© 1999 American Heart Association, Inc.
Atherosclerosis and Lipoproteins |
Efficient Nuclear Delivery of Antisense Oligodeoxynucleotides and Selective Inhibition of CETP Expression by Apo E Peptide in a Human CETP–Stably Transfected CHO Cell Line
From the Departments of Internal Medicine (K.L., J.O., K.S., B.Z., K.A.) and Pathology (S.J.), Fukuoka University School of Medicine, Fukuoka, Japan, and the Department of Internal Medicine (D.P.V., J.T.S., H.J.P. L.C.S.), Baylor College of Medicine, Houston, Tex.
Correspondence to Keijiro Saku, MD, PhD, FACP, Department of Internal Medicine, Fukuoka University School of Medicine, 7-45-1 Nanakuma Jonan-ku, Fukuoka 814-80, Japan. E-mail hh035399{at}msat.fukuoka-u.ac.jp
 |
Abstract |
|---|
 Top Abstract IntroductionMethodsResultsDiscussionReferences |
|---|
Abstract—N,N-Dipalmitylglycyl–apolipoprotein E (129–169) peptide (dpGapoE) is an efficient gene delivery system for both plasmids and antisense oligodeoxynucleotides (ODNs). To develop a new and efficient approach to the regulation of cholesteryl ester transfer protein (CETP) expression, we used dpGapoE to transfect phosphorothioate antisense ODNs against nucleotides 329 to 349 of human CETP cDNA into a human CETP–stably transfected Chinese hamster ovary (CHO) cell line (hCETP-CHO). After transfection, translocation to the nuclei and concentration in nuclear structures were observed in >95% of the cells at 6 and 12 hours by fluorescence microscopy. No membrane disruption was observed after transfection of ODNs by dpGapoE. Although the translocation stability of phosphorothioate ODNs in the nuclei continued for >48 hours, it had weakened after 24 hours. Cellular CETP mRNA levels gradually declined, and the maximum reduction in the mRNA level (>50%) was observed at 36 hours, after which the mRNA level started to recover. CETP activity in the culture medium declined over 72 hours. The maximum reduction in CETP activity was observed at 36 hours (53.8% of control). Neither CETP mRNA nor CETP activities changed throughout the experiment after the transfection of sense phosphorothioate ODNs delivered by dpGapoE complex or naked antisense ODNs. We conclude that (1) the novel synthetic dpGapoE was a highly effective and nontoxic vehicle for the nuclear delivery of antisense ODNs into hCETP-CHO cells and (2) antisense ODNs selectively inhibited both CETP expression and activity in an hCETP-CHO cell line. This approach may enable gene regulation in vivo and could possibly be used as an antiatherosclerotic agent to alter high density lipoprotein metabolism.
Key Words: peptide vectors • receptor-mediated gene transfer • LDL receptor • gene therapy • in vitro studies
|
Introduction |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Over the past decade, antisense oligodeoxynucleotides (AS-ODNs) have been recognized as a new generation of putative therapeutic agents.1 2 3 The use of ODNs as therapeutic tools requires that they enter target cells and interact with their cellular targets to have a therapeutic effect.2 However, ODNs are usually unable to enter most cell types with high efficiency. Therefore, some form of efficient nuclear delivery system is essential.3 The most commonly used nucleic acid delivery techniques, including microinjection, viral vector delivery, electroporation, and transfection with cationic lipids, are characterized by limitations and drawbacks.4 Most clinical trials of gene therapy make use of retroviral or adenoviral vectors, which are potentially very efficient. However, 2 factors suggest that nonviral gene delivery systems will be the preferred choice in the future, ie, safety and ease of manufacture.5 Cationic lipids have been used to successfully transfect plasmid DNA and AS-ODNs into various cells both in vitro and in vivo6 7 8 ; however, they are toxic and exhibit markedly decreased activity in the presence of serum.7 8 Recently, receptor-mediated gene transfer has been developed, in which DNA is conjugated with polylysine and a cell-specific carrier molecule that is the ligand for a surface receptor, such as transferrin,9 10 asialoglycoprotein,11 polymeric immunoglobulin,12 mannose,13 folic acid,14 and serpin enzyme complex receptors.15 Peptide-mediated gene transfer has also been developed, in which peptide condenses DNA or ODNs, which are then delivered into cells.4 16 17 18
Our primary hypothesis is that highly efficient gene delivery can be achieved by using receptor-mediated delivery. The liver is of central importance to metabolism and is an obvious target for gene therapy, because many human metabolic disorders are secondary to hepatic enzyme dysfunction. The liver has many LDL receptors that are potential sites for the delivery of exogenous DNA via an LDL receptor ligand. N,N-Dipalmitylglycyl–apo E (129–169) (dpGapoE) contains the minimum determinants for binding to both lipid surfaces and the LDL receptor.19 In preliminary studies, we demonstrated that dpGapoE could act as an efficient gene delivery system for both plasmids and AS-ODNs.20
Atherosclerosis is a multifactorial disease that encompasses both genetic and environmental factors. Numerous epidemiological studies have shown that lipids/lipoproteins play a major role in the evolution of atherosclerosis. HDL continues to attract attention and has assumed increasing importance in coronary heart disease. The protective effect of HDL is probably due to its role in reverse cholesterol transport from peripheral tissues to the liver,21 which is a key process in modulating cholesterol metabolism in peripheral tissues. Lecithin:cholesterol acyltransferase (LCAT) is the key enzyme in this process, whereas cholesteryl ester transfer protein (CETP) may be regarded as a potentially atherogenic factor that bypasses the reverse cholesterol transport pathway by transferring cholesterol ester molecules from antiatherogenic HDL to proatherogenic apo B–containing lipoproteins.22 To develop a new approach to the regulation of CETP expression in vivo, we investigated the translocation and gene regulation of human CETP AS-ODNs delivered by dpGapoE in a human CETP gene–stably transfected Chinese hamster ovary (CHO) cell line. This is the first demonstrated use of an LDL receptor–binding peptide in a nonviral gene delivery system. The combined use of this peptide and AS-ODNs achieved the effective downregulation of CETP expression in vitro.
|
Methods |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Cell Culture
A CHO cell line that was stably transfected with a human CETP gene was kindly provided by Dr Alan R. Tall, Columbia University, New York, NY. This cell line23 was produced by cotransfecting CHO cells with plasmids containing the cloned human CETP gene and the dihydrofolate reductase gene and was selected in medium that lacked thymidine and purines. CETP was produced in serum-free medium once the cells were confluent (or near confluence). The hCETP-CHO cells were cultured with growth medium containing Ham's F-12 medium, 10% FBS, 2 mmol/L ß-glutamine, and 50 µg/mL gentamicin under 5% CO2 at 37°C. Cells were seeded on a 100-mm dish and used for the gene regulation study and on a slide chamber (surface area, 400 mm2) for the translocation study. The cells were used for the experiments 2 days after reaching 90% confluence.
Oligonucleotides
The sequences of phosphorothioated ODNs against human CETP used
in this study were as follows: AS-ODNs, 5'-CTTGACTTGGCCAAGGAGCAT-3';
sense ODNs (S-ODNs), 5'-ATGCTCCTTGGCCAAGTCAAT-3'; positions +329 to
+349 of the human cDNA sequence.24 These selected target
sequences have relatively low homology with any other known sequences
found in the GenBank database. For the translocation study,
FITC-conjugated AS-ODNs (FITC-ODNs) were composed of phosphorothioated
sequences with 5'-end FITC conjugation.
Peptide Synthesis
Apo E peptide 129 to 169 and dpGapoE were synthesized as
previously described.19 25 The peptide isolated after
lyophilization was >99% pure as determined by analytical
reversed-phase high-performance liquid
chromatography, had the expected amino acid composition
as determined by amino acid analysis, and was of the expected
molecular mass as determined by fast atom bombardment mass
spectrometry. The sequence of nonacylated peptide was determined using
an Applied Biosystems 477A sequencer.
ODN/dpGapoE Complex-Mediated Transfection Into hCETP-CHO
Cells
The hCETP-CHO cells were washed 3 times with serum-free Opti-MEM
medium (GIBCO BRL) after they grew to 90% confluence. dpGapoE was
mixed with ODNs at a weight ratio of 53/4 (dpGapoE/ODN) in sterile,
distilled water under a micromixer. The mixtures were allowed to stand
at room temperature for 30 minutes. After being mixed with Opti-MEM at
a final ODN concentration of 0.1 µmol/L, the mixtures were added
to the culture dishes. After being transfected for 2 hours at 37°C,
the cells were washed twice with Opti-MEM medium and incubated with
Opti-MEM medium for the indicated times. Cells that were incubated with
Opti-MEM but without ODNs or dpGapoE were used as controls.
Cytotoxic Activity of dpGapoE and AS-ODN/dpGapoE Complex in
hCETP-CHO Cells
The cytotoxicity of dpGapoE and AS-ODN/dpGapoE complex was
investigated. Cells were treated as described above. Cell viability was
assessed by trypan blue exclusion after 24 and 48 hours.
Kinetics of FITC-Conjugated ODN Localization in the Cell
Nucleus
The hCETP-CHO cells were grown on a Dermanox plastic chamber
slide (Nunc, Inc). Cells were transfected with FITC-ODNs in the
presence or absence of dpGapoE for 2 hours and then maintained in
Opti-MEM at 37°C as described above. After incubation, the cells were
rinsed twice with PBS and fixed with 10% (vol/vol) formaldehyde/PBS
for 20 minutes at room temperature. After being rinsed again with PBS,
cells were stained with 4',6'-diamidino-2-phenylindole (DAPI) for 5
minutes at room temperature. The cells were then rinsed twice with PBS
and mounted in glycerol mounting medium. The translocation of FITC-ODNs
was determined by an Olympus fluorescence microscope with a
standard excitation/emission filter combination for FITC and DAPI.
Similar experiments were performed at low temperature (4°C) to block
the physiological cellular response.
To examine the effect of serum on the transfection activity of dpGapoE, ODN/dpGapoE complex was added to the transfection medium. To prove that the ODN/dpGapoE complex was binding to the LDL receptor, 30 µg of protein per milliliter of human LDL (1.019<d<1.063 g/mL) and 50 µg of protein per milliliter of human apo E (Research Diagnostics, Inc) bound to dimyristoylphosphatidylcholine were added to the transfection medium to compete with dpGapoE for cellular uptake. Anti–LDL receptor antibodies (chicken anti–human LDL receptor polyclonal antibodies, obtained from Research Diagnostics, Inc) were used to block the LDL receptor–mediated endocytosis for 1 hour before transfection.
Measurement of CETP Activity
The hCETP-CHO cells were transfected with AS-ODNs only,
AS-ODN/dpGapoE complex, or S-ODN/dpGapoE complex for 2 hours after they
reached 90% confluence and then maintained in Opti-MEM medium. The
activity of CETP in the medium was determined as described
previously.26 27 A reaction mixture containing 90 µL of
discoidal bilayer particles with
[3H]cholesterol oleate (39 nCi) as
a donor of CE and 110 µg of human LDL (1.019<d<1.063
g/mL) protein as an acceptor was incubated with 10 µL of culture
medium for 30 minutes at 37°C in the presence of 1.4 mmol/L
2,2'-dithio-bis-(5'-nitropyridine), an inhibitor of LCAT.
After incubation, LDL was precipitated by dextran sulfate and
MgCl2 and isolated by
centrifugation. The LDL precipitate was dissolved in
100 µL of 0.1N NaOH, and radioactivity in counts per minute was
determined using a Beckman liquid scintillation counter. CETP-mediated
CE transfer was calculated by subtracting the baseline (medium from 0
hours) values, which included spontaneous transfer, from the total
values for the sample at the indicated times. CETP activity was
expressed as CETP-mediated CE transfer per reaction time (hour) per
105 cells. To avoid interassay variation, all
samples were measured simultaneously in 1 assay system.
Preparation of cDNA Probe by Reverse Transcription–Polymerase
Chain Reaction (PCR)
Total cellular RNA was extracted from hCETP-CHO cells with
RNAzol B (Biotecx Laboratories, Inc), reversed-transcribed using
reverse transcriptase and oligo(dT)16 (Perkin
Elmer), and then used in a PCR. The PCR primers for human CETP were as
follows: 5'-CTTTCCATAAACTGCTCCTG-3' and 5'-CTGGTTGGTGTCGAAACCCT-3'.
PCR consisted of 35 cycles of 1 minute of denaturation at 95°C, 1
minute of annealing at 55°C, and 1 minute of extension at 72°C,
followed by 15 minutes of final extension. The PCR product was a
461-base fragment that was subcloned into pT7Blue vector to make
pT7-CETP3. The sequence of the PCR product was confirmed by
sequencing using an Applied Biosystems ABI 373A DNA sequencer. Human
CETP cDNA probe (363 bases) for Northern blotting was excised from
pT7-CETP3 after RsaI digestion and then labeled with
[32P]dCTP by using a random labeling kit
(Amersham).
Northern Blot Analysis
Total cellular RNA was extracted from hCETP-CHO cells with
RNAzol B (Biotecx Laboratories, Inc) at the indicated times after
transfection. Twenty microliters of total RNA was fractionated on a 1%
agarose formaldehyde gel and blotted onto a positively charged nylon
membrane (Amersham). The membrane was hybridized with
32P-labeled CETP probe and rehybridized with
32P-labeled human GAPDH (Clontech) as described
elsewhere.27 28 Densitometric analysis of
autoradiograms was performed by a densitometric scanner
with computer-assisted analysis (300A computing densitometer
and ImageQuant Software version 3.0 Fast scan, Molecular Dynamics). The
CETP mRNA levels were adjusted for GAPDH mRNA and expressed as the
percentage changes from baseline (0 hours).
Statistical Analysis
Data are expressed as mean±SD. Differences between treatment
groups were statistically evaluated by an ANOVA, followed by Fisher's
test by using Statview-J 4.11 for the Macintosh.
|
Results |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Translocation of FITC-ODNs Into the Cell Nucleus
In these experiments, FITC-labeled phosphorothioated ODNs were used to investigate the ability of dpGapoE to deliver short-strand AS-ODNs to hCETP-CHO cells. In a control experiment in which hCETP-CHO cells were incubated with 10 µmol/L FITC-ODNs, fluorescence microscopy revealed only weak cell-associated fluorescence, which was poorly internalized and localized in punctuate cytoplasmic regions (Figure 1A
and 1B). In sharp
contrast, cells that were incubated for 1 hour in the presence of
ODN/dpGapoE complex displayed a fluorescent nucleus, and this
fluorescence was colocalized with that of DAPI; a granular
accumulation of fluorescence was also found in the cytoplasm
(Figure 1C and 1D). After 6 hours (Figure 1E and 1F) and
12 hours (Figure 1G and 1H) of incubation, the
fluorescence in the cytoplasm and nucleus increased, and >95%
of the cells exhibited a dense fluorescent nucleus. The nuclear
translocation of FITC-ODNs was reduced after 24 hours of incubation
(Figure 1I and 1J), but some still remained after 48 hours of
incubation (Figure 1K and 1L).
|
At low temperature (4°C), the cellular uptake of FITC-ODNs in the
presence of ODN/dpGapoE complex (Figure 1M
and 1N) was the same
as that with "naked" ODNs (Figure 1A and 1B) after 1 hour of
incubation. In contrast, FITC-ODNs were translocated into the cytoplasm
and some nuclei at 37°C after 1 hour of incubation (Figure 1C and 1D). FITC fluorescence had scarcely accumulated, and it was
seen only on the membrane surface in the presence of dpGapoE at 4°C
(Figure 1M).
In the presence of serum, FITC-ODNs were translocated into >90% of
cell nuclei after 1 hour of incubation (Figure 1O and 1P). In
the presence of LDL (Figure 1Q and 1R) and
dimyristoylphosphatidylcholine-bound apo E (Figure 1S and 1T),
only a few cells were transfected, and very little granular
accumulation of fluorescence was seen in some cells. Marked
blockade of fluorescence translocation was observed in cells
that had been pretreated with anti–LDL receptor antibodies (Figure 1U and 1V).
Cytotoxic Activity of dpGapoE and AS-ODN/dpGapoE Complex in
hCETP-CHO Cells
The cytotoxic effects, including disruption of the cytoplasmic
membrane of the dpGapoE and OND/dpGapoE complex, were evaluated by
trypan blue exclusion after 24 and 48 hours of incubation. The
Table shows that in the absence of ODNs,
dpGapoE had slight cytotoxic effects; ie, cell viability was reduced by
only 4% to 5%. However, in the case of AS-ODN/dpGapoE complex, cell
viability was preserved to the same extent (the Table) as that
in controls.
|
Time Course of the Effect of ODNs on CETP Activity in Culture
Medium
CETP activity was assessed by using discoidal bilayer particles
with [3H]cholesteryl oleate and human LDL.
Figure 2 shows the time course of CETP
activity in the hCETP-CHO cell medium after incubation with AS-ODNs
only, AS-ODN/dpGapoE complex, or S-ODN/dpGapoE complex. A control
experiment, in which hCETP-CHO cells were incubated with Opti-MEM but
without dpGapoE or ODNs, showed that CETP accumulated in a
time-dependent manner in serum-free medium (Figure 2). Compared
with controls, after transfection with AS-ODN/dpGapoE complex, CETP
activity in the culture medium significantly decreased with time, ie,
to 46.4% (2 hours), 62.0% (6 hours), 58.3% (12 hours), 54.6% (24
hours), and 55.9% (36 hours) of controls. However, after 48 hours of
incubation, CETP activity started to recover, to 69.3% of controls at
48 hours, and to 87.6% of controls at 72 hours. However, CETP
activities were still lower than those in controls at 72 hours of
incubation. In contrast, no difference was found after transfection
with S-ODN/dpGapoE or naked AS-ODNs compared with controls (Figure 2).
|
), or AS-ODN/dpGapoE
(
), CETP activity in the medium was measured. A control
experiment (
) was performed using cells that were
incubated without ODNs or dpGapoE. *P<0.01 vs
control.
Time Course of the Effect of ODNs on CETP mRNA
In a control experiment, CETP mRNA levels of hCETP-CHO cells
slightly increased, but not significantly, during 48 hours of
incubation (Figure 3A). After hCETP-CHO
cells were incubated with AS-ODN/dpGapoE complex for 2 hours, cellular
CETP mRNA levels, as assessed by densitometric analysis of
Northern blots, significantly and gradually decreased with time, ie, to
82.8% (6 hours), 69.9% (12 hours), 52.9% (24 hours), and 41% (36
hours) of baseline (0 hours). Thereafter, the mRNA level started to
recover, to 57.9% (48 hours) and 72% (72 hours) of baseline
(Figure 3D). However, transfection with S-ODN/dpGapoE complex
(Figure 3C) or naked AS-ODNs (Figure 3B) did not change
the cellular CETP mRNA level compared with that in controls (Figure 3A).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
Most "information-rich" molecules, such as ODNs, genes, peptides, and proteins, are poorly taken up by cells because these molecules do not efficiently cross the lipid bilayers of the plasma membrane or the endocytic vesicles.29 This is considered to be a major limitation to their ex vivo or in vivo use in fundamental studies or in possible clinical applications. AS-ODNs have been recognized as a new generation of putative therapeutic agents.1 2 3 They are currently delivered by various techniques, including microinjection, electroporation, viral transfection, association with cationic lipids, liposome encapsidation, and receptor-mediated endocytosis. Various problems have been encountered in their use, including low transfer efficiency, complex manipulation, cellular toxicity, and immunogenicity, which would preclude their routine use in vivo.4 An ideal nonviral DNA delivery system would have the following characteristics: (1) structurally well characterized, nontoxic, biodegradable, and nonantigenic; (2) DNA protection from degradation and stability in biofluids; (3) cellular uptake controlled by cell-specific plasma membrane receptors; (4) rapid, pH-dependent endosomal release; (5) efficient dissociation of DNA from the complex in the cytoplasm for transport to the nucleus; and (6) efficient bioactivity of DNA.
dpGapoE (129–169) encompasses all of the putative LDL
receptor–binding region of apo E (amino acid residues 136 to 150),
appears to be highly 
-helical in an acidic or basic environment, and
seems not remarkably helical at neutral pH in the absence of
lipid.19 dpGapoE, which is a positively charged lipophilic
peptide, also condenses ODNs to give a particulate delivery system of
48.2±5.7 nm, as determined by submicron particle size measurement
(L.C.S., unpublished data, 1999). Gene delivery complexes were created
by stepwise self-assembly of ODNs and dpGapoE (Figure 4). As also shown in Figure 4, this system appears to have all of the determinants required for
transfection in vitro and in vivo. Because many lipoprotein receptors
and their ligands are well characterized, we chose dpGapoE as a
targeting ligand for the LDL receptor, which is abundant in the
liver.
|
In these experiments, FITC-labeled phosphorothioated ODNs were used to
investigate the ability of dpGapoE to deliver short-strand AS-DNA to
hCETP-CHO cells. Although they are negatively charged,
phosphorothioated ODNs are capable of entering cells by
endocytosis.30 However, this uptake is very inefficient,
as shown in Figure 1A and 1B. In sharp contrast,
dpGapoE-mediated transfection of as little as 0.1 µmol/L ODNs
resulted in bright fluorescence in virtually 100% of the
cells. These results show that dpGapoE was highly effective for the
delivery of AS-ODNs into hCETP-CHO cells. More than 95% of the cells
exhibited a dense fluorescent nucleus after 6 and 12 hours of
incubation. Thus, in this nucleic acid delivery system, there was
efficient release of ODNs from the liposome and efficient dissociation
of ODNs from the complex into the cytoplasm, after which the ODNs
migrated into the nucleus. The phosphorothioate AS-ODNs delivered by
this system can be accumulated and concentrated in the nucleus.
However, after 24 hours of incubation, the FITC-labeled ODNs began
disappearing. The mechanism of this change is still unknown. The
degradation of AS-ODNs in the nucleus may be involved.
Interestingly, dpGapoE had slight cytotoxic activity, whereas the ODN/dpGapoE complex did not produce any cytotoxic effects on hCETP-CHO cells. The reduction in cytotoxic activity may be due to the formation of a complex with ODNs, which decreases the perturbation activity of peptides toward the cell membrane.31
A low-temperature translocation study showed that FITC-ODNs were
remarkably bound to the cell membrane surface, and very little was
translocated into the cytoplasm or nucleus in the presence of dpGapoE
(Figure 1M and 1N). The addition of excess human LDL (Figure 1Q and 1R) and apo E coupled with dimyristoylphosphatidylcholine
(Figure 1S and 1T) to the transfection medium remarkably
inhibited the translocation of FITC-ODNs into the cytoplasm and nuclei
by dpGapoE at 37°C. With the blocking anti–LDL receptor antibodies,
transfection activity was remarkably inhibited (Figure 1U and 1V). These findings strongly suggest that nucleic acid delivery by
dpGapoE occurred mainly via LDL receptor–mediated endocytosis.
However, the addition of 5% FBS to the transfection medium had little
effect on nuclear delivery of ODNs into cultured cells (Figure 1O and 1P). This may be helpful for the combined use of dpGapoE
and ODNs in vivo.
The biological effect of AS-ODNs against human CETP in hCETP-CHO cells
was also examined. AS-ODNs delivered by dpGapoE dramatically suppressed
cellular CETP mRNA levels (Figure 3). A parallel reduction in
CETP activity in the medium was also observed (Figure 2). In
contrast, both S-ODNs delivered by dpGapoE and naked AS-ODNs had no
effect on cellular CETP mRNA (Figure 3) or CETP activity (Figure 2) in the medium. These results demonstrate that a human CETP
AS-ODN complex with dpGapoE, but not naked AS-ODNs, suppressed CETP
expression in hCETP-CHO cells in vitro. No effect was observed in cells
that had been incubated with S-ODNs complexed with dpGapoE. This result
strongly supports the idea that inhibition of the CETP gene was
sequence-specific. The maximum effects of AS-ODNs delivered by dpGapoE
on cellular CETP mRNA and CETP activity in the medium were observed
after 36 hours of incubation; thereafter, these effects grew weaker,
and CETP mRNA and activity began to recover. These changes may be due
to the disappearance of AS-ODNs, as observed in the translocation
study.
From these studies, we can conclude that the novel synthetic dpGapoE is highly effective for the nuclear delivery of AS-ODNs into hCETP-CHO cells. Moreover, AS-ODNs selectively inhibited CETP expression in an hCETP-CHO cell line. This approach may enable gene regulation in vivo. could be used to alter HDL metabolism, and thus, counteract atherosclerosis.
|
Acknowledgments |
|---|
This work was supported by grants-in-aid from the Ministry of Education, Science and Culture of Japan [Nos. 09670773 (K.S.), 10670221 (J.S.), 10670693 and 11670724 (K.S.)] and grants from the National Institutes of Health, Bethesda, Md IH (HL 30914 and 056865).
|
Footnotes |
|---|
K.L. and J.O. equally contributed to this article.
Part of this work was presented at the 70th Scientific Sessions of the American Heart Association, Orlando, Fla, November 9th, 1997, and published in abstract form (Circulation. 1997;96[suppl I]:I-108).
Received September 22, 1998; accepted February 3, 1999.
|
References |
|---|
|
TopAbstractIntroductionMethodsResultsDiscussionReferences |
|---|
- Hélène C, Toulmé JJ. Specific regulation of gene expression by antisense, sense and antigene nucleic acid. Biochim Biophys Acta. 1990;1049:99–125.[Medline] [Order article via Infotrieve]
-
Stein CA, Cheng YC. Antisense
oligonucleotides as therapeutic agents: is the bullet
really magical? Science. 1993;261:1004–1012.
[Abstract/Free Full Text] - Wagner RW. Gene inhibition using antisense oligodeoxynucleotides. Nature. 1994;372:333–335.[Medline] [Order article via Infotrieve]
-
Morris MC, Vidal P, Chaloin L, Heitz F, Divita G. A
new peptide vector for efficient delivery of
oligonucleotides into mammalian cells. Nucleic
Acids Res. 1997;25:2730–2736.
[Abstract/Free Full Text] - Anderson WF. Human gene therapy. Nature. 1998;392(suppl):25–30.
- Felgner PL, Ringold GM. Cationic liposome-mediated transfection. Nature. 1989;337:387–388.[Medline] [Order article via Infotrieve]
- Bennett CF, Chiang MY, Chan H, Shoemaker JEE, Mirabelli CK. Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides. Mol Pharmacol. 1992;41:1023–1033.[Abstract]
-
Hofland HEJ, Shephard L, Sullivan SM. Formation of
stable cationic lipid/DNA complexes for gene transfer. Proc Natl
Acad Sci U S A. 1996;93:7305–7309.
[Abstract/Free Full Text] -
Wagner E, Cotten M, Foisner R, Birnstiel ML.
Transferin-polycation-DNA complexes: the effect of polycations on the
structure of the complex and DNA delivery to cells. Proc Natl
Acad Sci U S A. 1991;88:4255–4259.
[Abstract/Free Full Text] -
Wagner E, Zenke M, Cotten M, Beug H, Birnstiel ML.
Transferrin-polycation conjugates as carriers for DNA uptake into
cells. Proc Natl Acad Sci U S A. 1990;87:3410–3414.
[Abstract/Free Full Text] -
Perales JC, Ferkol T, Beegen H, Ratnoff OD, Hanson RW.
Gene transfer in vivo: sustained expression and regulation
of genes introduced into the liver by receptor-targeted uptake.
Proc Natl Acad Sci U S A. 1994;91:4086–4090.
[Abstract/Free Full Text] - Ferkol T, Kaetzel CS, Davis PB. Gene transfer into respiratory epithelial cells by targeting the polymeric immunoglobulin receptor. J Clin Invest. 1993;92:2394–2400.
-
Ferkol T, Perales JC, Mularo F, Hanson RW.
Receptor-mediated gene transfer into macrophages. Proc
Natl Acad Sci U S A. 1996;93:101–105.
[Abstract/Free Full Text] - Ginobbi P, Geiser TA, Ombres D, Citro G. Folic acid-polylysine carrier improves efficacy of c-myc antisense oligodeoxynucleotides on human melanoma (M14) cells. Anticancer Res. 1997;17:29–35.[Medline] [Order article via Infotrieve]
-
Ziady AG, Perales JC, Ferkol T, Gerken T, Beegen H,
Perlmutter DH, Davis PB. Gene transfer into hepatoma cell lines via the
serpin enzyme complex receptor. Am J Physiol. 1997;273:G545–G552.
[Abstract/Free Full Text] - Gottschalk S, Sparrow JT, Hauer J, Mims MP, Leland FE, Woo SLC, Smith LC. A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells. Gene Ther. 1996;3:448–457.
- Wyman TB, Nicol F, Zelphati O, Scaria PV, Plank C, Szoka FC Jr. Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers. Biochemistry. 1997;36:3008–3017.[Medline] [Order article via Infotrieve]
-
Niidome T, Ohmori N, Ichinose A, Wada A, Mihara H,
Hirayama T, Aoyagi H. Binding of cationic -helical peptides to
plasmid DNA and their gene transfer abilities into cells. J
Biol Chem. 1997;272:15307–15312.
[Abstract/Free Full Text] -
Mims MP, Darnule AT, Tovar RW, Pownall HJ, Sparrow DA,
Sparrow JT, Via DP, Smith LC. A nonexchangeable apolipoprotein E
peptide that mediates binding to the low density lipoprotein receptor.
J Biol Chem. 1994;269:20539–20547.
[Abstract/Free Full Text] - Liu KX, Saku K, Via DP, Smith LC. Efficient delivery of plasmid and antisense oligo into a human CETP gene transfected cell line by a novel lipophilic apo E peptide. Circulation. 1997;96(suppl I):I-108. Abstract.
- Shepherd J, Packard CJ. High density lipoprotein metabolism. In: Catapano AL, Bernini F, Corsini A, eds. Atherosclerosis Reviews. New York, NY: Raven Press; 1993;24:17–41.
- Lagrost L. Regulation of cholesteryl ester transfer protein (CETP) activity: review of in vitro and in vivo studies. Biochim Biophys Acta. 1994;1215:209–236.[Medline] [Order article via Infotrieve]
-
Weinberg RB, Cook VR, Jones JB, Kussie P, Tall AR.
Interfacial properties of recombinant human cholesterol
ester transfer protein. J Biol Chem. 1994;269:29588–29591.
[Abstract/Free Full Text] - Drayna D, Jarnagin AS, McLean J, Henzel W, Kohr W, Fielding C, Lawn R. Cloning and sequencing of human cholesteryl ester transfer protein cDNA. Nature. 1987;327:632–634.[Medline] [Order article via Infotrieve]
- Sparrow JT. An improved polystyrene support for solid phase peptide synthesis. J Org Chem. 1976;41:1350–1353.[Medline] [Order article via Infotrieve]
-
Kato H, Nakanishi T, Arai H, Nishida HI, Nishida T.
Purification, microheterogeneity, and stability of
human lipid transfer protein. J Biol Chem. 1989;264:4082–4087.
[Abstract/Free Full Text] - Ou J, Saku K, Jimi S, Ohta T, Zhang B, Pownall HJ, Arakawa K. Mechanism of action of probucol cholesterol ester transfer protein (CETP) mRNA in a Chinese hamster ovary cell line that had been stably transfected with a human CETP gene. Biochim Biophys Acta. 1998;1393:153–160.[Medline] [Order article via Infotrieve]
- Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd ed. Plainview, NY: Cold Spring Harbor Laboratory Press; 1989.
- Lebleu B. Delivering information-rich drugs: prospects and challenges. Trends Biotechnol. 1996;14:109–110.[Medline] [Order article via Infotrieve]
- Beltinger C, Saragovi HU, Smith RM, LeSauteur L, Shah N, DeDionisio L, Christensen L, Raible A, Jarett L, Gewirtz AM. Binding, uptake, and intracellular trafficking of phosphorothioate-modified oligodeoxynucleotides. J Clin Invest. 1995;95:1814–1823.
- Ohmori N, Niidome T, Kiyota T, Lee S, Sugihara G, Wada A, Hirayama T, Aoyagi H. Importance of hydrophobic region in amphiphilic structures of 8706'3f-helical peptides for their gene transfer-ability into cells. Biochem Biophys Res Commun. 1998;245:259–265.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  R. W. Mahley, K. H. Weisgraber, and Y. Huang Inaugural Article: Apolipoprotein E4: A causative factor and therapeutic target in neuropathology, including Alzheimer's disease PNAS, April 11, 2006; 103(15): 5644 - 5651. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Chang, T. r. Ma, R. D. Miranda, M. E. Balestra, R. W. Mahley, and Y. Huang Lipid- and receptor-binding regions of apolipoprotein E4 fragments act in concert to cause mitochondrial dysfunction and neurotoxicity PNAS, December 20, 2005; 102(51): 18694 - 18699. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||