Gene Correction of the Apolipoprotein (Apo) E2 Phenotype to
Wild-type ApoE3 by in Situ Chimeraplasty*
Aristides D.
Tagalakis
§,
Ian R.
Graham§,
David R.
Riddell
,
J. George
Dickson§, and
James S.
Owen
¶
From the
Department of Medicine, Royal Free and
University College Medical School, London NW3 2PF and the
§ Department of Biochemistry, Royal Holloway
University of London, Surrey TW20 0EX, United Kingdom
Received for publication, December 14, 2000, and in revised form, February 2, 2001
 |
ABSTRACT |
Apolipoprotein (apo) E is a polymorphic
plasma protein, synthesized mainly by liver. Here, we evaluate whether
synthetic DNA-RNA oligonucleotides (chimeraplasts) can convert a
dysfunctional isoform, apoE2 (C
T, R158C), which causes Type
III hyperlipidemia and premature atherosclerosis, into apoE3. First, we
treated recombinant Chinese hamster ovary cells stably secreting
apoE2 with a 68-mer apoE2 to apoE3 chimeraplast. About one-third of
apoE2 was converted to apoE3, and the repair was stable through 12 passages. Subcloning treated cells produced both apoE2 and apoE3
clones. Direct sequencing and reverse transcription polymerase chain
reaction confirmed the genotype, whereas phenotypic change was verified
by isoelectric focusing and immunoblotting of secreted proteins.
Second, we established that the APOE2 gene can be targeted
both in vivo, using transgenic mice overexpressing human
apoE2, and in chromosomal context, using cultured lymphocytes from a
patient homozygous for the
2 allele. We conclude that
chimeraplasty has the potential to convert the apoE2 mutation in
patients with Type III hyperlipidemia to apoE3.
 |
INTRODUCTION |
Apolipoprotein (apo)1 E
is a 34-kDa polymorphic protein that has anti-atherogenic actions by
clearing remnant lipoproteins and promoting cholesterol efflux from
cells (1-4). Low apoE is a risk factor for coronary artery disease
(5), and apoE deficiency results in severe hyperlipidemia and
atherosclerosis (6, 7). By contrast, infusing apoE into hyperlipidemic
rabbits regresses atherosclerotic lesions (8), and apoE transgenic mice
resist diabetic or dietary hyperlipidemia (9, 10). The three common isoforms of apoE differ in two amino acid positions; apoE2 (Cys-112 and
Cys-158), apoE3 (Cys-112 and Arg-158), the most prevalent or wild-type
protein, and apoE4 (Arg-112 and Arg-158). The rarest variant, apoE2, is
the primary molecular cause of Type III hyperlipidemia (11), which is
characterized by accumulation of remnant lipoproteins and premature
heart disease.
Approaches to treat or prevent atherosclerosis include systemic gene
therapy, using viral or non-viral delivery strategies, to overexpress
proteins that inhibit atherogenesis or stabilize lesions. Indeed,
adenoviral-mediated apoE gene transfer ameliorates experimental
hyperlipidemia and atherosclerosis (12, 13). The recent development of
targeted gene repair, using synthetic DNA-RNA oligonucleotides
(chimeraplasts), offers another possibility (14-16). Chimeraplasts
contain short regions of correcting DNA bounded by long stretches of
2'-O-methyl RNA; hairpin loops and GC clamps are also
incorporated to make the structure self-associating. These features
promote strong, specific binding to the target genomic sequence and
allow the DNA repair machinery of the cell to identify and
correct the point mutation in situ (15).
Here, we apply chimeraplasty to recombinant Chinese hamster ovary (CHO)
cells secreting human apoE2 and show that the gene conversion of apoE2
to apoE3 is stable and permanent at the DNA, mRNA, and protein
level. In addition, we demonstrate gene repair in vivo,
using transgenic mice overexpressing human apoE2, and also verify that
the human genome can be targeted using lymphocytes from a patient
homozygous for the
2 allele. We conclude that it is feasible to
correct the apoE2 mutation in patients with Type III hyperlipidemia by chimeraplasty.
 |
EXPERIMENTAL PROCEDURES |
Recombinant CHO Cells--
CHOdhfr
cells were
cultured in Iscove's modified Dulbecco's medium with 10%
dialyzed fetal bovine serum (FBS; Sigma), supplemented with 2 mM glutaMAX, 100 µM hypoxanthine, 16 µM thymidine, and 1% non-essential amino acids (Life
Technologies, Inc.). Human apoE2 cDNA was cloned into p7055 (17),
an expression vector with the selectable DHFR gene,
and then 6 µg of purified p7055.E2 were complexed with cationic
liposomes to transfect CHOdhfr
cells (2 × 106). After 2-3 weeks of growth in Iscove's selection
medium, which lacks hypoxanthine and thymidine, clones of CHO-E2 cells
were isolated by limiting dilution; as controls, CHO-E3 cells were produced similarly.
Transgenic Mice Expressing Human ApoE2--
Male transgenic mice
expressing human apoE2 (E
/
/hE2) (18) and female
E
/
mice were mated. DNA was extracted from tail tips of
offspring and screened by PCR at three loci (19); animals positive for human APOE and the hygromycin-resistance marker, but
negative for murine APOE, were selected and maintained on
standard chow.
Cultured Human Lymphocytes--
Lymphocytes were isolated from
the heparinized blood of a patient homozygous for the
2 allele and
immortalized with Epstein-Barr virus (20). Transformed cells were
cultured in RPMI 1640 medium supplemented with 10% FBS (Sigma) and 2 mM L-glutamine.
Chimeraplasty--
The 68-mer oligonucleotides were synthesized
commercially by MWG-Biotech (Ebersberg, Germany) with ten
(fifteen for 88-mers) complementary 2'-O-methyl RNA residues
flanking each side of the 5-base-DNA stretch; four Thr residues in each
loop and a 5-bp GC clamp ensured that they self-associated into a
double-hairpin structure. 24 h prior to transfections cells were
seeded into 6-well plates (2 × 105 cells/well).
Various concentrations (100-1000 nM) of chimeraplasts, preincubated for 10 min with PEI (ExGen 500; TCS Biologicals Ltd., Botolph Claydon, United Kingdom), usually at a 5:1 amine to phosphate molar ratio and in 50 µl containing 150 mM NaCl, were
then added to each well of cells growing in 0.5 ml of serum-containing
medium. After 2-24 h, the monolayers were washed with PBS, 2 ml of
fresh medium was added for 24 h, and then cellular DNA was
extracted. In certain experiments, we continued to passage and culture
portions of the cells, in some cases to isolate clones by limiting
dilution. Gene correction of APOE2 was studied in
vivo using E
/
/hE2 transgenic mice. Here, the
68-mer chimeraplast (1000 nM) was preincubated with PEI
(ExGen 500; 20× concentrated) in a final volume of 100 µl containing
5% glucose, and then the complex was injected into the peritoneal
cavity of one mouse (treated); another mouse received PEI alone
(control). Seven days later, the mice were killed, and their livers
were snap-frozen before performing PCR-RFLP analysis on genomic DNA
extracted from random biopsies.
ApoE Genotyping and DNA Sequencing--
Genomic DNA (DNeasy kit;
Qiagen) was extracted for analysis by PCR-RFLP, amplifying a 244-bp
fragment by 35 cycles (97 °C for 1 min, 63 °C for 1 min, and
72 °C for 1 min) with the primer pair,
5'-GATCAAGCTTCCAATCACAGGCAGGAAG-3' (sense) and
5'-GATCCGGCCGCACACGTCCTCCATG-3' (antisense), and separating the
HhaI-digested products either on 4% agarose
gels or, more commonly, on 20% Tris-buffered EDTA polyacrylamide gels (NOVEX, Groningen, The Netherlands). Each genotype gave a specific combination of HhaI fragment sizes,
and they are as follows: apoE2, 91 and 83 bp; apoE3, 91 and 48 bp; and
a mixed population of apoE2 and apoE3 cells, 91, 83, and 48 bp (21).
The gene conversion frequency was estimated by scanning densitometry,
normalizing the DNA in each lane to the 91-bp band, and using DNA from
an E2/E3 blood donor as a standard. For automatic DNA sequencing of PCR
products, the above sense primer was used on material purified on a
1.5% agarose gel.
RT-PCR--
Poly(A+) RNA was extracted from cells
using the QuickPrep Micro mRNA purification kit (Amersham Pharmacia
Biotech), and after synthesizing first-strand cDNA with a
RETROscript kit (Ambion, Austin, Texas), PCR amplification was
carried out as described for genomic DNA.
Protein Analyses--
Human apoE secreted into culture medium
was measured by a two-antibody sandwich ELISA, using a goat polyclonal
anti-human apoE (DiaSorin Ltd., Wokingham, UK) for capture and
biotinylated goat polyclonal anti-human apoE (Biogenesis Ltd., Poole,
UK) for detection. The standard was calibrated human serum (YSI UK
Ltd., Farnborough, UK), whereas streptavidin-HRP (Amersham Pharmacia Biotech) and 3,3',5,5'-tetramethylbenzidine (TMB; Perbio Science UK Ltd., Tattenhall, UK) were used for color development. ApoE phenotyping was carried out by isoelectric focusing (i.e.f.) and immunoblotting (22), separating desialylated and delipidated proteins
from culture medium or control plasma in a pH 4.0-6.5 gradient using
3% agarose gels. After blotting the proteins onto nitrocellulose
membranes and immunolabeling with a monoclonal antibody against human
apoE, the bands were visualized using goat anti-mouse IgG-HRP conjugate
as secondary antibody and enhanced chemiluminescence (ECL; Amersham
Pharmacia Biotech).
 |
RESULTS |
Conversion of ApoE2 to ApoE3 in Recombinant CHO Cells by a
Synthetic Chimeraplast--
Monolayers of CHOdhfr
cells
were transfected with the selectable expression vector, p7055.E2, and
24 clones stably secreting human apoE2 (CHO-E2) were isolated.
Seventeen clones secreted 0.5-2.0 µg of apoE/ml/24 h, and the four
most productive were expanded (Fig. 1).
In initial experiments, reagent concentrations and transfection times
were varied, treating subconfluent CHO-E2-16 cells for 2-24 h with
100, 200, 400, and 600 nM of a 68-mer RNA-DNA chimeraplast (Fig. 2A), complexed with PEI
at an amine to phosphate molar ratio of 5:1. Cells were washed once
with PBS, fresh medium was added for 24 h, and then DNA was
extracted to assess apoE2 to apoE3 gene conversion by PCR-RFLP
analysis. Clear conversion was seen at each concentration tested,
although 400 nM was the most efficient (Fig. 2B)
with a conversion frequency of 37.7 ± 12.7% (mean ± S.D.;
n = 8; range 14.8-54.8%), as judged by scanning
densitometry. There was little difference between cells treated for 2 or 24 h, or on increasing the PEI to oligonucleotide ratio,
although ratios >10:1 were cytotoxic. Importantly, the PCR-RFLP
pattern was unchanged when CHO-E3 cells were treated with the
chimeraplast (Fig. 2B, lane 3), whereas an E3 to
E2 chimeraplast as negative control did not affect the CHO-E2-16
cells.

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Fig. 1.
Screening clones for secretion of apoE2.
CHO cells were stably transfected with the selectable expression
vector, p7055.E2. 24 clones were isolated by limiting dilution, and
after screening medium for apoE by ELISA, the four most productive
(clones 7, 16, 17, and 18) were expanded.
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Fig. 2.
Conversion of the apoE2 cDNA in
recombinant CHO cells to apoE3 by chimeraplasty. A, the
synthetic 68-mer chimeric oligonucleotide used is shown aligned with
the homologous sequence in the human apoE2 gene. The
2'-O-methylated modified RNA residues are
lowercase, and the DNA residues are capital; the
centre DNA mutator region in shown in bold, and the bottom
all-DNA strand is underlined. The apoE2 to E3 chimeraplast
had X = C and Y = G, and the control apoE3 to E2 chimeraplast
had X = T and Y = A; the single mismatched apoE2 to E3
chimeraplast had X = C and Y = A. The GC clamp is identified
on the left by a nick in the sequence complementary to the
3' to 5' bridge. The targeted nucleotides in apoE cDNA are
double-underlined, whereas the five nucleotides on each
side, which were added to make the 88-mer, are singly
underlined. B, a clear conversion of apoE2 to apoE3
(middle vs. CHO-E3 control cells on the right)
was seen 48 h after transfecting recombinant CHO-E2-16 cells
(left lane of this 4% agarose gel) with the 68-mer·PEI
complex. C, this conversion efficiency was substantially
increased by targeting CHO-E2-16 cells, previously treated with
68-mer·PEI (left lane of this 20% polyacrylamide gel), a
second time (right). D, three additional CHO-E2
clones (numbers 7, 17, and 18; see Fig. 1) were also readily
converted to apoE3.
|
|
When CHO-E2-16 cells, previously treated with chimeraplast (400 nM for 4 h), were targeted a second time the initial
conversion of 37.3% was increased dramatically to nearly 60% (Fig.
2C). To confirm that the apoE2 to apoE3 correction was not
clone-specific, three other CHO-E2 clones (numbers 7, 17, and 18; see
Fig. 1) were studied. Each clone was found to be efficiently converted to a mixture of apoE2/E3 cells with 400 nM of the
chimeraplast (Fig. 2D), and whereas treatment with 200 nM had little effect on line 7, the conversion of lines 17 and 18 was readily apparent (Fig. 2D).
Modifying the Chimeraplast Design--
Chimeraplasts of two
additional designs were evaluated. The first, an 88-mer had 10 extra
RNA residues to strengthen hybridization and potentially enhance
conversion by the enzymatic mismatch repair machinery. This 88-mer was
indeed found to give higher conversion, 23.3% at 400 nM
compared with 14.8% by our standard 68-mer (Fig. 3A). A
second chimeraplast with only a single mismatch (in the all-DNA strand)
against the targeted double-stranded apoE DNA (Fig. 2A) was
also examined, but, though effective at both 400 and 200 nM
(47.7 and 16.7%; see Fig.
3B), it proved inferior to the
standard double-mismatched 68-mer (54.8 and 45.8%; see Fig. 3B).

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Fig. 3.
Modifying the chimeraplast design.
A, a higher conversion efficiency was found using an 88-mer
chimeraplast, whereas in B, a single-mismatched 68-mer
(Single-68) was less efficient than our standard
double-mismatched 68-mer (Std-68).
|
|
Clonal Analysis of Chimeraplast-treated CHO-E2 Cells at the Level
of Genomic Sequence, mRNA, and Protein--
Chimeraplast-treated
CHO-E2-16 cells were passaged 12 times using 1:10 splits, and the apoE
DNA was reanalyzed by PCR-RFLP; the gene correction of apoE2 to apoE3
was unchanged (Fig. 4A, center lane). Next, the passaged cells were cloned, and
after expansion and analysis of nine clones, three different genotypes were found; four clones were apoE2/3, three were apoE2 (uncorrected), and two were apoE3 (corrected). ApoE2 (line 8) and apoE3 (line 3)
clones were then compared with the starting apoE2/apoE3 mixed cell
population (Fig. 4A). Direct DNA sequencing confirmed the genotypes, in particular that the apoE3 genotype had been created by
chimeraplasty (Fig. 4B). Moreover, the expected pattern for true apoE2 and apoE3 clones was found when polyadenylated RNA was
analyzed by RT-PCR-RFLP (Fig. 4C). Importantly,
PCR-independent confirmation of gene correction was obtained by
separating the secreted apoE isoforms using i.e.f. As expected,
non-cloned apoE2/3 cells gave two bands, identical to a plasma control
sample. By contrast, the apoE2 and apoE3 clones gave the predicted
single bands separated by one charge unit (Fig. 4D),
confirming that chimeraplasty had produced a phenotypic change.

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Fig. 4.
Conversion of apoE2 to apoE3 is permanent at
the genomic, mRNA, and protein level. A,
when chimeraplast-treated CHO-E2-16 cells were passaged 12 times (1:10
dilutions), the gene conversion was unchanged (middle), and
both uncorrected apoE2 cells (e.g. #8;
right) and corrected apoE3 cells (e.g.
#3; left) were isolated. B, gene
correction was confirmed by direct sequencing; the PCR product from the
passaged cells had a C nucleotide at position 170, as well as the T
expected for apoE2 (left), whereas the corrected apoE3 clone
(number 3; right) had only C with no evidence of T. C, we also confirmed the correction had occurred at the
mRNA level by RT-PRCR-RFLP analysis of poly(A+) RNA
extracted from the passaged apoE2/E3 cells and from the corrected
(#3) and uncorrected (#8) clones. D,
apoE isoforms secreted into medium were detected by isoelectric
focusing and immunoblotting, using plasma from apoE3/E3 and apoE3/E2
donors as controls. As expected medium from uncorrected cells
(#8) contained only apoE2, whereas the passaged apoE2/E3
cells secreted both apoE2 and apoE3, and the corrected cells
(#3) contained only apoE3. Gene correction by chimeraplasty
results, therefore, in the stable synthesis and secretion of wild-type
apoE3 protein.
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|
Targeting the APOE2 Gene in Transgenic Mice and Human
Lymphocytes--
The APOE2 gene was targeted in
vivo by injecting 1000 nM of the standard
68-mer·PEI complex into a transgenic mice overexpressing human
apoE2; a second animal received PEI alone. DNA extracted from liver was
analyzed 7 days later, and about 25% of hepatic APOE2 was
found to be converted to APOE3 (Fig.
5A). We also targeted APOE2 within the human genome, treating cultured lymphocytes
from a homozygous
2/
2 patient for 16 h with increasing
concentrations (0-1000 nM) of 68-mer·PEI complexes
(amine:phosphate molar ratios between 5:1 and 8:1). Although low
chimeraplast concentrations were ineffective, a clear conversion of
APOE2 to APOE3 was seen at 800 and 1000 nM with a 7:1 ratio of PEI to oligonucleotide giving the
highest conversion (for example, using 800 nM, 34% conversion was seen versus 22 and 3% at 8:1 and 6:1,
respectively; see Fig. 5B).

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Fig. 5.
Targeting the APOE2 gene in
transgenic mice and human lymphocytes. A, a clear conversion
of hepatic APOE2 to APOE3 was seen when the
68-mer chimeraplast was injected into an E / /hE2 mouse.
B, a similarly efficient APOE2 to
APOE3 gene correction occurred when EBV-transformed
lymphocytes from a patient homozygous for the 2 allele were treated
with the 68-mer·PEI complex (800 nM). The highest
conversion was seen at an amine to phosphate molar ratio of 7:1.
|
|
 |
DISCUSSION |
Diverse techniques are being developed to introduce transgenes
into various tissues to express therapeutic proteins. There are many
problems to overcome, achieving efficient gene transfer and specific
gene delivery to target tissues, sustaining levels of gene expression,
preventing adverse immunological responses, and ensuring safety,
particularly with viral vectors (22). By contrast, when defective
proteins result from point mutations in the wild-type allele, the genes
that encode them can potentially be repaired by synthetic RNA-DNA
oligonucleotides, termed chimeraplasts. Chimeraplasty, or targeted gene
correction, is an exciting alternative to viral gene therapy. It has
the great advantage of retaining existing gene promoters and enhancers
and cell-specific context. Although a new technology, there is already
evidence that chimeraplast-induced corrections are permanent and
produce phenotypic conversions. Examples in vitro include
the restoration of tyrosinase activity in melanocytes of albino mice
(23) and correction of the bs hemoglobin allele that causes
sickle cell anemia (24). Phenotypic changes are also reported in
vivo using natural and transgenic preclinical models of disease,
including rat and dog hemophilia B, which have defective clotting
factor IX (25), the Gunn rat model of Crigler-Najjar syndrome (26), and
mouse and dog models of muscular dystrophy (27, 28). As the technique
has also been used in bacteria (29) and plants (30), there appears no
obvious restriction to DNA sequences that can be targeted.
Here, we have used chimeraplasty to correct a dysfunctional variant of
apolipoprotein E, termed apoE2, that causes the genetic disorder of
Type III hyperlipidemia. As established human cell lines secreting
apoE2 are unavailable (we found that hepatoblastoma HepG2 cells (31)
and THP-1 monocyte macrophages (32) were derived from apoE3/E3 donors),
we studied recombinant CHO cells. Initial experiments with 400 nM of a standard 68-mer chimeraplast consistently gave
conversion efficiencies of about 30%, whereas retargeting
substantially increased this number. Moreover, the correction was
specific; CHO-E3 cells were unaffected, while an apoE3 to apoE2
chimeraplast did not convert CHO-E2 cells. Although short stretches of
DNA can enter cells through endocytosis, we used PEI as carrier. This
polycation is a very efficient delivery vehicle for plasmids and
oligonucleotides in vitro (33, 34) and in vivo
(35); it condenses the chimeraplast and inhibits lysosomal degradation
so that nuclear targeting is improved (33).
As the cells initially studied, CHO-E2-16, contained apoE2 cDNA
randomly integrated into the chromosome, it was important to discount a
positional effect as overtly influencing the correction. This was
affirmed by showing that three additional CHO-E2 clones were converted
at comparable rates to the CHO-E2-16 line. Design was important in
increasing efficiency, as a chimeraplast with a single mismatch in the
all-DNA strand was less effective than the standard double-mismatched
68-mer. However, this finding does not corroborate an earlier report
(36) suggesting that the mismatch most efficiently repaired is the one
created between the target and the all-DNA strand of the chimeraplast.
An 88-mer chimeraplast also gave a higher conversion, presumably
because the additional RNA bases strengthened hybridization and formed
a more stable complex with the target sequence (16). However, this does
not necessarily indicate greater practical value; the increased size of
an 88-mer means a more expensive reagent that is also probably of lower
purity than a 68-mer.
Having established that chimeraplasts can target the apoE gene in our
model system, we critically evaluated whether the technique might
repair the apoE2 mutation in patients with Type III hyperlipidemia. First, we determined whether the gene correction was permanent and
passed to future generations of cells and whether wild-type protein was
secreted. Chimeraplast-treated cells were maintained in continuous
culture for 2 months (12 passages at 1:10 splits) and then clones were
isolated. PCR-RFLP analysis showed an unchanged conversion efficiency
and the presence of corrected apoE3. Direct DNA sequencing and RT-PCR
analysis confirmed that chimeraplasty had produced permanent changes at
genomic and mRNA levels, respectively. Conveniently, the single
nucleotide polymorphism distinguishing apoE2 from apoE3 results in an
amino acid change, Cys-158 to Arg-158, and a charge difference between
the two isoforms. This is exploited in routine phenotyping using i.e.f.
to separate plasma apoE isoforms with detection by immunoblotting (37).
We used an identical protocol to detect apoE secreted into culture
medium and to monitor chimeraplast-induced conversion of apoE2 to
wild-type protein. Our analyses confirmed the genotyping data;
chimeraplast-treated CHO-E2 cells secreted both apoE2 and apoE3
isoforms, whereas the corrected apoE3 clone secreted only wild-type protein.
Next, we assessed whether our chimeraplast could efficiently target the
liver, which synthesizes >90% of plasma apoE (38). We used an animal
model of Type III hyperlipidemia, the doubly transgenic
E
/
/hE2 mouse, produced by crossing transgenic mice
overexpressing human apoE2 with apoE-deficient mice (18), and observed
clear conversion of hepatic APOE2 to APOE3. This
was important; it established that another cell type can be effectively
targeted and that the apoE2 gene, rather than apoE2 cDNA, can be
corrected. We now have studies underway to confirm that this finding is
consistent, to improve targeting efficiency, and to study any reversal
of the hyperlipidemia and associated atherosclerosis. Finally, we also wanted to show that our chimeraplast could target the APOE
gene in context within human chromosome 19. We achieved this by
successfully converting EBV-transformed lymphocytes from a homozygous
2/
2 patient to the apoE3 genotype.
Many inherited point mutations in liver-derived proteins are associated
with hyperlipidemia and premature cardiovascular diseases, whether
secreted like apolipoproteins, enzymes, and transfer proteins or
retained like the LDL receptor. Nevertheless, apoE is a good candidate
for generic gene therapy as it has multiple antiatherogenic actions,
both within the circulation and at lesion sites (39). Indeed,
adenoviral-mediated expression of apoE3 from liver significantly regresses early fatty streaks in the arterial wall, as well as advanced
lesions (40, 41). The apoE2 isoform has defective binding to the LDL
receptor and LDL receptor-related protein, leading to impaired
clearance of atherogenic remnant lipoproteins from the circulation
(42). ApoE2 is also much less efficient at sequestering cellular
cholesterol than apoE3 (3). We predict, therefore, that using
chimeraplasty to convert apoE2 into apoE3 will reduce progression of
atherosclerotic lesions or even promote regression in patients with
Type III hyperlipidemia; heterozygous apoE2/E3 subjects do not have
premature atherosclerosis. Thus, our present finding that the
APOE2 gene can be targeted and efficiently converted to
APOE3, resulting in secretion of apoE3 protein, is significant. Furthermore, it implies that apoE4 can be converted to
apoE3, an intriguing possibility as the
4 allele is strongly associated with Alzheimer's disease and a variety of other
neurodegenerative disorders (43). Clearly, further studies in
vitro and in preclinical models will be needed to realize such
long term goals.
 |
ACKNOWLEDGEMENTS |
We are grateful to several colleagues for the
generous provision of reagents; we thank L. Havekes and K. Willems van
Dijk for providing transgenic E
/
/hE2 mouse, P. Cullen
and B. Pottinger for EBV-transformed lymphocytes, J. Breslow for
human apoE cDNA, and B. Miloux for p7055 expression vector.
 |
FOOTNOTES |
*
This work was supported in part by Project Grant PG/99032
and by Ph.D. Studentship FS/97054 (to A. D. T.) from the British Heart Foundation.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Dept. of Medicine,
Royal Free and University College Medical School, University College
London, Royal Free Campus, London NW3 2PF, UK. Tel.: 44-207-4332853; Fax: 44-207-4332852; E-mail: j.owen@rfc.ucl.ac.uk.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.C000883200
 |
ABBREVIATIONS |
The abbreviations used are:
apo, apolipoprotein;
CHO, Chinese hamster ovary;
DHFR, dihydrofolate reductase;
FBS, fetal
bovine serum;
PCR, polymerase chain reaction;
bp, base pair;
PEI, polyethyleneimine;
PBS, phosphate-buffered saline;
RFLP, restriction
fragment length polymorphism;
RT, reverse transcription;
ELISA, enzyme-linked immunosorbent assay;
HRP, horseradish peroxidase;
i.e.f., isoelectric focusing;
EBV, Epstein-Barr virus;
LDL, low density
lipoprotein.
 |
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