(Circulation. 2001;103:1274.)
© 2001 American Heart Association, Inc.
Basic Science Reports |
Long-Term Stable Correction of Low-Density Lipoprotein Receptor–Deficient Mice With a Helper-Dependent Adenoviral Vector Expressing the Very Low-Density Lipoprotein Receptor
From the Departments of Molecular and Cellular Biology (K.O., I.-H.K., A.M., S.N., H.-J.L., M.M.-S., C.A.-R., L.C.), Molecular and Human Genetics (L.P., B.L., A.B.), Pathology (M.F.), and Medicine (L.C.), Baylor College of Medicine, Houston, Texas.
Correspondence to Dr Lawrence Chan, Departments of Molecular and Cellular Biology and Medicine, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. E-mail lchan{at}bcm.tmc.edu
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Abstract |
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Background—Familial hypercholesterolemia (FH) that results from LDL receptor (LDLR) deficiency affects
1 in 500 persons in the heterozygous state
and 1 in 1 million persons in the homozygous state. We tested
a novel gene therapy strategy for the treatment of FH in a mouse
model.
Methods and Results—We
delivered the VLDL receptor (VLDLR) to the liver of LDLR-deficient mice
and compared the effect of a helper-dependent adenoviral vector with
all viral coding sequences deleted (HD-Ad-mVLDLR) with a
first-generation vector (FG-Ad-mVLDLR), an HD-Ad (HD-Ad-0) that
contained no expression cassette, and dialysis buffer (DB). A single
intravenous injection of HD-Ad-mVLDLR led to a lowering of plasma
cholesterol that lasted 
6 months. Acute liver toxicity (as measured
with liver enzyme elevation) occurred after FG-Ad-mVLDLR but not after
HD-Ad-mVLDLR, HD-Ad-0, or DB treatment. At 6 months, VLDLR was detected
in the liver with Western blotting and with immunofluorescence staining
only in HD-Ad-mVLDLR–treated mice. Aortic atherosclerosis was almost
completely prevented in these animals.
Conclusions—HD-Ad–mediated intravenous delivery of VLDLR to hepatocytes is well tolerated. It produces long-term lowering of plasma cholesterol and prevents atherosclerosis development in LDLR-deficient mice. These data provide support for the feasibility and safety of this approach for therapy of human subjects.
Key Words: genes • adenovirus • receptors • lipoproteins • hypercholesterolemia
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Familial hypercholesterolemia (FH) is an autosomal dominant disorder that is caused by mutations in the LDL receptor (LDLR).1 2 With optimal pharmaceutical intervention, the plasma LDL cholesterol of the majority of patients with heterozygous FH can be maintained below 2.59 to 3.36 mmol/L (100 to 130 mg/dL). However, the majority of homozygous and a minority of heterozygous patients have proved to be resistant to treatment with conventional therapy. For these patients, the only other therapeutic option is LDL apheresis.3 However, there is rapid reaccumulation of the LDL, and the procedure must be repeated every 2 to 3 weeks to maintain the LDL cholesterol transiently below 2.59 to 3.36 mmol/L. Because of the difficulty in reversing the hypercholesterolemia of this subgroup of FH patients, somatic gene therapy has been examined as an alternative form of treatment.
The only clinical trial for somatic gene therapy of FH used a retroviral vector to deliver normal LDLR by transducing hepatocytes isolated from such patients and reimplanting them into the patients.4 Unfortunately, the therapeutic response in 5 patients treated by ex vivo gene transfer was only marginal, which "precludes a broader application of liver-directed gene therapy" via this approach.4
In vivo transfer of the LDLR gene with first-generation adenoviral vectors to LDLR-deficient mice5 and WHHL rabbits6 7 was highly effective in producing a transient lowering of the plasma cholesterol in these animals. Furthermore, the in vivo transfer of the VLDL receptor (VLDLR), a paralogue of the LDLR, to the liver of LDLR-deficient mice was also found to be effective in reversing the hypercholesterolemia of these animals.8 9
The VLDLR is a member of the LDLR gene family.10 It is normally expressed in heart, skeletal muscle, adipose, and other tissues but is essentially undetectable in liver. When expressed in cultured cells, the VLDLR binds to apoE-enriched lipoproteins but not to LDL, which contains only apoB-100.11 VLDLR gene therapy in LDLR-deficient mice produced a substantially more sustained hypocholesterolemic response than did LDLR. The superiority of the surrogate receptor (VLDLR) over the "natural" receptor (LDLR) was attributable to the fact that LDLR was recognized as a neoantigen by the LDLR-deficient animals, which mounted humoral and cellular immune responses to the LDLR, leading to the extinction of transgene expression, whereas ectopic expression of VLDLR did not induce an immune response.8 Therefore, VLDLR appears to be an excellent surrogate gene for the treatment of FH, especially in the context of complete LDLR deficiency.
Despite the much more protracted transgene expression, adenovirus-mediated VLDLR gene transfer resulted in metabolic correction that lasted only 3 to 9 weeks.8 9 This was not unexpected, because the vector that was used, a first-generation adenoviral (FG-Ad) vector, had been shown to induce inflammation and an immune response.12 13
HD-Ad vectors devoid of all viral coding regions were
developed in an attempt to reduce the immunogenicity of the vector and
to prolong transgene
expression.14 15
The recent development of helper-dependent systems that use a FG-Ad
helper virus to provide the necessary viral proteins in trans for the
packaging of the HD-Ad vectors greatly facilitates their large-scale
production.15 16
Gene delivery to the liver with 
1-antitrypsin
as a reporter gene in
mice17 18 and
baboons19 led to long-term
stable transgene expression that lasted for >1 year. In this
communication, we compared the use of a first-generation vector
(FG-Ad-mVLDLR) with a helper-dependent vector (HD-Ad-mVLDLR) to deliver
the VLDLR gene to the liver of LDLR-deficient
mice.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Recombinant Adenovirus
For the construction of all of the HD-Ad vectors, the p
28 plasmid
(Figure 1
) was used as backbone. To generate p28, we
started from the pSTK51 plasmid that contains the Ad5 left ITR and
packaging signals (Ad bp 1 to 440), a 16 054-bp
EclXI/PmeI
(bp 1799/center to bp 17 853/Left) from the hypoxanthine
phosphoribosyltransferase (HPRT) gene (GenBank:Humhprtb), and the Ad5
right ITR (Ad bp 35 818 to 35 935). To increase the size of the
stuffer DNA in the plasmid, we inserted a 11 105-bp
BamHI (bp 15 730/center to bp
26 835/Left) fragment from the cosmid C346 (GenBank L31948) to
derive the p28 plasmid, which has a size of 27.3 kb without
the pBluescript backbone. A unique
AscI site was retained between
the C346 DNA stuffer and the right ITR of p28 for the cloning of the
expression cassette. The right and left ITRs in p28 are flanked by 2
PmeI sites used to free the
adenoviral ends before transfection. (The complete sequence of the
backbone plasmid is available on request.)
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, Ad packaging sequence.
We generated a shuttle plasmid, pLPBL1, which was derived
from pGEM7 and contains a multicloning site flanked by 2
AscI sites. We produced the
construct HD-Ad-mVLDLR by subcloning a 2.15-kb
SstI/BglII
fragment of the phosphoenolpyruvate carboxykinase (PEPCK)
promoter,20 a fragment of
human apoA-I intron 1 with the immediately flanking region of exons 1
to 2 (bp 474 to 698, GenBank J00098.1), a 3.1-kb mouse VLDLR cDNA
fragment modified to have the
BglII site at the 5'-end and a
CCACC sequence immediately 5' to the ATG codon, and the bovine
ß-globin polyadenylation signal into the
SstI/HindIII
site of pLPBL1. We subcloned the PEPCK-mVLDLR expression cassette into
the AscI site of p28, which
yields a HD-Ad vector with a size appropriate for efficient and stable
packaging.21 The resulting
plasmid was linearlized by PmeI
digestion before transfection into 293Cre4 cells. Rescue and
amplification of the HD vector were performed with 293Cre4 cells
through coinfection with AdLC8cLuc1 helper-virus according to the
method of Parks et al.16 The
recombinant HD-Ad was purified twice through CsCl density
ultracentrifugation and dialyzed against 10 mmol/L Tris, pH 7.4, 1
mmol/L MgCl2, 10% (v/v) glycerol (DB) at 4°C,
and aliquots of purified vectors were stored at -85°C. FG-Ad-mVLDLR
was prepared as described
previously.8 The ratio of
particle to infectious unit was 40:1 with
FG-Ad-mVLDLR.
Animals
Female LDLR-deficient
mice5 (6 to 8 weeks of age)
on a C57BL/6 background were fed a diet containing 0.2% (wt/wt)
cholesterol and 10% (v/w) coconut oil, which maintained the plasma
cholesterol level at 9.05 to 14.22 mmol/L. After 4 weeks of the diet,
various vectors in dialysis buffer were injected into mice through the
tail vein. On the days indicated, mice were anesthetized with
Methofane (methoxy flurane) and blood was collected into tubes
containing EDTA from the retro-orbital plexus.
Polymerase Chain Reaction Analysis of Vector
DNA and Reverse Transcription–Polymerase Chain Reaction Analysis of
Vector-Derived mVLDLR Transcripts in Various Mouse Tissues
The presence of adenoviral ITR in various tissues was
detected with polymerase chain reaction (PCR). Primers used were
5'-AGCCAATATGATAATGAGGGGGTG-3' and
5'-TACGCGCTA-TGAGTAACACAAA-3'. The
apoe gene was used as an
internal control. PCR primers for
apoe were exon 3 forward,
5'-GAACCGCTTCTGGGATTACCT-3', and exon 4 reverse,
5'-GCGCTCACGGATGGCACTCAC-3'. To analyze for vector-specific VLDLR mRNA,
we used vector-specific PCR primers to perform reverse transcription
(RT)-PCR. Total RNA was isolated from mouse tissues with an RNeasy kit
(QIAGEN Inc); residual DNA was removed with DNase I (Promega Corp)
digestion. The forward primer was derived from the human apoA-I exon
sequences 5' to the mVLDLR cDNA (5'-GAAGGAGGTCCCCCACGGCCC-3'), and the
reverse primer was complementary to mouse VLDLR cDNA
(5'-TTCACCATCGCATCTCCAGGAC-3'). The endogenous GAPDH mRNA was used as
an internal PCR control with primers 5'-CCCCCTATTGACCTCAACTACATGG-3'
and 5'-CCTGCTTCACCACCTTCTTGAC-3'. The amplified products were
fractionated on 1.5% agarose gel.
Immunohistochemistry
Liver tissue was frozen in tissue freezing medium
(Triangle Biomedical Sciences) in liquid nitrogen. Cryosections (5 µm
thick) were fixed in cold acetone at -20°C and used for
immunofluorescence
staining.22 The primary
antibody was a rabbit anti-mouse C-terminal
peptide8 purified over a
peptide affinity column. The sections were incubated with a purified
antibody at a concentration of 2 to 10 µg/mL for 1 hour at 37°C.
The FITC-conjugated goat anti-rabbit immunoglobulin diluted to 1 to 5
µg/mL in saline was used for detection.
Other Procedures
Lipoprotein measurements, lipid and FPLC analyses,
and rate of 125I-IDL disappearance in plasma
were determined as previously
described.8 Southern blotting
and immunoblot analyses were performed as described
previously8 with probes and
restriction enzymes and antibodies specified in the figure legends.
Liver sections were prepared for histological examination as previously
described.23 Quantitative
analyses of aortic atherosclerotic lesion areas were performed as
previously described.24
Statistical analyses were made with ANOVA with SigmaStat
(SPSS).
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Construction and Amplification of VLDLR and Control Vectors
Three types of adenoviral vectors were constructed: a first-generation VLDLR vector (FG-Ad-mVLDLR), a helper-dependent VLDLR vector (HD-Ad-mVLDLR), and an empty HD-Ad vector (HD-Ad-0). During the production of HD-Ad-mVLDLR and HD-Ad-0, possible DNA rearrangement was monitored throughout the process. As shown in Figure 2a
, for HD-Ad-mVLDLR, we did not detect any
rearrangement of the DNA by restriction mapping with the 3 different
enzymes. Helper virus contamination was analyzed through Southern
blotting with an ITR probe. The
PstI digestion of the helper
virus produces a banding pattern that is distinct from that of the
HD-Ad-mVLDLR or HD-Ad-0. We did not detect any helper-virus bands in
the HD-Ad-mVLDLR
(Figure 2b) or HD-AD-0 preparations (data not shown). TaqMan
PCR analysis indicated that helper virus contamination was ~0.2%. We
performed two independent experiments with different doses of
HD-Ad-mVLDLR. We used a cytomegalovirus promoter–driven FG-Ad-mVLDLR
as a FG-Ad control, because a PEPCK promoter–driven FG-Ad-mVLDLR was
ineffective in reducing plasma cholesterol in LDLR-deficient mice (Oka
et al, unpublished results).
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Comparison of HD-Ad-mVLDLR, FG-Ad-mVLDLR, and
DB Injection in LDLR-Deficient Mice
In the first experiment (low dose), an equivalent dose
of HD-Ad-mVLDLR and FG-Ad-mVLDLR (1x1011
particles per mouse or 5x1012 particles/kg)
or an equal volume of DB was injected intravenously via the tail vein
into LDLR-deficient mice. Liver enzymes were monitored weekly after
treatment. As shown in
Figure 3 (left), marked elevations of plasma AST and ALT
levels were observed with FG-Ad-mVLDLR, but enzyme levels remained
unchanged after HD-Ad-mVLDLR or DB treatment.
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DB treatment was associated with a small dip in plasma
cholesterol levels during the first month, which we believe was caused
by the substantial blood loss that resulted from the repeated
phlebotomies in all 3 groups of animals
(Figure 4, top). The cholesterol levels in DB-treated mice
remained at 7.76 to 10.34 mmol/L (mean±SEM, n=10) thereafter.
FG-Ad-mVLDLR treatment caused an early drop in plasma cholesterol
levels that occurred within 1 week after vector injection. Levels
gradually returned toward pretreatment values thereafter and did not
differ from those of the DB-treated control animals after day 28.
HD-Ad-mVLDLR injection led to a more gradual but sustained lowering of
the plasma cholesterol level. The hypolipidemic effect was evident in 7
days, but cholesterol levels did not reach a nadir until 28 days after
treatment. The cholesterol levels remained significantly lower than
those in DB-treated animals at 161 days (6.28±0.98 and 8.26±1.37
mmol/L, respectively, for HD-Ad-mVLDLR– and DB-treated animals,
P<0.01) and significantly
lower than those of FG-Ad-mVLDLR–treated animals at 196 days after
injection (8.28±1.29 and 10.16±0.98 mmol/L, respectively, for
HD-Ad-mVLDLR– and FG-Ad-mVLDLR–treated animals,
P<0.05).
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, DB control, n=10; 
, FG-Ad-mVLDLR (1x10
P<0.001, HD-Ad vs the other 2 groups. 
P<0.001 HD-Ad vs DB and P<0.05 HD-Ad vs FG-Ad. ¶P<0.001 FG-Ad vs other 2 groups. Bottom, High-dose experiment: 
, HD-Ad-mVLDLR (3x10
Comparison of HD-Ad-mVLDR and HD-AD-0 Treatment
in LDLR-Deficient Mice
In the second experiment, we administered
3x1011 particles per mouse of HD-AD-0 or
HD-Ad-mVLDLR. There was no change in plasma ALT or AST activities in
either treatment group
(Figure 3
, right). The basal plasma cholesterol level
(Figure 4, bottom) was the same in the 2 groups (mean±SEM, 11.82±1.63 and
11.72±1.58 mmo/L, respectively, for the HD-Ad-0 and HD-Ad-mVLDLR
groups). For the HD-AD-0–treated mice, the level increased slightly
before it showed a slight decline, leveling off at 8.53±1.37 mmol/L
(n=12) at 161 days. In the HD-Ad-mVLDLR–treated mice, there was a
sharp decline in plasma cholesterol from 11.72±1.58 to 3.08±0.52
mmol/L (n=11) at 28 days. It then displayed a gradual upward trend; at
161 days, the plasma cholesterol level in HD-Ad-mVLDLR–treated mice
was 5.74±0.54 mmol/L, still significantly lower than that of
HD-Ad-0–treated control animals
(P<0.001).
At 140 days after treatment, plasma lipoproteins were
fractionated with FPLC
(Figure 5). In HD-Ad-0 control animals, there is a minor VLDL
peak, a prominent IDL/LDL peak, and a moderate HDL peak. In
HD-Ad-mVLDLR–treated animals there was a decrease in all 3 peaks. The
lowering was most marked in the IDL/LDL peak. Although considerable
lowering of the HDL peak was also evident, there was a major reduction
in the ratio of VLDL/IDL/LDL to HDL, suggesting that the HD-Ad-mVLDLR
treatment led to a marked reduction in the atherogenic potential of the
originally highly atherogenic plasma lipoproteins in these animals.
Such a reduction in HDL cholesterol associated with a decreased plasma
apoAI level has been observed in mice treated with a first-generation
virus.8 This may be secondary
to the marked reduction of IDL, which results in insufficient transfer
of the surface components for HDL assembly by triglyceride
lipase–mediated remodeling of IDL particles.
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We showed previously that induced hepatic VLDLR
overexpression specifically targets the removal of IDL compared with
other lipoproteins.8 At 63
days after HD-Ad-0 or HD-Ad-mVLDLR treatment, we measured the rate of
disappearance of 125I-IDL
(Figure 6). In the HD-Ad-0–treated mice,
125I-IDL disappeared from plasma with a
half-life of 57±2 minutes (mean±SD for 4 mice). In the
HD-Ad-mVLDLR–treated mice, 125I-IDL
disappeared with a half-life of 18±1 minutes. Therefore, HD-Ad-mVLDLR
treatment accelerates IDL removal from the circulation by 300%.
Because IDL is a precursor of LDL, both IDL and LDL are lowered as a
result of the therapy.
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Tissue Distribution of Vector and Transgene
Expression
We tested the distribution of HD-Ad-mVLDLR vector DNA
in the various mouse tissues at the end of the second experiment.
Through PCR analysis with vector-specific primers and with the
endogenous apoe locus used as a
single-copy gene control, HD-Ad-mVLDLR DNA was detected mainly in the
liver. It was also detected, albeit at much lower levels (substantially
<1 copy per cell), in lung, adipose tissue, and spleen. It was also
present, but barely detectable, in small intestine and muscle (data not
shown). We performed RT-PCR on RNA isolated from these tissues using
vector transcript-specific primers and found that of all of the tissues
that harbored vector DNA, only the liver expressed vector-derived
mVLDLR transcripts
(Figure 7a).
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VLDLR transgene expression was further examined in liver
samples through Western blotting
(Figure 7b). At the end of 196 days (experiment 1), VLDLR
protein expression was undetectable in DB or FG-Ad-mVLDLR animals. It
was clearly detected in the HD-Ad-mVLDLR–treated animals. In
experiment 2, at the end of 168 days, VLDLR protein expression was not
detected in HD-Ad-0 animals but was strongly positive in HD-Ad-mVLDLR
animals. We further analyzed for VLDLR protein expression through
immunofluorescence staining in liver sections from the second
experiment
(Figure 7c). We found that at 28 days after HD-Ad-mVLDLR
treatment, >95% of hepatocytes expressed VLDLR protein, and at 168
days, 30% to 35% of the liver cells displayed immunoreactive VLDLR
detectable with the monospecific polyclonal antibody. VLDLR was
undetectable in the liver of HD-Ad-0–treated
mice.
Liver Histopathology of HD-Ad-mVLDLR–Treated
and Control Mice
Liver pathology was examined by a pathologist (M.J.F.)
who was blinded to the treatment the mice received. Five groups of mice
were examined in the 2 sets of experiments. All except the DB control
animals displayed focal portal tract inflammation with minimal
differences among the groups. None of the livers displayed
architectural disturbances, and there was minimal hepatocellular
necrosis, consisting of focal cell dropout in association with
lymphocytic infiltrates. The latter were frequent among all groups (all
mice receiving vectors, 63% of DB controls in experiment 1, and
~55% of mice in experiment 2), possibly reflecting the endemic
presence of mouse pathogens in the mouse room used. Some livers in all
groups of mice also displayed focal proliferation of bile ductules in
the lobules, and in a few livers of each group, there was mild
hepatocyte anisocytosis. The histological alterations in some of the
mice exposed to Ad vectors suggest the possibility that entry of the
vector, whether FG-Ad or HD-Ad, may produce some injury to the liver
that is still evident after several months. Finally, the average amount
of hepatocellular steatosis in the groups was slightly less in the
controls than in the VLDLR vector recipients, which may have been
caused by efficient uptake of lipoprotein. However, this was not
statistically significant.
Induced Hepatic VLDLR Expression Prevents
Aortic Atherosclerosis Development in LDLR-Deficient Mice
We measured the extent of aortic atherosclerotic lesion
involvement by quantitative
morphometry.24 At 196 days
(Figure 8, left), the DB-treated control group had the most
extensive lesions, averaging 7.77±1.50 mm2
(mean±SD, n=9). The FG-Ad-mVLDLR group, at 5.07±2.29
mm2 (n=7), displayed a mild (35%) but
significant reduction in lesion area. The HD-Ad-mVLDLR group had an
even smaller lesion area of 3.38±1.10 mm2
(n=9), a 57% protection. In the second experiment
(Figure 8, right), at the end of 168 days, the aortas of the
HD-Ad-0 group displayed an average lesion area of 5.41±1.03
mm2 (n=12). In the HD-Ad-mVLDLR group,
atherosclerosis lesion development was almost completely prevented. The
lesion area of 0.69±0.29 mm2 (n=11) in our
experience is only slightly above that seen in wild-type mice and
represented an 87% protection compared with the extensive lesions seen
in HD-Ad-0 animals.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Liver-directed LDLR gene transfer in vivo5 6 7 was found to be an effective means to reverse the hypercholesterlemia in 2 FH animal models: the WHHL rabbit25 and the LDLR-deficient mouse.5 However, the effect of in vivo LDLR gene transfer was transient because a FG-Ad was used in these studies. In addition, the LDLR induced de novo is recognized as a neoantigen in LDLR-deficient mice, and the resultant humoral and cellular immune response led to the extinction of transgene expression.9 The use of VLDLR instead of LDLR circumvents this problem. Another potential advantage of VLDLR over LDLR in specific clinical situations is that VLDLR binds to apoE226 27 and some dominant apoE mutants27 with higher affinity than does LDLR. It may be more effective than the latter in reversing the abnormal lipids in the dyslipidemias associated with some apoE mutants.27 Unlike humans, mice have substantial hepatic apoB mRNA editing activity, resulting in IDL that contains both apoB100 and apoB48. Hepatic expression of the VLDLR was found to be more effective in lowering apoB100 than in lowereing apoB488 ; thus, VLDLR gene therapy may be more effective in humans who produce only apoB-100 in the liver. Our observations, and those in 2 previous reports,8 9 support the efficacy of the VLDLR as an effective surrogate lipoprotein receptor gene for the treatment of FH.
As a liver-directed gene-transfer vector, Ad offers some
advantages as well as
drawbacks.14 The major
advantages include the high efficiency of transduction and the
propensity of the vector administered intravenously to reach
hepatocytes. The liver selectivity is not absolute, however, as
evidenced by the presence of small amounts of vector DNA in tissues
other than the liver. The PEPCK promoter used for the HD-Ad-mVLDLR
construct confers an additional level of specificity. In
HD-Ad-mVLDLR–treated mice, we could detect by RT-PCR vector-directed
VLDLR mRNA exclusively in the liver
(Figure 7a) and not in any other tissues tested. These data
indicate that the combination of liver-specific uptake and the
promoter-mediated tissue specificity led to an essentially absolute
liver specificity of HD-Ad transgene expression. All previously
reported studies on FH animal models have used ubiquitously expressed
promoters. We believe that use of a liver-specific promoter such as
PEPCK would avoid some of the problems, such as enhanced immune
stimulation,28 that are
associated with promiscuous transgene expression.
The high efficiency of transduction is another desirable
property of Ad vectors. We found that the use of HD-Ad-mVLDLR at a dose
of 3x1011 particles per mouse produced
VLDLR transduction in >95% of the hepatocytes at 28 days. At the end
of a 6-month period, 30% to 35% of the liver cells still expressed
immunoreactive VLDLR. This contrasts with 
5% hepatocyte transduction
after the direct intraportal administration of an adeno-associated
viral vector in
mice.29
Many of the drawbacks of FG-Ad vectors have been
circumvented by the generation of HD-Ad
vectors.14 Despite the
relatively prolonged expression of the VLDLR after a single injection
of HD-Ad-mVLDLR, there was a gradual decline in both the level of
expression (with decreasing proportion of liver cells expressing the
VLDLR with time, from >95% at 28 days to 30% to 35% at 168 days)
and the plasma lipid-lowering effect. A major reason for this finite
period of transgene expression is the fact that the Ad chromosomal
integration is extremely
low.30 Reinjection of the
same HD-Ad vector is generally ineffectual because of the induction of
neutralizing antibodies to the Ad vector. However, this problem can be
circumvented by the use of HD-Ad generated with a helper Ad virus of a
different serotype, because neutralizing antibodies to Ad are serotype
specific. In fact, Parks et
al31 recently demonstrated
the feasibility of such an approach. Theoretically, if each HD-Ad
vector injection produces a therapeutic effect that lasts 6 months,
reinjections of HD-Ad vectors of different serotypes should enable the
same HD-Ad vector to be readministered multiple times (only the helper
virus has to be changed; the therapeutic vector HD-Ad remains the same)
and the maintenance of a therapeutic response that lasts for years. The
use of different helper serotypes might also permit reinjection in
patients who received subtherapeutic doses of vectors early in clinical
trials to later receive higher doses of HD-Ad vectors.
In conclusion, we have shown that HD-Ad–mediated gene transfer of VLDLR to the liver of a mouse model of FH produces long-term correction of the hypercholesterolemia and prevention of aortic atherosclerosis. The use of HD-Ad in this study greatly attenuated the acute liver injury associated with FG-Ad.32 The results are encouraging, and further evaluation of this therapeutic strategy for the treatment of FH is warranted.
While this article was under review, Chen et al33 reported delivering the VLDLR to the liver of LDLR-deficient mice using an adeno-associated viral vector. They injected intra-portally via the spleen 1x1012 particles per mouse. The plasma cholesterol levels were as follows (mean±SEM): preinjection, 1404±166 mg/dL; 3 months after injection, 1126±486 mg/dL; and 6 months after injection, 864±204 mg/dL. Immunocytochemical staining showed that 2% to 5% of the liver cells expressed VLDLR at 3 and 6 months. After 6 months, aortic atherosclerotic lesion area was reduced 33% compared with PBS controls.
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Acknowledgments |
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This work was supported by grant HL-59314 from the National Institutes of Health. Dr Lee was supported by KOSEF (Korean Science and Engineering Foundation). Dr Merched was supported by ARCOL (French Committee for the Coordination of Research on Atherosclerosis and Cholesterol). We thank Merck & Co. for providing reagents developed by Dr Frank Graham of McMaster University, Hamilton, Canada; Dr Guojun Bu of Washington University, St Louis, Mo, for providing a monospecific rabbit anti-VLDLR antibody (used for Western blotting); Ekaterina Antonova for technical assistance; and Dr Lanny Haverkamp for assistance with capture of the immunohistochemical images.
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Footnotes |
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Guest Editor for this article was Peter Libby, MD, Brigham and Women’s Hospital, Boston, Mass.
Received June 12, 2000; revision received September 27, 2000; accepted September 29, 2000.
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References |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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