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Vol. 58, Issue 2, 380-387, August 2000
Chain Expression through Antisense
Oligonucleotide-Mediated Redirection of Pre-mRNA Splicing
Departments of Molecular and Cellular Pharmacology (J.G.K., R.A.M., B.P.M.) and Pharmacology (N.M.D.), ISIS Pharmaceuticals, Carlsbad, California
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Abstract |
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 Top
 Abstract
 Introduction
Materials and Methods
Results
Discussion
References
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Expression of the interleukin-5 receptor-
(IL-5R) chain is
thought to play an important role in the pathogenesis of asthma and
other eosinophilic diseases. With antisense oligonucleotides (ASOs)
chemically modified to provide increased hybridization affinity for RNA
but that do not support RNase H-mediated cleavage (2'-O-methoxyethyl-modified ASOs), we show that
constitutive splicing of murine IL-5R mRNA can be modulated in cells
such that individual exons may be selectively deleted from mature
transcripts. Specific deletion of individual exons and redirection of
alternative splicing of the IL-5R mRNA have been achieved with this
approach, by targeting 3'-splice sites or exon sequences immediately
downstream of an alternative splice site. ASO targeting with these
strategies resulted in inhibition of mRNA and protein levels of the
membrane IL-5R isoform capable of signaling IL-5-mediated growth and
antiapoptotic signals to eosinophils. Membrane isoform IL-5R
inhibition was coupled with an increase in expression of mRNA for the
alternatively spliced soluble isoform, which binds IL-5 extracellularly
and may block its function. These observations suggest the potential general therapeutic use of an antisense approach to increase expression of variant RNA transcripts and to thereby produce proteins devoid of
specific functional domains that may impact disease processes, as well
as its specific utility for modulating expression of a key cytokine
receptor implicated in allergic inflammation.
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Introduction |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Antisense
oligonucleotides (ASOs) have received increased attention due to their
potential for treating human disease (Crooke, 1992
; Stein and Cheng,
1993; Crooke and Bennett, 1996). DNA-phosphorothioate and "second
generation" chemically modified oligonucleotides have been shown to
provide pharmacological benefit and reduce target gene expression in
vivo (Bennett et al., 1995; Altmann et al., 1996; Monia, 1997; Orr and
Monia, 1998). The chemical modifications incorporated into
antisense design have been driven by the need to increase nuclease
resistance as well as improve hybridization efficiency and
pharmacokinetic profile. Although ASOs entered in clinical trials to
date have been designed to support enzyme-mediated mechanisms of RNA
degradation, continued interest has been directed to the development of
novel mechanisms of oligonucleotide interference with gene expression.
The regulation of signal transduction through cytokine receptors in
vivo is complex and is determined in part by relative expression of
different isoforms that arise from alternative splicing. In the murine
interleukin-5 receptor- (IL-5R) gene, there is evidence that
soluble forms of the receptor arise by exclusion of exon 9, the
transmembrane coding region, or alternatively, of exon 9 and exon 10 (Imamura et al., 1994). These soluble receptor proteins can bind and
neutralize IL-5 bioactivity, presumably by preventing interaction of
the cytokine with the membrane-bound form of the receptor -chain
(Kikuchi et al., 1994; Takatsu et al., 1994). Only the membrane form is
complexed with the common 
-chain, which is required for signaling
and also forms heterodimers with IL-3 and granulocyte-macrophage colony
stimulating factor (GM-CSF) -chains (Hara and Miyajima, 1992; Park
et al., 1992). Thus, expression of the soluble forms of IL-5R may be
beneficial in disease states where IL-5 has been shown to play a
prominent role, such as eosinophilic syndromes and asthma.
It has been previously shown that chemically modified ASOs can induce
correction of aberrantly spliced mutant human -globin constructs
stably expressed in mammalian cells (Sierakowska et al., 1996). To
determine whether selective modulation of endogenous constitutive or
alternative mRNA splicing in cells could be achieved with an ASO
approach, we designed uniformly 2'-O-methoxyethyl (MOE)-modified ASOs to sequences either within the alternatively spliced exon 9 or across various intron-exon boundaries in the mouse
IL-5R gene and tested their activity in the
IL-5R+ mouse BCL1 B cell
lymphoma. The patterns of IL-5R mRNA expression were then examined
with Northern blotting and an RNase protection assay with probes that
distinguish the membrane and soluble forms of the receptor mRNA as well
as individual exons within the gene. With this strategy,
oligonucleotides were discovered that both selectively inhibited
expression of the membrane form of the IL-5R and simultaneously
superinduced transcript levels of the soluble form. Additionally,
specific loss of the immediate downstream exon was observed with
oligonucleotides targeted to several different 3'-splice sites within
the mouse IL-5R pre-mRNA, suggesting that a rational strategy of
targeted exon deletion in mRNA transcripts may be possible with
chemically modified ASOs.
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Materials and Methods |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cell Culture. The B lymphoma cell line BCL1 was purchased from the American Type Culture Collection (Rockville, MD). BCL1 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (Sigma Chemical Co., St. Louis, MO); 10 mM HEPES, pH 7.2; 50 µM 2-mercaptoethanol; 2 mM L-glutamine; 100 U/ml penicillin; and 100 µg/ml streptomycin (Life Technologies, Grand Island, NY).
ASO Synthesis and Cell Transfection.
2'-O-Methoxyethylribose-modified phosphorothioate
oligonucleotides were synthesized on an automated DNA synthesizer
(Applied Biosystems model 380B) as described previously (Monia et al., 1992). Both uniformly 2'-MOE-modified and chimeric
oligonucleotides were used in these studies (see Table 1). The chimeric
oligonucleotides contain 2'-MOE-modified residues flanking a
2'-deoxynucleotide/phosphorothioate region (gap) that supports RNase-H
activation (Monia et al., 1993). Oligonucleotides were analyzed by
capillary gel electrophoresis and judged to be at least 85%
full-length material. BCL1 cells (1 × 107 cells in PBS) were transfected with
oligonucleotides by electroporation at 200 V, 1000 µF with a BTX
Electro Cell Manipulator 600 (Genetronics, San Diego, CA).
Northern Blotting and Probe Design.
Total cellular RNA was
isolated with the RNeasy kit (Qiagen, Santa Clarita, CA). Northern
blotting was performed as previously described (Monia et al., 1992)
with cDNA probes generated from oligonucleotides matching the exon
sequences of either exons 8, 9, or 10. Signals were quantitated with a
Molecular Dynamics PhosphorImager (Sunnyvale, CA).
Riboprobe Design and RNase Protection Assay. RNase protection experiments were conducted with Riboquant kits according to the manufacturer's instructions (Pharmingen, San Diego, CA). A custom riboprobe was designed to protect the mRNA sequence corresponding to the distal half of exon 6, all of exons 7 and 8, and the proximal half of exon 9, and purchased from Pharmingen. Signals were quantitated with a Molecular Dynamics PhosphorImager.
Western Blot Analysis
Western blotting was
performed as described previously (Dean and McKay, 1994). For Western
blotting, membrane-enriched fractions were prepared as Triton X-100
insoluble material (Dean and McKay, 1994) and separated by
SDS-polyacrylamide gel electrophoresis with 8% gels. Antibody to mouse
IL-5R was purchased from Santa Cruz Biotechnology (Santa Cruz, CA)
and used at 1:1000 dilution for Western blotting.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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RNase H-Dependent ASOs Do Not Differentially Inhibit Murine
IL-5R mRNA Membrane and Soluble Isoforms.
Exon 9 of the murine
IL-5R chain represents the only nucleotide sequence unique to the
membrane form transcript and encodes the transmembrane domain (Imamura
et al., 1994). Evidence indicates that two soluble forms of IL-5R
mRNA are produced by alternative splicing events, splicing exon 8 to
exon 10 or 11, respectively (Takaki et al., 1990; Imamura et al., 1994;
Fig. 1A).
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chain, as analyzed
by RNase protection assay (RPA; Fig. 1B; see Fig. 1A for probe
placement). Because the RPA probe does not extend to exon 10 sequence,
only one species of soluble receptor mRNA was observed in RPA analysis of BCL1 RNA. To develop an ASO that specifically
inhibits the membrane-bound form of the receptor, a series of 20mer
2'-MOE ribonucleotide "gapmers" were synthesized, overlapping every
10 nucleotides across the 90-base pair exon (Fig.
2A). These ASOs contain uniform
phosphorothioate backbones and a stretch of 10 2'-deoxy residues in the
center of the molecule that supports RNase H cleavage flanked by five
bases at each of the 5' and 3' ends that are MOE modified and thus
provide greater stability against exonuclease activities and higher
affinity for hybridization to RNA (Monia et al., 1993; Altmann et al.,
1996; Dean and Griffey, 1997). Oligonucleotides containing uniform
2'-MOE modification have previously been shown to be poor substrates
for RNase H-mediated cleavage in cells (Monia et al., 1993). The exon 9 spanning ASOs were electroporated into BCL1 cells
and RPA was used to examine inhibition of membrane and soluble IL-5R
mRNA. As shown in Fig. 2B, several of the oligonucleotides reduced mRNA
levels but none showed specificity for the membrane isoform, suggesting
that the pre-mRNA species is targeted.
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Targeting 3'-Splice Sites with Uniformly 2'-MOE-Modified ASOs
Induces Specific Exon Deletion.
In a second attempt to selectively
inhibit the membrane-bound isoform of IL-5R, we compared the effects
of a 3'-splice site targeting approach, with uniformly 2'-MOE-modified
ASOs, on splicing of exons 9 and 10, as well as the constitutively
spliced exon 8. Because only four bases of intron sequence are known
flanking exons 8, 9, and 10 of the mouse IL-5R gene (Imamura et al.,
1994), uniform 2'-MOE ASOs were synthesized that would hybridize to
these four bases and the adjoining 16 bases of exon sequence at the 3'-splice site. In contrast to MOE gapmers, these oligonucleotides do
not support RNase H activity but exhibit enhanced hybridization affinity for target mRNA (Monia et al., 1993; Altmann et al., 1996).
transcript that is reflected by all three probes (Fig.
3). Importantly, transfection with
uniformly modified 2'-MOE oligonucleotides targeted to the 3'-splice
sites of the introns preceeding exons 8, 9, or 10 resulted in specific
exclusion of that respective downstream exon but not that of the other
two following, adjacent, or upstream exons. Hence, targeting the
3'-splice site intron/exon junctions with high-affinity ASOs can yield
selective deletion of individual exons within the mRNA transcript,
including exclusion of an exon that is constitutively spliced.
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, a uniform 2'-MOE ASO was
designed for the 5'-intron 8 splice site. After transfection of
BCL1 cells with oligonucleotides hybridizing to
either the 3'- or 5'-splice sites, mRNA was prepared and analyzed by
RPA. Treatment with the 3'-splice site-targeted oligonucleotide
resulted in loss of the full-length membrane and soluble IL-5R bands
and the appearance of a new band of smaller size (Fig.
4, lanes 4 and 5). Because the RPA probe
spans part of exon 6, all of exons 7 and 8, and part of exon 9, this
novel band most likely corresponds to the protected fragment
constituting sequence from exons 6 and 7. This result suggests that
treatment with this oligonucleotide produces a transcript that lacks
exon 8 and that the protected fragment of exon 9 is too small to be
visualized on the gel. These results are in agreement with Northern
data shown in Fig. 3. In contrast, treatment with the 5'-intron 8 splice site oligonucleotide partially inhibited full-length membrane
and soluble IL-5R transcripts and produced another novel mRNA
species of a size in agreement with the induced selection of a cryptic
splice site within exon 8 (Fig. 4, lanes 6 and 7). Thus, our data
suggest that placement of an ASO with high affinity for its target mRNA
sequence on a 5'-splice site can generate a novel mRNA transcript that
results from use of a nearby upstream cryptic splice site.
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ASOs targeted to the 3'-Splice Site of Exon 9 Modulate Alternative
Splicing of Murine IL-5R.
To more fully address optimal
placement of uniform 2'-MOE ASOs to modulate alternative splicing of
mouse IL-5R, additional phosphorothioate 20mers were designed
across the 5'-splice site of exon 9, as well as to the same target
sequences tested previously as MOE chimeric gapmers (Fig. 2A). The
activities of these ASOs in BCL1 cells were then
assessed by RPA to directly compare effects on expression of the
membrane and soluble receptor isoforms. Strikingly, electroporation of
BCL1 cells with these oligonucleotides not only
resulted in reduced levels of the membrane form of IL-5R mRNA but
also in the concomitant superinduction of expression of soluble form
transcript (Fig. 5). Although these
activities were observed for all of the oligonucleotides targeting exon
9 and the known intron sequence, the potencies of both of these concurrent effects were coordinately diminished by moving the targeted
sequence toward the 5'-splice site. The oligonucleotide used in the
Northern blotting experiments shown in Fig. 3 (ISIS 21750) also
inhibited membrane IL-5R expression by RPA, as expected; however,
ASOs targeting nearby exon sequences were of similar potency. These
data suggest that targeting of the 3'-splice site, or of sequences
immediately downstream and completely within the alternatively spliced
exon 9 of the murine IL-5R mRNA, with a uniformly 2'-MOE modified
ASO results in redirection of splicing activity, such that membrane
IL-5R mRNA expression is inhibited whereas levels of the soluble
form are increased.
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mRNA expression are
consistent with a hybridization-dependent mechanism.
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protein levels because ISIS 21752 was found
to diminish the ~60-kD form of IL-5R in Triton X-100 insoluble
membrane fraction-enriched cell extracts from BCL1 cells as determined by immunoblotting (Fig.
7). Control ASOs, targeted at
sequences present only in processed soluble isoform mRNA (bridging exon 8 to exon 10, ISIS 21853, or exon 8 to exon 11, ISIS 21855) did not produce inhibition of membrane IL-5R protein
(lane 4), suggesting specificity of action of ISIS 21752. As supporting
evidence that the observed ~60-kD band represents the IL-5R
membrane-bound form, an antisense inhibitor that targets sequence
within the 3'-untranslated region and hence inhibits all forms
of the IL-5R mRNA (ISIS 16949) also reduced expression of this band.
Soluble IL-5R was not detectable in this assay (data not shown) and
has not historically been measurable in vitro or in vivo without the
use of recombinant DNA techniques (Kikuchi et al., 1994; Takaki et al.,
1990). However, an antisense approach similar to that described
in our work targeting exon sequence near the 5'-splice site of the
bcl-x pre-mRNA produced increased expression of
bcl-xs protein (Taylor et al., 1999), indicating that
this strategy can result in up-regulation of mRNA and its corresponding
protein product.
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Discussion |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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We have observed that targeting 3'-splice sites of the murine
IL-5R subunit pre-mRNA with uniformly 2'-MOE-modified ASOs produced
skipping of the immediate downstream exon. In addition, hybridization
of 2'-MOE ASOs to sequences in the proximity of the 3' splice site of
exon 9 of the IL-5R blocked inclusion of exon 9 and redirected the
splicing machinery to the next downstream splice acceptor site (in
intron 9). These effects result in the loss of the membrane form mRNA
for the IL-5R chain as well as the superinduction of the soluble
form mRNA. These observations potentially extend the therapeutic
utility of antisense technology in that oligonucleotides may now be
rationally designed to inhibit expression of disease-specific domains
in proteins and loss of expression of the whole protein may be avoided.
This strategy may be useful for systems in which alternatively spliced
transcripts convey opposing function in vivo or where loss of the
architectural or adaptor roles of proteins may be detrimental.
That the exon 9 targeted gapmers equally inhibited expression of both
IL-5R isoforms suggests pre-mRNA is the site of hybridization, consistent with earlier studies (Condon and Bennett, 1996). In addition, the ability of uniformly modified 2'-MOE ASOs to almost completely inhibit expression of membrane-encoding IL-5R mRNA while
simultaneously increasing levels of transcript that encodes soluble
IL-5R by nearly 100% suggests that these oligonucleotides are
redirecting the splicing machinery such that the overall rate of
transcription is not affected. The observation that 2'-MOE-modified ASOs hybridizing across or near 3'-splice sites induced skipping of the
following exon suggests that the mechanisms of oligonucleotide-mediated modulation of alternative and constitutive splicing may be similar. A
potential mechanism of interference of oligonucleotides bordering intron-exon boundaries involves blocking the binding of proteins that
regulate splice-site selection, as observed in Drosophila where Sxl binding blocks use of the strong default 3'-splice site in
tra, thus forcing selection of the weaker distal 3'-splice site (Sosnowski et al., 1989). As a consequence, the resulting processed mRNA would lack a single exon in each case. However, alternatively, hybridization of oligonucleotides to exon sequences may
block the function of exonic enhancers (Hertel et al., 1996; Manley and
Tacke, 1996; Schaal and Maniatis, 1999) or communication across the
exon, as proposed by Berget and colleagues (Robberson et al., 1990;
Niwa et al., 1992).
Previous work has shown that 2'-O-methyl ASOs produce
inhibition of constitutive splicing of adenovirus pre-mRNAs inserted into luciferase reporter plasmids when weak splice sites are created in
the pre-mRNA by mutation (Hodges and Crooke, 1995). In this study, it
was found that weak consensus branch point and 3'-intron splice-site
sequences were particularly good targets for ASOs. However, because
limited intron sequence is known for the mouse IL-5R chain gene, the
pertinent consensus splicing sequences are not readily available. It is
tempting to speculate that the unusually large size of intron 8 estimated by polymerase chain reaction (Imamura et al., 1994) and the
equal frequency of alternative splicing as opposed to constitutive
splicing of exon 9 foretells the existence of a weak splice site at
this location. Partial sequencing of intron 8 will be required to
address this possibility.
Kole and colleagues have previously used an ASO approach to correct
aberrant splicing caused by intron mutation in thalassemic -globin
mRNA (Sierakowska et al., 1996). In these studies, phosphorothioate 2'-O-methyl oligoribonucleotides were targeted to an
aberrant 5'-splice site of the pre-mRNA expressed in stably transfected HeLa cells and blocked aberrant splicing, apparently by an occupational mechanism. This strategy is potentially important for treatment of
diseases where known mutations are mapped. Additional studies in this
system with -globin constructs and splicing extracts showed that
aberrant splicing of mutant -globin mRNAs could be blocked by
antisense targeting within the terminal 25 bases of exon sequence at
either the 5' or 3' ends (Dominski and Kole, 1994). Moving the
oligonucleotides toward the middle of the exon resulted in a less
potent inhibitory effect. Our observations extend the practical
applicability of antisense-mediated modulation of splicing demonstrated
in these studies by illustrating effects on wild-type, endogenously
expressed pre-mRNAs. Recently, in agreement with our data, modulation
of bcl-x mRNA isoform expression has been observed
with 2'-MOE ASOs targeted to regions near a 5'-splice site, by inducing
the recognition of a nearby cryptic splice site (Taylor et al., 1999).
The observed differences between our study and the -globin mutant
pre-mRNAs suggest that splicing regulation by protein factors and/or
small nuclear ribonucleoprotein particles is
likely to be complex and perhaps different between in vitro splicing
systems and intact cells. Importantly, 3'- and 5'-splice site targeting
provides a more general strategy for globally influencing splicing
patterns and producing modular transcripts from normal genes.
For IL-5R, differences in mRNA regulation exist between the mouse
and human genes. Although modulating alternative splicing of murine
IL-5R is necessary to specifically target the membrane-bound receptor, in the human, in-frame stop codons in exons 10 and 11 produce
soluble forms of the receptor, in the absence of alternative splicing
(Tavernier et al., 1991, 1992; Tuypens et al., 1992; Murata et al.,
1992). Thus, targeting of exons downstream from exon 10 with RNase
H-supporting oligonucleotides may yield human membrane-specific
oligonucleotides of potential therapeutic utility. Additionally, in
contrast to BCL1 cells as well as enriched
populations of mouse eosinophils harvested from the peritoneum of
allergen-challenged mice that express approximately equal levels of
mRNA for the membrane and soluble forms of the receptor, human
TF-1 cells greatly overexpress the soluble form of the receptor
mRNA (data not shown). This also has been reported for human
eosinophils (Tavernier et al., 1991). Evidence exists that human
soluble IL-5R can inhibit IL-5 signaling responses by binding IL-5
in solution to prevent its interaction with -chain-associated
membrane IL-5R chains. Thus, a membrane-specific ASO approach may be
more therapeutically useful than a small-molecule drug that prevents
ligand binding.
In conclusion, this study has shown that ASOs designed to bind with
high affinity to 3'-splice sites or flanking exon sequences in pre-mRNA
result in specific deletion of individual exons and modulation of
transcript isoform expression, without affecting the overall rate of
splicing of the pre-mRNA. These splicing effects were observed for the
normal murine IL-5R gene in unmanipulated intact cells, whereas
previous work used splicing extracts or gene overexpression. Thus, ASOs
are potentially useful for controlling mRNA processing in vivo and
should provide novel strategies for creating useful therapeutics that
take advantage of our increased understanding of the distinct biology
of related proteins that arise from differential splicing.
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Acknowledgments |
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We thank Kathy McGraw and Tao Lu for excellent technical assistance, Joe Johnston and Ed Wancewicz for input, and Drs. Sue Freier and Jackie Wyatt for helpful discussions and review of the manuscript. We are grateful to Tracy Reigle for help with preparation of figures.
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Footnotes |
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Received January 20, 2000; Accepted May 11, 2000
Send reprint requests to: James G. Karras, Ph.D., Department of Molecular and Cellular Pharmacology, Isis Pharmaceuticals, 2292 Faraday Ave., Carlsbad, CA 92008. E-mail: jkarras{at}isisph.com
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Abbreviations |
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ASO, antisense oligonucleotide;
IL-5R, interleukin-5 receptor-;
GM-CSF, granulocyte-macrophage colony
stimulating factor;
2'-MOE, 2'-O-methoxyethyl;
RPA, RNase protection assay.
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References |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides.
Proc Natl Acad Sci USA
91:
11762-11766 subunit gene: Characterization of the gene structure and chromosome mapping.
DNA Cell Biol
13:
283-292[Medline]. (sIL-5R). Development of an ELISA system for detection of sIL-5R.
J Immunol Methods
167:
289-298[Medline].-globin mRNA in mammalian cells by antisense oligonucleotides.
Proc Natl Acad Sci USA
93:
12840-12844-chain and a -chain shared with the receptor for GM-CSF.
Cell
66:
1175-1184[Medline]. subunit.
Proc Natl Acad Sci USA
89:
7041-7045 chain gene.
Eur Cytokine Network
3:
451-459[Medline].
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) versus its 1 (
), 3 (
), or 5 (×) base mismatch
control oligonucleotides. BCL1 cells were electroporated
with either ISIS 21752 or its mismatch controls and RNA harvested and
analyzed by RPA 24 h later. Data shown are from phosphorimage
analysis of RPA gel and are normalized to L32 levels. C, kinetic
analysis of inhibition of membrane (
) IL-5R
) IL-5R