a), who showed that in vitro
treatment of rabbit fi globin mRNA with a 17-mer that had
perfect complementarity to one part of the mRNA
sequence generated two overlapping fragments in a ratio
of about 4:1. Although the minor fragment could have
been a post-cleavage degradation product, its size was
consistent with ODN-mediated cleavage at a site in the
mRNA to which the ODN had partial (13/17 with 7
contiguous base pairs) complementarity.
Overall, results with ODNs have been quite encouraging in terms of the magnitude of the inhibition (Krol et al.
1988a). However, some of the most impressive results have
relied on bioassays and concern viral infectivity and
growth. The circumstances accompanying viral replication are rather unusual and successes in this area are
thought by many to be a rather inadequate guide to the
general efficacy of the technology. The short size of ODNs
and the irreversible nature of the cleavage reaction that
certain ODNs ( P - 0 and P-S) mediate make the issue of
specificity particularly acute. We are forced to the
conclusion that, however judicious is the choice of ODN
sequence, it is impossible to exclude the gratuitous
cleavage of unknown mRNAs through partial hybrid
formation, and this could complicate interpretation. In
addition, very high ODN concentrations seem to cause
completely non-specific RNA cleavage (Cazenave et al.
1987a), and in some circumstances ODNs can directly
inhibit the activity of specific enzymes in a process not
involving hybridisation (Matsukura et al. 1987). In view of
all these potential problems the only rigorous way of
definitively attributing a function to an mRNA by these
methods, would be to show that the effects of ODN
ablation are reversed by the reintroduction of fresh target
mRNA. Failing this, evidence should be produced showing
that more than one ODN directed against the same target
mRNA is capable of the same biological effect, since it is
402
A. Colman
most unlikely that ODNs of radically different sequence
would have similar secondary targets (see, for example,
Minshull et al. 1989).
Antisense RNAs
Antisense RNAs have been known for some time to occur
naturally in prokaryotic cells where they can interfere
with gene expression at the levels of transcription,
translation and DNA replication (Green et al. 1986;
Simons and Kleckner, 1988). Specific antisense RNAs
have also been found in several eucaryotic systems,
including Drosophila (Spencer et al. 1982), mouse (Farnham et al. 1985; Nepveu and Marcu, 1986) and barley
(Rogers, 1988). The function of these sequences remains
obscure. The first artificial use of antisense RNAs in
eucaryotes was reported by Izant and Weintraub (1984,
1985), who described the coinjection of cultured mouse L
cells with plasmids containing the herpes simplex thymidine kinase gene (HSV-TK) in a sense or antisense
orientation with respect to the TK promotor. Using
[3H]thymidine incorporation and autoradiography of
injected cells, they achieved four- to fivefold suppression of
TK activity with the antisense construct. As a test for
specificity, they also showed that under the same circumstances a chicken TK activity remained unaffected.
Subsequently antisense RNA-mediated inhibition of gene
activity has been demonstrated in stably transformed cell
lines and after microinjection of cells with synthetic
antisense RNAs (see below).
How do antisense RNAs work? Possible mechanisms are
shown in Fig. 2B. Early experiments by Melton (1985) and
Harland and Weintraub (1985) using microinjected Xenopus oocytes demonstrated that antisense RNAs can work
by interfering with mRNA translation. Both these studies
were performed with antisense RNAs directed against
non-Xenopus transcripts. Wormington (1986) showed that
oocyte endogenous mRNAs were also susceptible and that
a 98 % reduction of the ribosomal protein LI synthesis was
obtained after antisense RNA injection. Significantly,
these studies showed that stable duplexes were formed
between antisense and sense RNAs and that antisense
RNAs directed to the 3' portion of transcripts could be
effective. In all cases a large antisense/sense ratio was
needed for effective action. Translational interference has
also been demonstrated using stably integrated antisense
genes. Ch'ng et al. (1989) used retroviral-mediated gene
transfer to introduce antisense sequences directed against
creatine kinase B (CK-B) into human U937 lymphoma
cells. They found that splicing, 3' end processing and
export of sense RNA from the nucleus, were unaffected by
the production of approximately equimolar amounts of
antisense RNA, despite the fact that the antisense RNA
was complementary to parts of the last coding exon,
adjacent intron, and 3' flanking sequences. Export of the
antisense RNA into the cytoplasm was less efficient than
that of sense RNA (70% versus 95%, respectively).
Nevertheless, a 40 % reduction of creatine kinase activity
was obtained and further studies indicated that an
antisense region complementary to the last 50 bases of
coding sequence and 160 bases of non-coding region were
responsible for the translational suppression of CK-B
production.
The results presented above are most easily accommodated into a model where duplex formation between
antisense and sense RNAs interferes with either the
binding or the translocation of ribosomes on the mRNA.
Whilst there has been much conjecture that antisense
transcripts can interfere with transcription or posttranscriptional processing, in most antisense experiments
the mode of antisense inhibition remains obscure. Often
antisense transcripts are extremely difficult to detect and,
even where seen, in only three examples were duplexes
reported (Kim and Wold, 1985; de Benedetti et al. 1987;
Yokoyama and Imamoto, 1987). In one case the duplexes
were only seen in the nucleus, leading Kim and Wold
(1985) to speculate than the 80-90% reduction in TK
activity they obtained in antisense-HSV-TK transformed
mouse L cells, was primarily due to the formation of RNA
duplexes in the nucleus, which acted to trap sense RNA
there, since, unusually in these experiments, they found a
300-fold excess of antisense over sense transcripts.
Yokoyama and Imamoto (1987) also detected RNA duplex
formation in their study of the transformation of the
human promyeloleukaemic HL60 cells with an antisense
c-myc gene. The transformed lines showed an increased
commitment toward monocyte differentiation. Nuclear
run-off experiments showed that c-myc gene transcription
was reduced in the antisense clones; however, since
monocyte differentiation is associated with down regulation of c-myc expression, the effect on transcription could
be a secondary event. Attempts to demonstrate antisense
interference directly in vivo at the level of RNA processing
(as can occur in vitro, Munroe, 1988) have been likewise
unsuccessful (Chang and Stoltzfus, 1985).
The dearth of information on the way antisense RNAs
work obviously undermines a rational approach to reagent
design and, as with ODN strategies, this problem is
compounded by ignorance of secondary structures (of sense
and antisense RNAs) and regions coated in protein. These
concerns might seem esoteric if antisense strategies
generally worked well, but this is not the case. A survey of
17 reports for which quantitative data were available
indicated a degree of inhibition ranging from 0 to 99 %
(Krol et al. 1988a). The best results have been obtained
where either multiple copies of the antisense gene have
been incorporated into a cell or where a strong promotor
has been used to drive antisense transcription. The
simplest explanation of these findings is that a high
antisense to sense transcript ratio is important; however,
as indicated above, this is not always the case and is
difficult to prove anyway. Often a failure to obtain
inhibition is either not reported or not very informative.
There is one case, however, where the absence of
inhibition proved the starting point of a novel and exciting
discovery. On investigating the failure of injected antisense RNAs to inhibit expression from specific mRNAs in
Xenopus embryos, two groups (Rebagliati and Melton,
1987; Bass and Weintraub, 1987) independently discovered that injected RNA duplexes were unwound by an
'unwindase' activity present in the cytoplasm of the
embryonic cells. Apparently the activity is segregated in
the nucleus of the oocyte, is released into the cytoplasm
when the nucleus breaks down during maturation, and
finally returns to a nuclear location at the gastrula stage
of development. It was shown that this enzyme modifies as
many as 50 % of adenine (A) residues in the duplex to
inosine (I), and this destabilises the duplex (Bass and
Weintraub, 1988). Since inosine base pairs with guanosine, an inevitable consequence of this modification would
be the corruption of translational reading frames in any
coding region involved in the duplex structure (Fig. 2C).
Recently, Kimelman and Kirschner (1989) found a
naturally occurring antisense RNA in Xenopus oocytes
that is complementary to a 900 bp region of basic fibroblast
growth factor (bFGF) mRNA, which is also present.
Surprisingly this 'antisense' RNA has an open reading
frame encoding a 25xlO 3 M r protein. It appears that all
the bFGF mRNA is in hybrid form, since on maturation of
the oocyte all the bFGF mRNA molecules were shown to
become modified by the unwindase that is released into the
cytoplasm by the events of maturation. This unwindase
activity is now believed to be of widespread occurrence in
eucaryotic cells (Wagner and Nishikura, 1988), but
presumably is absent from the cytoplasm of mouse
(Bevilacqua et al. 1988) and Drosophila (see below)
embryos where antisense RNA-mediated inhibition has
been shown to occur. The activity remains an enigma. If it
is present and active in the cell nucleus of most cells, one
might expect it to interfere with various processing
events, e.g. splicing, that involve the transient formation
of RNA duplexes. However, experiments involving the
splicing of extensively folded pre-mRNAs in vitro and in
vivo led Solnick and Lee (1987) to suggest that nuclear
proteins present in vivo can melt RNA duplexes. Ironically
it would now seem that the failure to detect hybrids in the
original Xenopus experiments cannot explain the lack of
inhibition seen, since any A-I modifications should lead to
truncated and/or inactive proteins. A more likely explanation for the failure is that some maternal mRNAs
(though not all, see Wormington, 1986) cannot form
hybrids with injected antisense RNAs.
Ribozymes
The excision of the intron of the large ribosomal subunit
RNA in Tetrahymena thermophila occurs by self splicing
(Kruger et al. 1982). One of the reaction products a 393nucleotide linear RNA called L-19, is a so-called ribozyme.
It can promote transesterification and cleavage of phosphodiester bonds, and can act as a site-specific endonuclease (Zaug et al. 1986). Shortened versions of the intron
are now available and will cleave single-stranded RNA
substrates at a site following a tetranucleotide recognition
motif that is complementary to the 5' end of the ribozyme
(Fig. 3A). Changing the 5' sequence of the ribozyme alters
its specificity; however, mismatched sequences can be
cleaved to some extent, and this type of ribozyme works
optimally only on naked single-stranded RNA regions, at
high temperatures (50 °C).
Several other types of ribozyme are now known and all
have the potential to act catalytically (Fig. 2D). The
RNase P of bacteria is the only naturally occurring transacting ribozyme known (Guerrier-Takada et al. 1983). The
self-cleaving satellite RNA of tobacco ringspot virus, a socalled 'hairpin' ribozyme (Fig. 3B), has been adapted for
transactivity (Tritz and Hampel, 1989). However, the
ribozyme showing the most promise at present is the type
developed by Haseloff and Guerlach (1988) from studies of
the self-cleaving plant viroids and satellite RNAs. These
'hammerhead' ribozymes (Fig. 3© are considerably
smaller than the Tetrahymena-based ribozymes and will
cleave target RNAs after the sequence GUX (X=A, C or U)
although certain other sequences are tolerated (Koizumi et
al. 1988). The specificity of the cleavage is imposed by the
base pairing of the two molecules in the regions flanking
the cleavage site. This region, which provides the basis for
the de novo design of ribozymes, can be 11-18 bases long
and therefore has much greater specificity than the
Tetrahymena-based ribozyme. Recently, Birnsteil and coworkers (Cotten et al. 1989; Cotten, 1990) synthesised two
ribozymes whose hammerhead regions were complementary to sites within the murine U7 small nuclear RNA.
Antisense strategies in cell and developmental biology
403
formed into monkey COS cells and found to cause
inhibition of CAT activity (Cameron and Jennings, 1989).
A . Tetrahymena type
3.NNNNNNCA
X
C.
NNNNNNNN 5 .
C
Hairpin type
\
INNN
3-AU
e
GU
NNNNNN
ANNNN
G :g
c
'
*AG
NNNNNN
A
U-A
C-G
i^
GU
CA
AC CA' G
A GA A
C UALJAUGGLI
Fig. 3. Ribozyme structure. The recognition sites in target
(upper) RNAs for three types of ribozyme are shown with the
site of cleavage indicated by an arrow. Putative regions of
secondary structure are shown for the hairpin and
hammerhead ribozymes. The structure shown for the
Tetrahymena ribozyme is only meant to imply that the RNA is
tightly folded.
They then compared the abilities of these reagents to
catalyse the in vitro cleavage of U7 RNA in simple buffer
solution or in a nuclear extract from mouse hybridoma
cells. Both ribozymes cleaved efficiently in the salt
solution under relatively mild (for this type of ribozyme)
conditions (37°C, 5mM Mg 2+ ); however, only one of the
two worked at all in the extract, and then with a
dramatically reduced efficiency (10 -fold). This decrease
was attributed to the interaction of U7 RNA with specific
proteins in the extract to form small nuclear ribonucleoprotein particles (snRNPs). Using the inhibition of U7
snRNP-mediated histone pre-mRNA processing as an
assay, the same workers also compared the relative
efficacies in nuclear extracts of antisense RNAs, ribozymes and ODNs, all of which contained sequences
complementary to a 5' exposed region of the U7 RNA in
the snRNP. The ribozyme was least efficient, needing a
1000-fold molar excess over U7 RNA to achieve the same
degree of inhibition as a 600-fold excess of ODN and a 60fold excess of antisense RNA. However, all these reagents
were unstable, and to differing extents in the extract,
making interpretation difficult. Despite these unfavourable results in vitro, Cotten and Birnsteil (1989) have
managed to demonstrate ribozyme activity in vivo. They
inserted ribozyme-coding sequences near to the anticodon
sequence of a Xenopus tRNAMet gene and expressed this
gene in the Xenopus oocyte nucleus. Injected U7 RNA
transcripts, though not control (EGF receptor) transcripts,
disappeared in oocytes expressing this ribozyme. However,
in vivo as in vitro, the reaction was rather inefficient, with
an estimated 200-fold excess of cytoplasmic ribozyme over
substrate being necessary. Similar inefficiency was shown
in studies where genes encoding ribozymes directed
against chloramphenicol transacetylase (CAT) were trans404
A. Colman
Application of antisense technology to problems
in cell and developmental biology
Production of phenocopies
For the developmental biologist, antisense offers in
principle an approach by which to test the function of
cloned genes that are known to be expressed during
development. This principle has been exemplified in part
by the publication of a number of studies where injected
antisense RNAs were used to produce phenocopies of
known mutants. The first such study by Rosenberg et al.
(1985) showed that injection of fragmented, uncapped
kruppel (Kr) antisense RNA into heterozygous (Kr/Kr+)
Drosophila embryos at the syncitial blastoderm stage
caused lesions in segmentation similar to, though not as
extreme as, those occurring in Kr/Kr mutant embryos.
Similar experiments were performed by Boulay et al.
(1987) and Cabrera et al. (1987) to confirm that particular
cloned cDNAs encoded the protein products whose absence
was responsible for the snail and wingless phenotypes,
respectively.
Phenocopies have also been created using transformation of cells with antisense genes. Crowley et al. (1988)
transformed cells of the slime mould, Dictyostelium
discoideum with antisense discoidin 1 genes. Discoidin 1 is
a developmentally regulated lectin involved in the process
of aggregation of cells during starvation. In transformed
cells (~150 copies of antisense gene), production of
antisense transcripts results in the steady state levels of
sense mRNA decreasing by 90%, with a corresponding
reduction in discoidin 1 protein. As a consequence
(presumably), the cell streaming process, which precedes
aggregation, is disrupted in a similar fashion to that
occurring in discoidin 1-minus mutants. In this study the
discoidin 1 promotor was used to drive the antisense
transcription. This has the advantage that spatial and
temporal regulation of sense and antisense expression
should be identical but could have the disadvantage that
an excess of antisense genes will be required to produce
sufficient antisense RNA. In addition, the possibility
arises that the down-regulation of discoidin 1 expression
could result from intra-nuclear competition between
antisense and sense genes for specific transcription
factors. However, in this case, nuclear run-off assays
indicated that transcription of the endogenous discoidin 1
genes was unaffected.
In the mouse, levels of the mRNA for the myelin basic
protein, MBP, were reduced by up to 80 % in the transgenic
progeny of a mouse containing over 10 copies of a MBP
antisense gene (Katsuki et al. 1988). Some but not all
(10/21) of these individuals were converted from the
normal to the shiverer phenotype. This phenotype, characterised by shivering 2 weeks after birth, was first
described in mice containing a homozygous autosomal
recessive mutation in the MBP gene. A surprising feature
of this transgenic line is the uneven penetrance of the
effect as seen by phenotypic variation, differences in the
steady-state levels of MBP mRNA and protein, and finally
the mosaic distribution of MBP-containing cells in the
brains of affected individuals. It is not clear whether this
variability can be attributed to the unique genetic make
up of each individual; however, it certainly highlights a
worrying aspects of this approach.
The above studies, which relied upon the availability of
characterised mutants, establish that the antisense approach can generate phenocopies although there is still
some concern in the scientific community about the
specificity of the lesions caused. Antisense has also been
used to create, a priori, mutant phenotypes where genetic
mutants had not previously been described. Knecht and
Loomis (1987) investigated the role of myosin in Dictyostelium discoideum by transforming the cells with a
myosin heavy chain (MHO antisense gene driven by an
actin-6 promotor. Expression from this construct led to
reduced amounts of MHC protein and caused defects in
cytokinesis with the formation of large, multinucleated
cells. Starvation-induced aggregation was also impaired.
Significantly, these effects were overcome when the
endogenous MHC gene expression was boosted by a
change in the food source from liquid culture to bacteria. A
similar, though not identical, phenotype was obtained by
Lozanne and Spudich (1987), who used homologous
recombination to delete partially the MHC A gene; the
resulting mutant produced only a MHC A fragment.
In Drosophila, Qian et al. (1988) found that transformation with an rpAl antisense gene could result in severely
disrupted oogenesis. RpAl is a ribosomal protein and no
mutant rpAl locus has yet been found although mutations
of another ribosomal protein, Rp49, lead to a minute
phenotype. The antisense construct was driven by an
inducible heat shock promotor. At elevated temperature
(37°C), over 80% of the eggs laid by transgenic females
were abnormal, whilst at 18°C, where very little antisense
transcription occurred, the number of defects was greatly
reduced. The severity of the phenotype was dependent on
the time of induction and also the antisense gene dosage
and level of transcription. For reasons that are not
understood, high antisense expression led to decrease in
several unrelated mRNAs, an effect that was not observed
with an analogous control construct containing the D-raf
antisense gene. Although RpAl protein synthesis was not
monitored in this study, the authors reinforce the point
first made by Kim and Wold (1985) that, for certain genes,
a complete loss of function is not necessary for the
emergence of a noticeable phenotype. The use of an
inducible promotor enables the contribution of a gene in a
chosen developmental window to be examined. This
property might prove especially useful where a gene is
required for different purposes at different developmental
stages; an example of this in Drosophila is the decapentaplegic gene product, which is required for dorsal-ventral
pattern formation in the early embryo (Gelbart et al.
1985), for larval viability (Segal and Gelbart, 1985) and
cuticle imaginal disc cell growth (Spencer et al. 1982).
second is that the strong murine sarcoma virus long
terminal repeat (LTR) promotor used in this study has
been shown to only be transiently active in Xenopus
development and like other injected promoters is expressed in a mosaic fashion (R. Harland, personal
communication). A mosaic pattern of expression is
difficult to reconcile with the uniform reduction of 4.1
protein seen throughout the retinal cells of the infected
individuals unless, as suggested by the authors, the
immunological detection methods are quantitatively
insensitive or, alternatively, the effect of 4.1 expression is
a secondary consequence of some earlier (and less
specific?) perturbation in development.
Attempts to interfere with early developmental processes using injected antisense RNAs (see above) or ODNs
have generally proved disappointing. We (Shuttleworth et
al. 1988) and others (Woolf et al. 1990) have attempted to
use ODNs to remove Vgl mRNA from Xenopus oocytes.
This mRNA, which is localised to a vegetal position in
oocytes and embryos, has homology to TGF/31 and may be
involved in mesoderm induction (Weeks and Melton,
1987). However, the amounts of either P - 0 or P-S ODNs
necessary to remove the maternal Vgl mRNA have proved
toxic to subsequent development, for unknown reasons,
although we have speculated that the expansion of the
deoxynucleotide pool as a result of ODN degradation
might be a contributory factor. These toxic effects might be
ODN-sequence specific, since Kloc et al. (1989) showed
recently that they could deplete embryos of the maternal
sequence, Xlgv 7, and still obtain normal development. In
the case of Vgl mRNA it appears that ODNs (Shuttleworth and Colman, 1988; Woolf et al. 1990) but not
antisense RNAs (Rebagliati and Melton, 1987) can gain
access (i.e. form hybrids) to the mRNA. This might be a
consequence of the relative length of these reagents. A
compromise approach might be to use small RNAs
synthesised by the method of Milligan and Uhlenbeck
(1989). These RNAs, if stable for long enough, should
hybridise with Vgl mRNA and therefore present a target
for the unwindase with the resultant corruption of the Vgl
mRNA reading frame. In Xenopus, maternal mRNAs like
Vgl offer a particularly attractive target for injected
antisense reagents because new transcription only becomes significant after the mid-blastula stage of development. Unfortunately even if the mRNA ablation works in
oocytes as we have found with a cyclin B mRNA (Colman,
Hunt and Minshull, unpublished), interpretation is complicated by the prior accumulation of the cyclin B protein
(T. Hunt, personal communication). Full-size oocytes also
contain Vgl protein (Dale et al. 1989; Tannahill and
Melton, 1989).
Gieblehaus et al. (1988) injected Xenopus embryos with
an antisense gene directed against the membrane skeleton
protein 4.1. They observed a very rapid reduction of
endogenous transcripts after the mid-blastula transition
at which time transcription from the injected genes takes
place. They were able to detect the antisense RNA but
could not find any RNA duplexes. The fall in sense
transcript levels was reversed by a second injection of
sense DNA. Injection doses of 100 pg antisense DNA/embryo led to embryonic death at neurulation; however,
smaller doses generated specific retinal defects, leading
the authors to conclude that the observed reduced
expression of the 4.1 protein is sufficient to perturb normal
cellular interactions in the retina. One puzzling aspect of
this work is that some embryos have normal levels of 4.1
protein despite having greatly reduced mRNA levels. A
A final note of caution that has emerged particularly
from Xenopus studies is that it is sometimes easy to
generate apparently specific developmental lesions by the
antisense approach. This point, made forcefully by
Rebagliati and Melton (1987), probably reflects the acute
sensitivity of certain developmental processes to the
slightest peturbation, and highlights the need for carefully designed controls. As stated above the ultimate
control involves mRNA replacement therapy. Unfortunately, this is rarely done and is often not technically
possible anyway. Consequently, a combination of factors the non-specific toxicity, the interdependence of different
regions of the embryo, the variable levels of antisense
expression - all conspire to make the early embryo a
particularly unsuitable target for this technology. These
factors are probably less relevant where cell-autonomous
Antisense strategies in cell and developmental biology
405
events are studied in cell culture, since the scope for
'knock-on' effects is more limited, and variable responses
are submerged under a statistical average.
Cell proliferation
Cell proliferation has proved a particularly useful model
for gauging the utility of the antisense approach. The
contribution of a variety of proto-oncogenes (c-fos:
Nishikura and Murray, 1987; Mercola et al. 1987; c-myc:
Yokoyama and Imamoto, 1987; Holtef al. 1988; Wickstrom
et al. 1988; c-src: Amini et al. 1986; p53: Khochbin and
Lawrence, 1989 and growth factors (bFGF: Becker et al.
1989). The case of c-myc is particularly instructive because
its cellular role has been examined using both antisense
RNAs and ODNs. c-myc gene expression is higher in
proliferating cells than differentiated ones. Gene transfer
experiments indicate that constitutive expression of c-myc
inhibits induced differentiation of certain cell lines.
Differentiation of HL60 cells with antisense P - 0 ODNs
resulted in a reduction of the 49xlO 3 M r c-myc protein and
was associated with a decreased growth rate (Holt et al.
1988; Wickstrom et al. 1988) and increased myeloid
differentiation (Holt et al. 1988). Similar results were
obtained with stable HL60 lines containing a c-myc
antisense gene (Yokoyama and Imamoto, 1987).
Interestingly, natural antisense c-myc RNAs have been
detected in transcripts from murine cells (Nepveu and
Marcus, 1986).
Cell cycle
Inevitably there is an intimate relationship between cell
proliferation and the cell cycle, so it is no surprise that
antisense-mediated interference with the cell cycle will
affect proliferation. Anti-c-wyc ODNs reduce human T
lymphocyte growth by inhibiting entry into S phase from
Gr (Heikkila et al. 1987), whilst c-fos antisense RNA
prevents re-entry of quiescent (Go) mouse 3T3 cells into
the cell cycle (Nishikura and Murray, 1987). A fascinating,
if perplexing, insight into the operation of meiotic and
mitotic cell cycles has come from ODN studies in Xenopus
oocytes and embryos. Stage VI oocytes are arrested in
prophase of meiosis 1. After hormonal stimulation,
meiosis resumes and proceeds until metaphase of meiosis
2 is reached, whereupon a further arrest occurs. This
process is called maturation. A major regulator of the
process is maturation promoting factor (MPF). Recently,
one component of MPF has been shown to be a heterodimer
consisting of cyclin B and p34cdc2, the Xenopus homologue
of the yeast cell cycle control protein, cdc2 (see Gautier et
al. 1990). p34cdc2 is a serine/threonine kinase and in its
inactive state contains phosphorylated serine, threonine
and tyrosine residues. Activation is associated with
dephosphorylation of tyrosine. Although many details of
maturation are poorly understood, it is clear that a cascade
of phosphorylation and dephosphorylation reactions occur.
Sagata et al. (1988) found that ODN-mediated depletion of
the mRNA encoding the p39c'"los proto-oncogene product,
resulted in inhibition of maturation. This result was
interpreted as demonstrating the involvement of p39c"mos
protein, a known protein kinase, in the activation of MPF.
Although correction of this inhibition by a subsequent
injection of c-mos mRNA was not attempted, injection of cmos RNA into untreated oocytes causes them to mature in
the absence of progesterone (Sagata et al. 1989a). This
result reinforces the view that c-mos has a crucial early
role to play in maturation; however, it also shows that the
injection of saturating amounts of c-mos mRNA can
406
A. Colman
overcome the normal repression of c-mos translation in
non-stimulated oocytes. Similar ODN experiments have
been performed in mouse oocytes, where O'Keefe et al.
(1989) showed that the c-mos product was necessary for
initiation of meiosis II, though not for completion of
meiosis I. The Xenopus and mouse results can be
reconciled by the observation that new protein synthesis is
required for completion of meiosis I only in the frog. It is
not known whether c-mos protein is needed for the
initiation of meiosis II in Xenopus; however, it certainly
plays a role in that meiotic division. A most surprising
recent finding is that the c-mos protein is also the frog
cytostatic factor, an elusive activity responsible for
maintaining metaphase II arrest in the matured oocyte
(Sagata et al. 19896). Thus the same protein appears to
have a role in both accelerating and retarding the meiotic
cell cycle.
Recently, we have cloned the cDNA, p40MO15, corresponding to an oocyte mRNA encoding a putative cdc2related protein kinase (Shuttleworth et al. 1990). When
oocytes were injected either of two complementary ODNs,
up to 80 % of the MO15 mRNA was cleaved. A control ODN
complementary to histone H4 had no effect. When the
kinetics of progesterone-induced maturation were monitored using the assay of germinal vesicle (oocyte nucleus)
breakdown or the appearance of histone HI kinase
activity, it was found that MO 15 RNA depletion caused
maturation to occur earlier. This effect was prevented by a
subsequent injection of synthetic MO15 mRNA. We
conclude from these observations that p40 MO15 is involved
in negatively regulating meiosis in Xenopus oocytes.
The Xenopus mitotic cell cycle has also been investigated with antisense ODNs, but this time in an in vitro
extract. Minshull et al. (1989) used ODN-mediated
depletion of mRNA encoding cyclins Bl and B2 to test the
participation of these proteins in mitotic events in
activated egg extracts. They found that whilst depletion of
each mRNA separately had no effect, their joint depletion
prevented entry into mitosis. Interestingly, Xenopus
cyclin B2 has recently been found to be a template, at least
in vitro for the c-mos protein kinase (Roy et al. 1990).
Conclusions and future prospects
I have reviewed the uses of three types of antisense
reagent: ODNs, antisense RNAs and ribozymes. Each has
been demonstrated to work in vivo although complete
target inactivation has proved a pious hope. That this
approach works even inefficiently is remarkable. It is one
thing to anneal complementary sequences in vitro as in
hybrid-arrested translation, and quite another to expect
similar effects with nucleoprotein complexes in vivo. The
choice of reagent is a complex one dictated by considerations of efficacy, purpose and practicality. ODNs offer the
advantage that target mRNAs can be irreversibly cleaved;
however, for most purposes they need to be continually
administered from the outside so that problems of tissue
accessibility, tissue targeting, potential toxicity, and
permeability compromise their general use in wholeorganism studies. Antisense RNAs and ribozymes offer
the great advantage of constitutive or inducible production in the cell or tissue of choice, after integration of
their genes into the genome of the host cell or organism.
However, because it does not mediate RNA cleavage,
antisense RNA does not have the catalytic potential of the
other reagents, and even when its genetic presence is
stabilised and uniform throughout the cells of Gl progeny,
inexplicable differences in expression occur, as most
vividly demonstrated in variegated, transgenic petunias
(Krol et al. 19886). Although this problem might also
afflict transgenic ribozyme genes there is no doubt that
their future development is an exciting proposition. The
successful incorporation of the active ribozyme sequences
into tRNA genes (Cotten and Birnsteil, 1989) offers hope
that RNA instability problems can be overcome via the
sanctuary of tightly folded RNA structures. If the same
principle can be applied to snRNA genes then the nuclear
location of snRNP particles could be exploited to target
ribozymes to unspliced pre-mRNAs (P. Turner, personal
communication). Finally, methods are now evolving that
allow for selection in vitro of ribozymes showing greater
catalytic activity on pre-chosen sites in RNA as well as
DNA (North, 1990) and, significantly, for selection of the
best ribozyme/target site combination for a chosen
substrate. This latter feature could remove some of the
present empiricism in the choice of target sequence.
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(Received 4 July 1990 - Accepted 25 July 1990)
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