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INTRODUCTION |
Cystic fibrosis (CF)1 is
an inherited disorder characterized by multi-organ involvement and
substantial heterogeneity in the presentation of disease (1). At a
molecular level, the pathogenesis of CF is attributable in part to over
800 known mutations2 within
the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which interfere with the processing or
integrity of the CFTR protein, a cAMP-activated chloride channel
(3-6). To the extent that mutations within the CFTR gene
permit residual chloride channel activity, a milder phenotype typically results.
Approximately 14% of the deleterious mutations known to cause cystic
fibrosis interfere with mRNA splicing, a frequency comparable to
that reported for other inherited disorders (3, 7).2 Of the
splicing mutations reported, the great majority disrupt either the
splice acceptor or splice donor sites that demarcate the 5' and 3' ends
of each exon, respectively, and drive the exclusion of that exon from
the mature transcript.
Splicing may also be derailed by mutations within introns that create
novel splice sites, resulting in the inappropriate inclusion of
non-coding sequence. This often occurs close to exons, but may also
occur deep within introns, creating either a novel donor or acceptor
site that, in conjunction with a nearby cryptic splice site of the
opposite polarity, defines a novel, aberrant exon that the spliceosome
recognizes and includes into the mature message. Several examples of
this mutational mechanism have been shown to underlie inherited
diseases, such as 
-thalassemia (8-12), CF (13, 14),
neurofibromatosis type 1 (15), multiple breast tumors (16),
dihydropteridine reductase deficiency (17), maple syrup urine disease
(18), Alport syndrome (19), ornithine 
-aminotransferase deficiency
(20), -glucuronidase deficiency (21), ataxia-telangiectasia (22),
congenital lipoid adrenal hyperplasia (23), and obesity in mice (24,
25). The true prevalence of mutations of this kind is probably
underestimated since few laboratories analyze intron sequences far from
coding regions.
One of the deep intron mutations that cause CF by the above mechanism,
3849 + 10 kb C 
T, is relatively common, with an overall frequency
of 1-2% and an elevated prevalence in individuals of Ashkenazi Jewish
ancestry (26).2 It is also the most common mutation among
CF patients with normal sweat electrolytes and mild disease (13), as
well as among fertile CF men (13, 27). This mutation generates an
aberrant 5' splice site deep in intron 19 of CFTR pre-mRNA and
activates a cryptic 3' splice site 84 nucleotides upstream.
Importantly, 3849 + 10 kb C T and other alleles of this class do
not alter wild-type splice sites. With these sequences intact, the
mRNA involved can be regarded as retaining the potential for normal
splicing, if usage of the aberrant splice sites could be inhibited.
Kole et al. (28-30) have previously demonstrated that
antisense oligonucleotides or RNAs with an affinity for aberrant 5' and 3' splice sites underlying -thalassemia deterred usage of these elements by the spliceosome in favor of the intact, wild-type splice
sites, promoting normal splicing patterns and correct expression of the
-globin gene. Here we report the applicability of this approach to
the correction of aberrant splicing in the context of CF with enhanced
synthesis and processing of CFTR protein.
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EXPERIMENTAL PROCEDURES |
Vector Construction--
CFTR expression vectors were
constructed that contain a truncated intron 19 harboring the 3849 + 10 kb C T locus. The mini-intron was assembled from three PCR
fragments of genomic sequence derived from the native CFTR
intron 19: an XhoI-HindIII fragment encompassing the 3' end of exon 19, the splice donor site, and the initial sequences
of intron 19; a HindIII-SalI fragment containing
the central region of intron 19 including the 3849 + 10 kb C T and its upstream cryptic acceptor site (this fragment was amplified with or
without the 3849 + 10 kb C T mutation); a
SalI-BamHI fragment straddling the intron 19-exon
20 junction that contains the native acceptor splice site and the
polypyrimidine tract preceding exon 20. To improve the efficiency of
splicing of the constructs in cell culture, the polypyrimidine tract
was optimized by the substitution of pyrimidines for three interrupting purines.
Ordered ligation of these fragments into a 415-bp mini-intron was
achieved as depicted in Fig. 1A. The mini-intron 19 was subsequently incorporated into the LCFSN CFTR vector
containing the intact coding region of CFTR and the
Geneticin (G418) resistance genes, controlled by a retroviral LTR
promoter (31) using a PCR-based mutagenesis protocol (32, 33). The
details of the construction are available on request.
Intron-containing constructs were digested with HphI to
detect the presence or absence of the mutation (13) and further by
direct sequencing (34). The modified vectors selected for further
studies were LCFSN-3849wt (wild-type mini-intron) and LCFSN-3849mut
(mutant mini-intron). The wild-type and mutant mini-introns were also
inserted in the cytomegalovirus-driven, CFTR expression vector, pCF1-CFTR (35) by similar procedures.
Cell Culture--
Mouse NIH 3T3 fibroblasts were grown in DMEM-H
(high glucose) supplemented with 10% fetal calf serum. The
immortalized human tracheal epithelial line, CFT1 (36), was grown in
serum-free Ham's F-12 supplemented with factors described elsewhere
(37). C127 mouse mammary epithelial cells were grown in DMEM-L (low glucose) supplemented with 10% fetal calf serum. Human T84 colorectal carcinoma cells, which express high levels of CFTR, were
used for controls and were grown in DMEM/F-12 supplemented with 5% newborn bovine serum. All culture media contained
penicillin/streptomycin.
Construction of Stable Cell lines--
3T3- or CFT1 cells were
stably transfected with either LCFSN-3849wt or LCFSN-3849mut plasmids
using LipofectAMINE (Life Technologies, Inc.) and selection with G418
(580 and 150 µg/ml G418, respectively, for the selection and
maintenance of 3T3- and CFT1-derived cell lines). 3T3 and CFT1 clonal
cell lines containing the wild-type and mutant mini-introns ((3T3-WT8,
3T3-M11, CFT1-WT, CFT1-M15) were selected for further study.
Construction of C127-derived mouse mammary epithelial cell lines was
accomplished by co-transfection of native or modified pCF1-CFTR
expression constructs with the pMEP4 plasmid (Invitrogen), which
confers hygromycin resistance. Selection proceeded in 500 µg/ml
hygromycin. The C127 cell line stably expressing the 
F508
CFTR mutant was a gift from Genzyme Genetics (Framingham,
MA) (38).
Antisense Oligoribonucleotides: Design and Usage--
Antisense
oligonucleotides were 18 or 19-mer 2'-O-methyl
oligoribonucleoside phosphorothioates (Hybridon Inc., Milford, MA, and
Midland Certified Reagent Co. Woodland, TX) (Table I). They were
targeted to either the novel splice donor site, the cryptic splice
acceptor site or to a region within the 84-bp aberrant exon that
includes an in-frame stop codon (Fig. 1B). Anti--globin or anti-CFTR oligonucleotides with two mismatched nucleotides were used
as negative controls (Table I).
Transfection with Antisense Oligonucleotides
(29)--
50-100 × 103 of 3T3-M11 cells were plated
in each well of a 24-well plate 48 h prior to treatment with the
oligonucleotide. Antisense oligonucleotides were complexed at room
temperature with either 6% LipofectAMINE or 4% DMRIE-C solutions in
200 µl of serum-free Opti-MEM (Life Technologies, Inc.) for 15-30
min. After dilution to 1 ml with Opti-MEM, each mixture was added to cells (pre-washed with Opti-MEM to remove serum) and incubated for 3-5
h. Subsequently, the transfection mixture was replaced with serum
containing growth media and cells were harvested 24-48 h after
transfection. The antisense oligonucleotide concentrations ranged from
0 to 1.0 µg/ml.
Oligonucleotide treatment of CFT1 cell lines with antisense
oligonucleotides was similar and used 5% DMRIE-C in Opti-MEM, 3-h
transfections, and harvesting 24 h after transfection. Sodium butyrate, previously shown to boost CFTR transgene
expression in 3T3-derived cells (39), was added (30 mM) to
CFT1 cells for 24 h after transfection. For C127 cell lines,
transfection was performed with 2.5% ExGen500 (MBI Fermentas) in
Opti-MEM for 3 h. Cells were harvested 24 h after transfection.
RNA Isolation and RT-PCR Analysis--
Cells were rinsed once
with Hank's balanced salt solution and lysed at room temperature for 5 min with 1 ml of TRI reagent (MRC, Inc.; Cincinnati, OH) per well of
transfected cells. Total cellular RNAs were purified according to
manufacturer's instructions, and concentrations were measured via
spectrophotometry at 260 nm. All RNAs were stored at 
20 °C.
RT-PCR was performed using the rTth kit (Perkin-Elmer) with
0.2-0.3 µg of RNA per reaction. The RT reaction was performed in 10 µl for 15 min at 70 °C. [
-32P]dATP was
incorporated during the subsequent PCR, performed in 50 µl (95 °C
for 3 min, followed by 18-24 cycles of 65 °C for 1 min and 95 °C
for 1 min).
Primers (A, 5'-ATCCAGTTCTTCCCAAGAGGC-3'; B,
5'-TCTTCC-CAAGAGGCCCACCCTCTG-3', C,
5'-TCTTCCCAAGAGGCCCACCAT-TTT-3'; D,
5'-CCAAATGACTGTCAAAGATCTCACAGCA-3') were designed to detect splicing
isoforms arising from processing of the mini-intron as depicted in
Figs. 2-7. RT-PCR products (typically 20 µl from the 50-µl RT-PCR
sample representing reaction products derived from 120 ng of RNA
template) were electrophoresed on 8% polyacrylamide (29:1,
acrylamide:bisacrylamide) non-denaturing gels (29), which were dried
and autoradiographed for 1-24 h. RT-PCR products derived from T84 RNA
template served as a positive control for wild-type splicing. If
necessary for further analysis, bands of interest were excised from
dried polyacrylamide gels, eluted, re-amplified using primers A and D
and sequenced (34).
Western Blots--
C127-derived stable cell lines were grown to
~80% confluence on 10-cm plates and transfected with antisense
oligonucleotides as described above. For protein isolation, cells were
rinsed twice with Hank's balanced salt solution and lysed with 1 ml of
3% SDS, 60 mM Tris, pH 6.8, 1 mM EDTA, 6%
sucrose, 100 µg/ml phenylmethylsulfonyl fluoride, 1-2 µg/ml
aprotinin, and 1-2 µg/ml leupeptin. Samples were made up to 50 mM dithiothreitol, and Pyronin Y tracking dye was added
prior to electrophoresis on 7% SDS-polyacrylamide gels. Proteins were
electroblotted to a nitrocellulose membrane and probed with a
polyclonal anti-CFTR antibody (-1468) (40) and a horseradish
peroxidase-conjugated secondary antibody (Amersham Pharmacia Biotech).
Detection utilized the ECL kit (Amersham Pharmacia Biotech) and luminography.
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RESULTS |
Restoration of Correct CFTR Splicing in 3T3-CFTR Cell Line--
In
order to model the 3849 + 10 kb C T splicing mutation in cell
culture, a 415-bp mini-intron was created by the ordered ligation of
three fragments derived from the native 22-kb intron 19 of CFTR (see
Fig. 1A and "Experimental
Procedures"). The construct retained intron 19 native splice sites
and approximately 240 bp of sequence bracketing the mutation locus and
the cryptic acceptor splice site activated by the mutation. The ligated
fragment was accurately inserted at the exon 19-exon 20 junction of the
CFTR cDNA portion of the LCFSN expression vector (31).
The expression of CFTR gene from this vector is driven by retroviral
(murine leukemia virus) LTR promoter. The resulting constructs were
used to generate 3T3- and CFT1-based cell lines, which constitutively expressed either the wild-type or the mutant mini-intron containing CFTR genes.
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Fig. 1.
A, modification of LCFSN CFTR
expression vector to include mini-intron 19. Three fragments of genomic
sequence derived from the native or 3849 + 10 kb C T mutated
CFTR intron 19 were obtained by PCR. The fragments were
cleaved with appropriate restriction enzymes (indicated at the
upper part of panel A), ligated,
amplified with tailed primers complementary to exon sequences near the
native splice junctions (5'ss, 3'ss, short
vertical lines), and inserted into LCFSN vector
(lower part of panel A)
linearized at the exon 19-exon 20 border with PflM1 (see
"Experimental Procedures" for more details). The position of the
mutation locus (*), the cryptic 3' splice site (3'cr), and the
nucleotide lengths of appropriate fragments are indicated. The
open boxes of the lower
part of panel A represent the CFTR
cDNA and Geneticin resistance gene. LTR, murine leukemia
virus LTR promoter. B, splicing pathways of LCFSN-3849mut
(mutant mini-intron) pre-mRNA. Boxes, exons;
heavy line, intron. Thin
solid and dashed lines represent
aberrant and corrected splicing pathways, respectively.
Heavy short bars, antisense
oligonucleotides targeted to the aberrant 3' splice site, the stop
codon, and the aberrant 5' splice site, respectively (see
"Experimental Procedures" for more details and Table I for
oligonucleotide sequences).
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Fig. 2 shows RT-PCR analysis of total RNA
from a 3T3-based stable cell line (3T3-M11) expressing the 3849 + 10 kb
C T CFTR construct. The cell line did not express detectable levels
of correctly spliced CFTR mRNA (Fig. 2A,
lanes 2 and 8) as shown by a lack of
the RT-PCR band co-migrating with that of the control CFTR mRNA
from T84 cells (Fig. 2A, lane 1). The
correctly spliced fragment was generated after treatment of the 3T3-M11
cells with antisense oligonucleotides (Table
I) complexed with cationic lipid carriers
and targeted to either the aberrant cryptic acceptor site (Fig.
2A, lanes 2-7) or the donor site
(Fig. 2A, lanes 8-13). For both
oligonucleotides the effects were dose-dependent.
Dose-dependent correction of CFTR pre-mRNA splicing was
also observed when both 3' and 5' splice site targeted antisense
oligonucleotides were combined and used for cell treatment (Fig.
2B, lanes 2-5). Lack of correctly
spliced CFTR mRNA in cells treated with an
oligonucleotide antisense to the human thalassemic -globin IVS2-654
aberrant donor site (29), which has no affinity for the CFTR
transgene's aberrant splice sites, shows sequence specificity of the
effects (Fig. 2B, lanes 6-8). These
results indicate that the antisense oligonucleotides prevented
utilization of the aberrant splice sites by the spliceosome and forced
it to reutilize the existing correct splice sites, thereby restoring
correct splicing of CFTR pre-mRNA.
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Fig. 2.
Correction of aberrant CFTR splicing in
3T3-M11 cells by antisense oligonucleotides. A, 3T3M11
cells were treated with antisense oligonucleotides (3'AS or 5'AS)
targeting the aberrant acceptor and donor splice sites, respectively.
Total RNA was isolated and subjected to RT-PCR, and the products were
analyzed by polyacrylamide gel electrophoresis. The products and the
primers used in RT-PCR are depicted below the panels (see
"Experimental Procedures"). Lane 1, RNA from
T84 cells, positive control for wild-type RNA splicing
(T84); lanes 2-7, correction of
aberrant splicing by 3'AS; lanes 8-13,
correction of aberrant splicing by 5'AS; lane 14,
water blank. The length, in nucleotides, of RT-PCR products derived
from correctly (119) and aberrantly (243, 203) spliced CFTR mRNAs
is indicated. Concentrations (µM) of antisense
oligonucleotides are shown at the top. Unless otherwise
indicated similar designations are used in the remaining figures.
B, lanes 2-5, correction of aberrant
splicing in 3T3-M11 cells in response to dual antisense oligonucleotide
treatment targeting both the aberrant splice donor or cryptic splice
acceptor sites; lanes 6-8, absence of splicing
correction upon treatment with antisense oligonucleotide targeting the
-globin, IVS2-654 aberrant donor site (29).
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Table I
Sequences of antisense oligonucleotides
AS, antisense. Asterisks (*) indicate mismatched bases relative to
target sequences. Underlined bases mark the location of the in-frame
ochre stop codon within the 84-bp aberrant insert. -Globin IVS2-654
antisense oligonucleotide as reported (29).
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Characterization of RT-PCR Products; Identification of a Cryptic 5'
Splice Site in Intron 19 of CFTR Gene--
RT-PCR of total RNA
expressed in 3T3-M11 cell line should in principle produce a single
band of 203 bp representing aberrantly spliced 3849 + 10 kb C T
CFTR mRNA (Fig. 2A, primers D and
A). After correction of splicing with antisense
oligonucleotides, a 119-bp band representing correctly spliced mRNA
should also appear. However, in oligonucleotide-treated cells, an array
of bands was generated, complicating identification of aberrant
products. Thus, we have designed a series of primers, diagrammed in
Fig. 3, intended to amplify either all
CFTR mRNAs, only correctly spliced mRNAs, or only aberrant
mRNAs.
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Fig. 3.
Differential RT-PCR analysis identifies
wild-type and aberrant CFTR splicing products. Lanes
1 and 3, T84 RNA; lanes 2,
4, and 5, RNA from 3T3-M11 cells treated with 0.3 µM 5' AS (same RNA as in lane 13,
Fig. 2A). Primers D and A (lanes marked
A), all CFTR mRNA isoforms are amplified.
Primers D and B (lanes B), RT-PCR specific for
correctly spliced CFTR mRNA. Primers D and C (lane
C), aberrant-specific RT-PCR. The length, in nucleotides, of
the resulting RT-PCR products is indicated.
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RT-PCR (primers D-A, Fig. 3) of RNA from
oligonucleotide-treated cells (the same RNA as analyzed in Fig.
2A, lane 13) detected both the
correctly spliced CFTR mRNA that co-migrates with T84-derived mature splicing products (Fig. 3, lane 1) and
additional species, which presumably included the aberrantly spliced
mRNAs (Fig. 3, lane 2). The
wild-type-specific primers, D-B, amplified only the correctly spliced
mRNA, as expected (Fig. 3, lane 4), while the aberrant specific primers, D-C, amplified a doublet of bands
migrating, at ~240 and ~200 bp, respectively, the smaller of which
closely approximated the 196-bp fragment detected in vivo
(13) (Fig. 3, lane 5). The nature of the heavier
of the two fragments was not immediately known.
To clarify the identity of the products arising from splicing of the
CFTR transgenes, RT-PCR generated bands were re-amplified with primers A and D and sequenced. The fragment that co-migrated with
the control for wild-type splicing was confirmed to be the product of
correct splicing, whereas the smaller of the two heavy bands included
the 84-bp insertion reported in vivo (13) and therefore
represented the expected aberrantly spliced CFTR mRNA. The heavier
fragment with an effective mobility of ~240 bp contained, in addition
to the sequences found in the smaller aberrant fragment, the first 40 bp of intron 19 inserted between the 3' end of exon 19 and the 5' end
of the 84-bp aberrant insertion. Analysis of the sequence at the 5'
region of native intron 19 of CFTR gene showed that a previously
unrecognized cryptic splice donor site (CA|GTAAGT) resides at
position 3849 + 41. Thus, in the 3T3-M11 cell line, the mini-intron
undergoes two aberrant splicing events, inclusion of the 84 nucleotide
"exon" and utilization of a cryptic 5' splice, resulting in a
243-bp splice isoform.
The products of correct splicing generated in 3T3-M11 cell line by
antisense oligonucleotide treatment were additionally identified by
RT-PCR analysis with primers D-B (Fig.
4, lanes 2-7). The
RNAs analyzed in this figure are the same as those in Fig.
2A (lanes 8-13). A single prominent
band co-migrating with the RT-PCR product of T84-derived native CFTR
mRNA (Fig. 4, lane 1) represents correctly spliced CFTR mRNA and shows with great clarity the dose response of
CFTR splicing correction to antisense treatment.
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Fig. 4.
Dose responsiveness of antisense-mediated
correction of aberrant splicing in 3T3-M11 cells demonstrated with
wild-type specific RT-PCR. Lanes 2-7,
correction of aberrant splicing in 3T3-M11 cells in response to
antisense treatment with oligonucleotide 5'AS, complementary to the
aberrant 5' donor splice site. Same RNAs as in lanes
8-13 of Fig. 2A.
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Correction of CFTR Splicing in CFT1 Cells--
To more closely
approximate the cells that would constitute actual targets if antisense
oligonucleotides were used for treatment of cystic fibrosis, the
CFT1-based cell lines carrying the modified CFTR transgenes,
LCFSN-3849wt or LCFSN-3849mut, were developed. The CFT1 cells, a human
papilloma virus-immortalized human tracheal epithelial cell line
derived from a CF patient, have retained their differentiated phenotype
yet lost the endogenous CFTR expression, eliminating a
background that might otherwise have obscured detection of
antisense-mediated effects (36).
Similar to 3T3-M11 cells, the monoclonal CFT1-M15 cell line exhibited
the dose- and sequence-dependent correction of modified CFTR pre-mRNA splicing by antisense oligonucleotide targeted to the
3849 + 10 kb C T donor splice site (Fig.
5, lanes 2-6). The
CFT1-M15 line differed from the 3T3-M11 counterpart in that the pair of
bands regarded as products of aberrant splicing was either absent or
present at much lower levels. It seemed possible that in these cells
the aberrantly spliced CFTR mRNA was much less stable than the
correctly spliced one because the ochre stop codon within the aberrant
exon provided a recognition element for the nonsense-mediated RNA decay
machinery (41). Thus, an antisense oligonucleotide targeted to the
sequence surrounding the premature stop codon was designed to test if
blocking this codon might promote appearance of the aberrant bands. An
alternative possibility that correct splicing might be promoted by an
antisense oligonucleotide that prevents binding of the splicing factors to the 84 nucleotides of the aberrant exon, thereby interfering with
the definition of the exon by the splicing machinery (42), was also
considered.
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Fig. 5.
Antisense-mediated correction of aberrant
splicing in the CFT1-M15 cell line. Lanes
2-6, RNA from cells treated with oligonucleotide 5'AS,
complementary to the aberrant 5' donor splice site.
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Although the "anti-stop" oligonucleotide showed no utility for
increasing the stability of aberrant transcripts, it did promote correct splicing as efficiently as the antisense oligonucleotides targeted to aberrant splice sites, i.e. equivalent
concentrations (0.3 µM) of the 5' AS, 3'AS, or anti-stop
oligonucleotides were comparably effective (Fig.
6, lanes 3-5).
Interestingly, dual (or even triple) treatment with all possible AS
oligonucleotide combinations appeared more effective than single
oligonucleotide treatment, suggesting additive or synergistic effects
among the oligonucleotides (Fig. 6, lanes
6-9).
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Fig. 6.
Increased correction of CFTR pre-mRNA
splicing in CFT1-M15 cells by combined antisense oligonucleotides.
Lanes 1-9, CFT1-M15 cells treated with different
combinations of antisense oligonucleotides (described above the
lanes).
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Restoration of Correct CFTR Gene Expression in C127
Cells--
Although expression of aberrant CFTR mRNA and
correction of splicing of CFTR pre-mRNA were clearly detectable in
3T3 and CFT1 cell lines, we were unable to detect in
oligonucleotide-treated cells the expected correct CFTR protein.
Assuming that this was due to a low level of expression of the
transgenes in both cell lines, we have explored another cell
line-vector combination as a means of attaining sufficiently high
levels of CFTR expression.
C127 mouse mammary epithelial cells divide rapidly and strongly express
CFTR protein when transfected with the pCF1-CFTR construct, in which
expression of CFTR cDNA is driven by a strong immediate early
cytomegalovirus promoter (35, 40). Transfection of these cells with
either native pCF1-CFTR (no intervening introns) or its modified
counterpart harboring the wild-type mini-intron 19 (pCF1-CFTRwt)
resulted in one prominent RT-PCR product representing mature
CFTR transcripts (data not shown). A stable cell line
(C127-M7) harboring the pCF1-CFTRmut construct (with mutated
mini-intron 19) expressed the aberrant CFTR mRNA with greater
efficiency than either the 3T3- or CFT1-derived cell lines and produced
minimal levels of correct mRNA in the absence of antisense
oligonucleotides (Fig. 7, lane
2).
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Fig. 7.
Antisense-mediated correction of aberrant
splicing in C127-M7 cells. The cells were treated with the
following: lanes 2-7, 5'AS oligonucleotide;
lane 8, 0.3 µM mismatched 5' AS
(negative control); lane 9, 0.3 µM
anti-stop AS; lane 10, 0.15 µM each
5'AS and anti-stop oligonucleotide.
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The C127-M7 cells were transfected with varying concentrations (0-0.5
µM) of the 5' AS or anti-stop antisense oligonucleotides using ExGen500 as delivery agent. As shown with the previous cell lines, C127-M7 cells produced correctly spliced CFTR
mRNA in a dose-responsive manner when treated with the
oligonucleotides individually (Fig. 7, lanes 2-7
and 9) or in combination (Fig. 7, lane
10). Additionally, treatment with a mismatched
oligonucleotide, with partial homology for the aberrant donor site, was
much less effective at promoting correct splicing, confirming sequence
specificity of the effect (Fig. 7, lane 8).
Identical results were attained using a mismatched oligonucleotide with
partial homology for the cryptic splice acceptor site (data not shown).
Expression of the aberrant transgene in C127-M7 cells as well as its
responsiveness to antisense treatment were sufficiently robust to
assess the synthesis of CFTR protein in oligonucleotide-treated cells
by Western blot analysis (see "Experimental Procedures"). Fig.
8 demonstrates an increase in CFTR
protein production with increasing dose of antisense oligonucleotides
targeted to the aberrant 5' splice site (Fig. 8, lanes
2-4). As with RT-PCR analysis, it is apparent that the
treatment with a mixture of two individually effective antisense
oligonucleotides, 5' AS and anti-stop, is superior, on an equimolar
basis, to single oligonucleotide treatment (Fig. 8, lane
5). As expected, neither the mismatched oligonucleotide, nor
antisense oligonucleotide targeted to the human -globin IVS2-654 splice site were effective at promoting translation of CFTR protein (Fig. 8, lanes 6 and 7, respectively).
C127 parental cells expressing modified pCF1-CFTR harboring a wild-type
mini-intron (pCF1-CFTRwt) were also shown to permit CFTR protein
formation (Fig. 8, lane 8).
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Fig. 8.
Detection in C127-M7 cells of appropriately
glycosylated CFTR protein generated by antisense oligonucleotides.
Total cellular protein was analyzed by immunoblotting and detection
with a polyclonal anti-CFTR antibody (see "Experimental Procedures"
for details). Lane 1, M, protein size
markers (molecular mass in kDa indicated on the left).
Lanes 2-4, C127-M7 cells treated with 0, 0.02, and 0.2 µM 5' AS, respectively. Lane
5, treatment with 0.1 µM each, 5' AS
oligonucleotide targeting aberrant 5' site and the in-frame stop codon
oligonucleotide (anti-stop). Lanes 6 and
7, negative controls with 0.2 µM mismatched
5'AS and -globin IVS2-654 (beta) oligonucleotides, respectively.
Lane 8, C127 cells transfected with pCF1-CFTRwt
construct. Lane 9, T84 cells (positive control).
Lane 10, C127 cells expressing pBPV-CFTR-F508
(38).
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The CFTR protein generated in oligonucleotide-treated cells co-migrated
on the gel with CFTR expressed in C127 cells harboring the pCF1-CFTRwt
construct or in T84 cells (Fig. 8, lanes 8 and 9, respectively). The migration of CFTR protein derived from
a C127 cell line expressing the common F508 CFTR mutant was faster (Fig. 8, lane 10). This result indicates that, in
contrast to F508 CFTR, glycosylation of which is known to be
defective (43), the CFTR protein generated by antisense
oligonucleotides is wild-type and fully glycosylated.
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DISCUSSION |
3849 + 10 kb C T is a member of a class of splicing mutations
that, rather than disrupting existing splice sites, create novel ones
that are erroneously recognized by the splicing machinery (8-25).
Usage of such splice sites results in the abnormal inclusion of intron
sequences in the mature transcripts with a wide range of clinical
consequences. The exon-shuffling theory (44) proposes that introns
serve as reservoirs of new coding sequence and evolutionary potential
and mutations like 3849 + 10 kb C T are mechanistically compatible
with this theory. From the standpoint of immediate clinical
consequences, however, molecular derangements of this class must be
detrimental in the vast majority of individuals.
The likelihood that an RNA sequence will be recognized by the
spliceosome depends chiefly on how well it approximates the consensus
splice site sequence. The relation between the consensus sequence and a
given splice site may be expressed numerically using the scoring system
of Shapiro and Senapathy (45). Relative to the consensus splice site
score of 100, 3849 + 10 kb C T creates a novel donor site with a
score of 88.9; the score of the non-mutated sequence at this site is
70.6. By comparison, the native intron 19 donor and acceptor splice
sites have scores of 83.2 and 86.7, respectively. This analysis
suggests that the novel splice site should be preferred over the native
ones by the splicing machinery. Moreover, and probably more
importantly, the novel splice site in conjunction with the cryptic
splice site located 84 nucleotides upstream in the intron creates an
exon-like sequence. The exon definition mechanism predicts that such
sequence should be efficiently included in the spliced mRNA (42).
Interestingly, despite the high score of the novel donor splice site
and creation of the aberrant exon, the impediment to normal splicing is
not absolute. In vivo, but not in vitro, as shown
by our results, approximately 8% of the CFTR transcripts
are normal length (13). This low level of wild-type mRNAs is
thought to account for the mild, if variable, phenotypes in patients
with the 3849 + 10 kb C T mutation (13, 27, 46, 47). The retention
of wild-type splice sites on 3849 + 10 kb C T alleles not only
permits low levels of normal splicing but also suggested a mechanism by
which wild-type splicing could be restored. If usage of the aberrant splicing elements were prevented by antisense oligonucleotides, it
seemed likely that the spliceosome will revert to normal utilization of
the native splice elements.
Using two different CFTR cDNA expression vectors, LCFSN and
pCF1-CFTR, in three different cell lines (3T3, CFT1, and C127), we have
demonstrated that antisense oligonucleotides complementary to either
the aberrant donor or the cryptic acceptor splice sites promoted the
formation of correctly spliced CFTR mRNA. This
corrective effect was specific for the oligonucleotides with full
complementarity to the 18 bp of sequence bracketing each novel splice
site. Oligonucleotides with two nucleotide mismatches and an
oligonucleotide of wholly unrelated sequence showed no efficacy for the
correction of splicing.
The correction of aberrant splicing in multiple cellular contexts
speaks to the potential broad applicability of this therapeutic modality. This approach has been also shown to be effective in correction of splicing of human thalassemic -globin pre-mRNAs (28-30) and in modification of splicing of dystrophin genes (48, 49).
Patients with 3849 + 10 kb C T reportedly produce one major
aberrant mRNA isoform reflecting the inclusion of 84 bp of intron sequence (13). RT-PCR analysis of splicing in the 3T3- and C127-derived mutant cell lines, however, identified two bands associated with aberrant processing. One represented the expected 84-bp insertion, while the second band incorporated the first 40 bp of intron 19 due to
splicing at a cryptic splice donor site (CA||GTAAGT) at position
3849 + 41, which, surprisingly, is present but does not appear to be
active, in native intron 19 sequence. This site has a Shapiro-Senapathy
score of 79.4 in primates and 80.1 in rodents (45) and, if activated,
should disrupt the reading frame of the mature message. It is possible
that the novel sequence context within the mini-intron may be
responsible for its use and/or that the heterologous expression of the
constructs in murine cells that do not normally express CFTR
gave rise, in vitro, to this unexpected splicing pathway.
However, since this splice site is present in the native
CFTR intron 19, one must consider that its activation may
occur in some patients or in specific tissue contexts. If this is true,
the level of usage of this cryptic donor site may contribute to
variable disease severity in patients with identical mutational
genotypes of the CFTR gene.
The anti-stop oligonucleotide that anneals to the vicinity of the ochre
stop codon in the aberrant exon promoted wild-type splicing. This
result, which was equally apparent in both CFT1- and C127-derived
mutant cells, should be viewed in light of the exon definition model
that postulates interaction between spliceosome components bridging an
exon (42). An oligonucleotide that sterically inhibits this bridging
would impair recognition of the exon as sequence to be retained in the
mature message. Blocking of the premature stop codon itself was
probably not pertinent to this effect.
Superior correction of splicing was seen when two rather than one
antisense oligonucleotide were transfected in concert, e.g. targeting the 5' and 3' splice sites simultaneously. Presumably one
oligonucleotide corrects splicing among those transcripts left
untouched by the other oligonucleotide. A rigorously quantitative assessment will be required to determine if the effect is synergistic or merely additive.
It is logical to assume a priori that correction of splicing
would be accompanied by increased production of normal protein. The
ribosome should translate correctly processed CFTR mRNAs
equally without regard to how they came to be correctly spliced. C127 cells stably transfected with the aberrant pCF1-CFTR transgene produced
CFTR protein upon treatment with the same oligonucleotides shown to
correct splicing. The increase in detectable protein was both sequence-
and dose-dependent, with dual oligonucleotide treatment
associated with the highest level of protein. Furthermore, we note that
the CFTR protein formed in response to antisense oligonucleotide
treatment was appropriately glycosylated, suggesting that CFTR
polypeptide processing beyond simple translation has also occurred,
presumably leading to cell surface expression and chloride conductance.
The potential for antisense-mediated approaches to achieve utility in
the clinical arena will depend on several factors. Splicing mutations
that evoke aberrant inclusion of intron sequence are currently regarded
as infrequent. Although several mutations that fit this category have
been reported (8-25), there is likely a bias against their
identification. Typically, mutation screening targets coding regions
and adjacent splice sites, whereas more cumbersome RNA-based analyses
are best suited to discern deep intron mutations. The mutations
identified thus far may represent the tip of the iceberg.
Each of the examples cited (8-25), including 3849 + 10 kb C T,
involve a novel splice site in proximity to a cryptic splice site of
the opposite polarity. In principle, these can occur at anyplace within
an intron. In contrast, many other intronic mutations create splice
donor and acceptor sites proximal to exons, leading to the inclusion of
intron sequences adjacent to those exons into the mature message
(50-57). As with 3849 + 10 kb C T, wild-type splicing elements
remain intact, making these aberrant alleles potential substrates for
antisense-mediated correction as well, as long as no steric hindrances
with the spliceosome occur. This approach has been tested in
vitro in the context of thalassemic mutations of human -globin
gene (28, 58).
2'-O-Methyl oligoribonucleoside phosphorothioates as
antisense oligonucleotides have high affinity for their target
sequences, are markedly resistant to intracellular degradation, and,
when complexed to their target, will not promote RNase H-mediated
cleavage of targeted RNA (59). With such obvious technical utility, it is important that neither the oligonucleotides nor the delivery vehicle
is unduly detrimental to the treated cells. Strong antisense-mediated correction has been demonstrated without a dramatic impact on cell
morphology or viability. Furthermore, an argument that antisense oligonucleotides might have unforeseen, nonspecific effects on splicing
and, consequently, the expression of a multitude of other genes is not
supported by gross assessment of cell viability, morphology, and growth
rates, all of which appear normal. In view of these promising results,
further experimentation and optimization should encompass ex
vivo or in vivo studies in animal models.
The potential for antisense pharmaceuticals to treat human disease
depends upon the safe delivery of oligonucleotides at therapeutic doses
to appropriate tissues. Many animal studies have demonstrated efficacious delivery of oligonucleotides through systemic treatment (reviewed in Ref. 60), and this approach might prove equally valid for
treatment of CF. Alternatively, aerosol delivery of the antisense
oligonucleotides directly into CF lungs may efficiently target the
impaired tissues. Importantly, treatment with antisense oligonucleotide
aerosols markedly reduced allergic response in lungs of rabbits
modeling adenosine-mediated hyper-responsiveness (61). The efficient,
low dose delivery of potentially therapeutic antisense appeared to be
facilitated by weakly cationic lung surfactants, which are known to
recycle between type II pneumocytes and the air-liquid interface in the
lung (2).
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