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
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.
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.
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.
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 β-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.
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.
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).
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.
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⇓).
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⇓).
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 α-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.
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).
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