1 Department of Pharmacology and 2 Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
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ABSTRACT |
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Cationic lipid-mediated intravenous gene
delivery shows promise in treating pulmonary diseases including lung
tumor metastases, pulmonary hypertension, and acute respiratory
distress syndrome. Nevertheless, clinical applications of
cationic lipidic vectors via intravenous administration are limited by
their transient gene expression. In addition, repeated dosing is not
effective at frequent intervals. In an effort to elucidate the
mechanism of gene inactivation, we report in this study that cationic
lipid-protamine-DNA (LPD) complexes, but not each component alone, can
induce a high level of cytokine production, including interferon-
and tumor necrosis factor-
. Furthermore, we demonstrate that LPD
administration triggers apoptosis in the lung, a phenomenon that may be
mediated in part by the two cytokines. Treatment of mice with
antibodies against the two cytokines prolongs the duration of gene
expression and also improves lung transfection on a second
administration of LPD. Although the mechanism underlying LPD-induced
cytokine production is unclear, methylation of the DNA significantly
decreased the level of both interferon-
and tumor necrosis
factor-
, suggesting that unmethylated CpG sequences in plasmid DNA
play an important role. These data suggest that decreasing the
CpG-mediated immune response while not affecting gene expression may be
a useful therapeutic strategy to improve cationic lipid-mediated
intravenous gene delivery to the lung.
cationic lipids; inflammation
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INTRODUCTION |
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RECENTLY, several groups (2, 9-11, 14, 15, 18, 19,
27, 29, 30, 36) have reported systemic gene expression by intravenous
administration of cationic lipidic vectors. Several parameters have
been identified as important for achieving a high level of gene
expression. These include structures of cationic and helper lipids and
the charge ratio between the cationic lipid and DNA (2, 9-11, 14,
15, 18, 19, 27, 29, 30, 36). The use of a cationic polymer such as
protamine sulfate can also improve intravenous lipofection (14, 15).
Although all organs can be transfected, the lung is the organ with the highest level of gene expression after intravenous administration. The
cells that are transfected are primarily lung endothelial cells,
although some monocytes are also transfected (9, 14, 19). This unique
pattern of intravenous lipofection suggests the possibility of using
cationic lipidic vectors to deliver a gene to treat pulmonary diseases.
Indeed, intravenous delivery of a retinoblastoma (RB) tumor suppressor
gene by lipid-protamine-DNA (LPD) resulted in a
significant decrease in the number of pulmonary metastases in
RB(+/) mice (20). Delivery of a prostaglandin G/H synthase gene
or a neutrophil inhibitory factor gene to pulmonary endothelial cells
by cationic liposomes can also protect rabbit lungs from endotoxin
injury (3, 35).
Despite this promise, the clinical applications of cationic lipidic vectors via intravenous administration are hampered by their toxicity, including the lethal effects in experimental animals (11). In addition, repeated injections are ineffective at frequent intervals. Typically, 7-14 days are required for recovery before a second injection can be effective (11, 27). Neither the toxicity nor the refractoriness to repeated injections is due to the cationic lipid itself because intravenous injection of cationic liposomes was not associated with lung injury and preinjection of cationic liposomes did not affect transfection of lungs by subsequent administration of cationic liposome-DNA complexes (11, 27). The toxicity is also not related to expressed gene products because injection of degraded DNA fragments formulated in lipidic vectors is as toxic as liposome-intact DNA complexes (11, 27). Clearly, toxicity is largely induced by cationic lipid-DNA complexes, yet the mechanism by which cationic lipid-DNA complexes generate toxicity remains unknown.
In this study, we demonstrate that cationic lipid-DNA complexes, but not each component alone, can induce the production of large quantities of proinflammatory cytokines that play an important role in complex-induced toxicity. This cytokine production is primarily triggered by unmethylated CpG sequences in the plasmid DNA. Thus decreasing the CpG-mediated immune response while not affecting gene expression may represent an important approach to improve cationic lipid-mediated intravenous gene delivery.
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MATERIALS AND METHODS |
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Chemicals. 1,3-Dioleoyl-3-trimethylammonium propane (DOTAP) was purchased from Avanti Lipids (Alabaster, AL). Cholesterol was obtained from Sigma (St. Louis, MO). Protamine sulfate was from Eli Lilly (Indianapolis, IN). Luciferase assay kit was obtained from Promega (Madison, WI). Indocarbocyanine (Cy3)-dCTP and Cy5-dCTP were from Amersham (Arlington Heights, IL). All other chemicals were of reagent grade.
Plasmids. Plasmids pCMVL and pEGFP-N1,
which contain, respectively, the cDNA of firefly luciferase and
enhanced green fluorescence protein (GFP) driven by a human
cytomegalovirus (CMV) immediate-early promoter, were amplified in the
DH5 strain of Escherichia coli, isolated by alkaline lysis, and then purified by cesium chloride centrifugation (23). The purified DNA was further removed from endotoxin by passing it through a QIAGEN-tip 500 column (QIAGEN, Valencia, CA). The endotoxin level in all plasmids used in this study
was <0.01 endotoxin unit/µg DNA.
Fluorescence labeling of plasmid DNA. Labeling of pCMVL with Cy3-dCTP or Cy5-dCTP was performed with the nick translation system (Promega). The labeled DNA was extracted with phenol-chloroform, precipitated with ethanol, and further purified by being passed through a Bio-Gel P-30 spin column (Bio-Rad Laboratories, Hercules, CA).
Plasmid methylation. pCMVL was methylated with Sss I CpG methylase (New England Biolabs, Beverly, CA) with 2 U enzyme/µg DNA for 2 h and incubated for an additional 2 h after the further addition of S-adenosylmethionine substrate with the use of buffer conditions suggested by the manufacturer. DNA was extracted with phenol-chloroform, precipitated with ethanol, and further purified by being passed through a Bio-Gel P-30 spin column (Bio-Rad Laboratories). The efficiency of methylation was confirmed to be >95% by enzymatic digestion.
Preparation of liposomes. Liposomes containing DOTAP in a 1:1 molar ratio with cholesterol were prepared as follows. The lipid mixture in chloroform was dried as a thin layer in a 100-ml round-bottomed flask that was further vacuum desiccated for 2 h. The lipid film was hydrated in 5% dextrose in water to give a final concentration of 10 mg DOTAP/ml. Preparation of small unilamellar vesicles by extrusion was performed as follows. The lipid solution was briefly sonicated, followed by incubation at 50°C for 10 min and then sequentially extruded through polycarbonate membranes with pore sizes of 1.0, 0.6, and 0.1 µm.
Preparation of LPD. For preparation of LPD, diluted DNA was added to the mixture of DOTAP-cholesterol liposomes and protamine at a final ratio of 0.6 µg of protamine to 12 nmol DOTAP to 1 µg of DNA (15). The mixture was incubated at room temperature for 10 min before use.
Simultaneous detection of Cy3-DNA and GFP in lungs after a single injection of LPD. Female CD-1 mice, 4-6 wk of age, were purchased from Charles River Laboratories (Wilmington, MA) and housed in accordance with institutional guidelines. The mice were injected intravenously with LPD containing 25 µg each of Cy3-labeled pCMVL DNA and pEGFP-N1 DNA. At different intervals after injection, mice were killed, and the lungs were perfused intravascularly with 2% paraformaldehyde and inflated with this solution to near total lung capacity. After the lungs were rinsed with cold PBS, the lungs were quickly frozen in optimum cutting temperature embedding medium with dry ice. Ten-micrometer lung cryosections were then prepared. To quantify the number of cells binding Cy3-tagged DNA or expressing the enhanced GFP encoded by pEGFP-N1 DNA, it was necessary to develop quantitative algorithms that identify DNA binding or expression at the single-cell level in sectioned material. Cells were defined by the presence of nuclei that were counterstained with the DNA-specific dye 4',6-diamidino-2-phenylindole. Nuclear profiles were collected with fluorescence microscopy (Olympus Provis) and imaged with an integrating Sony 3 chip color charge-coupled device camera, a Coreco frame grabber board (Coreco), and Optimas software (Optimas). The final images were collected with a ×40 Planapo objective, numerical aperture 1.0, with an image resolution of 700 × 600 pixels. The image was converted to gray scale and passed through a 3 × 3 Gaussian filter to remove noise, and nuclear profiles were extracted by selection of an appropriate threshold. The subsequent image was then converted to a binary image. Because EGFP expression is located in the cytoplasm and DNA binding and passaging are predominantly seen at the cell surface and in the cytoplasm, it was necessary to transform the nuclear image such that the EGFP and DNA association with each cell could be measured. We have found that a dilation of individual nuclear profiles such that these compartments are included was the most direct and simple method to do this. Thus Cy3 and EGFP images were collected as described above, and rendered to binary images. Subsequently, each binary image was compared with the nuclear images with an AND comparison, which will extract pixels that are only positive for the nuclear dye and either Cy3 or EGFP. For each specimen, ten randomly collected fields in the distal lung were collected.
Gene expression in the lung after repeated injections. pCMVL was used as a reporter gene in this experiment. Groups of five mice received intravenous administration of LPD at a dose of 50 µg DNA/mouse. At different times after the first injection, mice were given a second injection of LPD. Twenty-four hours later, mice were bled from the retroorbital sinuses under anesthesia and killed by cervical dislocation. Lungs were collected and washed twice with cold saline. Lungs were homogenized with lysis buffer (0.05% Triton X-100, 2 mM EDTA, and 0.1 M Tris, pH 7.8) with a tissue tearor (BioSpec Products, Bartlesville, OK). After two cycles of freezing and thawing, the homogenates were centrifuged at 14,000 g for 10 min at 4°C, and 20 µl of the supernatant were analyzed for luciferase activity with an automated LB 953 luminometer equipped with an automated injector (Berthod, Bad Wildbad, Germany). Gene expression is expressed as relative light units per milligram of protein.
Biodistribution of LPD in the lung after repeated
injections. Groups of three mice received intravenous
injection of LPD containing Cy5-labeled DNA at a dose of 25 µg/mouse.
At indicated times after the first injection, mice were given an
injection of LPD containing Cy3-labeled DNA at the same dose. Thirty
minutes later, mice were killed. Lungs were fixed by cardiac perfusion
with 2% paraformaldehyde in PBS, dissected out, inflated to normal
size through the trachea with the same fixative and held in this
condition, immersed in fixative for 1 more h, and then washed in 0.1 M
PBS. Subsequently, the lung was cut into thick (5-mm) slices and
cryoprotected by overnight immersion in 10% sucrose in PBS. Samples
were then shock frozen in liquid nitrogen-cooled isopentane and stored
at 80°C. Detection of Cy3- and Cy5-labeled DNA was performed
as described in Simultaneous detection of Cy3-DNA and
GFP protein in lungs after a single injection of
LPD.
Immunoassays. At different times after
the injection of free liposomes, free DNA, free protamine, or LPD, mice
were bled from the retroorbital sinuses under anesthesia. The blood was
allowed to stay at 4°C for 4 h and then centrifuged at 14,000 g for 10 min at 4°C. Serum was
collected and kept at 80°C until used. Cytokine levels in
mouse serum were determined with specific immunoassay kits for mouse
interferon (IFN)-
and mouse tumor necrosis factor (TNF)-
(Genzyme, Cambridge, MA). Samples were analyzed in triplicate on a
plate reader, and cytokine levels were calculated by linear regression
analysis on the basis of values obtained from a standard curve. To test
the effect of DNA methylation on cytokine production, a group of five
mice was given 50 µg of methylated pCMVL formulated in LPD.
Collection of serum samples and immunoassays were performed as
described above. The result was compared with that with unmethylated DNA.
Effect of antibody treatment on intravenous
lipofection. To test whether treatment of mice with
antibodies against cytokines can prolong the duration of gene
expression, a group of four mice received a daily intravenous
administration of a mixture of hamster anti-IFN- and anti-TNF-
antibodies (Genzyme), each at a dose of 100 µg/mouse for 3 days,
starting the day before the administration of LPD. Control mice were
treated with normal hamster IgG. At 2 and 3 days after injection of LPD
(25 µg DNA/mouse), mice were killed and lungs were collected. Gene
expression was assayed as described in Gene expression
in the lung after repeated injections. To test the
effect of antibody treatment on repeated injections, mice were treated
with anti-cytokine antibodies as described above. Four days after the
first injection of LPD (50 µg DNA/mouse), mice were given another
injection of LPD at the same dose. Gene expression was assayed 24 h
later as described in Gene expression in the lung
after repeated injections.
Apoptosis assay. Groups of three mice were given intravenous administration of free liposomes, free protamine, free DNA, or LPD. Control mice received only 5% dextrose. Twelve hours after the injection, mice were killed. Lungs were collected and cyrosections were prepared as described in Simultaneous detection of Cy3-DNA and GFP in lungs after a single injection of LPD. Detection of apoptotic cells in each tissue section with ApopTag peroxidase kit (Oncor, Gaithersburg, MD) was performed according to a protocol described by the manufacturer.
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RESULTS |
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Transient gene expression is not due to rapid
elimination of plasmid DNA from transfected cells. As
shown in Fig.
1A, gene expression can be detected as early as 2 h after the injection of LPD.
Gene expression peaked at 6 h and gradually declined
thereafter. The fluorescence intensity in transfected
cells is much weaker at 24 h compared with the fluorescence intensity
at 6 h. This pattern of gene expression is similar to that when a
luciferase plasmid was used as previously reported (14). To determine
whether transient gene expression is due to rapid elimination of
plasmid DNA from transfected cells, we simultaneously quantified
DNA-Cy3- and GFP-positive cells at different times after the injection of LPD. As shown in Fig. 1B, the
number of GFP-positive cells decreased rapidly, whereas the percentage
of DNA-Cy3-positive cells remained at a high level over 24 h,
suggesting that inactivation of gene expression is unlikely due to
rapid elimination of plasmid DNA from transfected cells. We are aware
of the possibility that the number of DNA-Cy3-positive cells might be
overestimated due to the fact that Cy3 can still be detected even after
DNA is degraded and/or inactivated inside cells. Indeed, this is a
difficult limitation in the current technology in detecting intact DNA
inside living cells.
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Repeated injections are not effective at frequent
intervals. To test whether long-term gene expression
can be achieved by repeated injections of LPD, mice were given a second
injection at different times after the first administration of LPD, and gene expression was assayed 24 h later. The result is shown in Fig.
2. The second injection is not effective
until 2 wk after the first injection. The length of refractory period
is closely related to the dose of DNA. Decreasing the dose of DNA from
50 to 25 µg/mouse shortened the duration of unresponsiveness from 10-14 days to ~1 wk (data not shown).
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Treatment of mice with LPD does not affect the uptake
of LPD by lungs after the second injection. To test
whether the refractoriness to repeated injections was due to the
inability of lungs to take up LPD on a second injection, we prepared
two different LPDs containing Cy5- or Cy3-labeled DNA and injected both
LPDs into the same mice at different intervals. The distribution of
Cy5- and Cy3-labeled DNA in lungs is shown in Fig.
3. When the two LPDs were injected at short
intervals, such as 0 or 30 min, Cy5- and Cy3-labeled DNAs were
intensively colocalized in lungs of treated mice (Fig. 3,
A and
B). With the prolongation in the
interval between the two injections, Cy3-labeled DNA became dominant in
lungs of treated animals. Yet colocalization of the two plasmids was
still observed even when the interval between two injections was 16 h
(Fig. 3C). The above results suggest
that treatment of mice with LPD does not affect the uptake of LPD by
lungs after a second injection.
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Intravenous administration of LPD induces a high level
of cytokine production. Figure
4A shows
the level of IFN- in mouse serum 5 h after the injection of free
liposomes, free DNA, free protamine, protamine-DNA complexes, or LPD.
Administration of LPD resulted in a high level of IFN-
production,
whereas free liposomes, free DNA, free protamine, or protamine-DNA
complexes had only a minimal effect in inducing the production of
IFN-
. The LPD-induced IFN-
was detected as early as 30 min after
injection, peaked at 5 h, and declined thereafter (Fig.
4B). IFN-
was hardly detectable
in mouse serum 48 h after the administration of LPD (Fig.
4B). LPD also triggered a high level
of TNF-
production (Fig. 5). Again,
TNF-
is mainly induced by LPD complexes because each component alone
was not effective in inducing TNF-
production. The kinetics of
cytokine expression after intravenous administration of LPD is similar
to that when CpG-containing phosphorothioate oligonucleotide was
intraperitoneally injected into mice (34). Cationic lipid-DNA complexes
without protamine are as active as LPD in inducing the production of
IFN-
and TNF-
(data not shown).
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Intravenous administration of LPD induces apoptosis in
lungs. Previous studies (8, 17) have shown that
cytokines, especially TNF-, can cause apoptosis of vascular
endothelial cells including lung endothelial cells. To determine
whether LPD-induced cytokines have a similar effect on lung endothelial
cells, we collected lungs at different times after intravenous
administration of LPD, and the presence of apoptotic cells was examined
by indirect immunohistochemistry. The results are shown in Fig.
6. About 10% of the cells in lungs were
identified to be apoptotic 12 h after intravenous injection of LPD. In
contrast, apoptotic cells were barely detectable in lungs of mice
treated with free liposomes, free DNA, or free protamine. This is in
agreement with the fact that LPD, but not each component alone, induces
a high level of cytokine production. DOTAP-cholesterol liposome-DNA
complexes without protamine are as active as LPD in
inducing apoptosis in the lung (data not shown).
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Treatment of mice with anti-cytokine antibodies
improves intravenous lipofection by LPD. To determine
whether gene inactivation after intravenous injection of cationic
lipidic vectors is due to the toxic effect of cytokines, mice were
given a daily intravenous administration of a mixture of anti-IFN-
and anti-TNF-
antibodies for 3 days, starting the day before the
administration of LPD. Gene expression in the lung on
days
2 and
3 was then evaluated and compared with
that in mice treated with irrelevant IgG. The result is shown in Fig.
7. The levels of lung transfection in the
mice treated with anti-cytokine antibodies were significantly higher
than those in mice treated with irrelevant IgG (Fig. 7). Treatment of
mice with anti-cytokine antibodies also improves lung transfection on
repeated injections. As shown in Fig. 8, a
low level of gene expression was found in the lungs of mice that
received nonspecific antibody treatment. However, mice that received
specific antibody treatment showed an ~20-fold increase in the level
of lung transfection. The above results indicate that the two cytokines
play an important role in inactivation of gene expression after
intravenous injection of LPD.
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Methylation of plasmid DNA reduces the level of
cytokine production. Recent studies (24, 33) have shown
that the unmethylated CpG sequences in bacterial plasmid DNA are highly
potent in inducing cytokine production including IFN- and TNF-
.
To determine whether LPD-induced cytokine production is also related to
the unmethylated CpG sequences in plasmid DNA, pCMVL plasmid was
randomly methylated by CpG methylase, and its ability to induce
cytokine was then compared with that of unmodified DNA. The result is
shown in Fig. 9. Methylation of plasmid DNA
significantly decreased the level of both IFN-
and TNF-
.
Sonicated pCMVL formulated in LPD triggered the cytokine production at
a level similar to that of intact pCMVL.
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DISCUSSION |
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Despite the potential of intravenous lipofection in treating lung tumor metastases (20) and other pulmonary diseases (3, 35), current vectors need to be improved to resolve two major problems: rapid decrease in gene expression and refractoriness to repeated injections at short intervals. Short duration of gene expression is a general problem for all nonviral vectors; however, gene expression declines much more rapidly in intravenous lipofection than in gene transfer via other routes of administration such as intratissue injection (4, 32). Furthermore, in intravenous lipofection, a duration of ~2 wk is required for lungs to recover before a second injection can be effective, another problem not shared by gene transfer via other routes of administration. These differences suggest a different mechanism of gene inactivation in intravenous lipofection compared with other gene transfer methods.
Biodistribution study (Fig. 3) suggests that unresponsiveness to repeated injections is unlikely due to the inability of lungs to take up lipid-DNA complexes on a second injection. It is also not likely due to lipid-induced damage to lung endothelial cells because injection of free liposomes alone did not prevent lung transfection after a second administration of cationic lipid-DNA complexes. In addition, Li and Huang (14) and others (27) have shown that intravenous injection of LPD, lipoplexes, or free liposomes does not generate noticeable necrosis in the lung. These studies suggest that inactivation of gene expression in lung endothelial cells might be caused by an indirect mechanism rather than by direct toxic effects of cationic lipid-DNA complexes on endothelial cells.
Immune responses have been shown to have an inhibitory effect on in
vivo gene transfer. For example, a specific immune response mounted
against vectors or gene products can cause elimination of transfected
cells or even prevent repeated dosing. Recently, nonspecific immune
responses have also been shown to have an inhibitory effect on gene
expression (6, 7, 21). In particular, IFN- and TNF-
could inhibit
gene transcription or decrease the stability of mRNA, resulting in
inactivation of gene expression (6, 7, 21). The inhibitory effect is
more pronounced when the gene is driven by a viral promoter. Such an
inhibitory effect was demonstrated in either an in vitro transfection
or an in vivo setting where a viral vector was used. Therefore, it is
important to determine whether nonviral vectors can also induce
nonspecific immune responses and how these responses affect gene expression.
Results from this study clearly show that cationic lipidic vectors
induce nonspecific immune responses: substantial amounts of IFN- and
TNF-
were induced after intravenous administration of LPD. In
separate studies, Scheule et al. (25) and Freimark et al. (5) have
shown that intratracheal administration of cationic lipid-DNA complexes
also induces cytokine production. Cytokines might cause inactivation of
gene expression through two different mechanisms. First, as discussed
above, cytokines might directly inhibit transgene expression in lung
endothelial cells. This was recently confirmed in an in vitro study
where addition of IFN-
or TNF-
to cultured mouse lung endothelial cells inhibited lipofection even at doses that were not toxic to
treated cells (Y. Tan and L. Huang, unpublished data).
This is also supported by the fact that neutralization of cytokines by
specific antibodies significantly prolonged the duration of gene
expression (Fig. 7). Second, many studies (8, 17) have shown that
endothelial cells, including lung endothelial cells, are highly
sensitive to cytokines, particularly TNF-
. Exposure of endothelial
cells to TNF-
could result in apoptosis in a dose-dependent manner
in vitro and in vivo. We also demonstrated in this study that
intravenous administration of LPD induces apoptosis of lung endothelial
cells. Apoptosis is unlikely due to direct toxic effects from cationic
lipids because intravenous administration of cationic liposomes alone
did not cause any apoptosis in lungs. This is in agreement with the
fact that injection of cationic liposomes alone generated only a low
level of cytokine production. All of the results above suggest that
cytokines play an important role in inactivation of gene expression in
intravenous lipofection. Inactivation of gene expression either caused
by direct inhibitory effect of cytokines or subsequent to cell
apoptosis could be responsible for both the short duration of gene
expression and the unresponsiveness of lungs to repeated injections at
short intervals.
It is generally regarded that proinflammatory cytokines such as IFN-
and TNF-
are produced in immune cells including natural killer
cells, T cells, and macrophages. Yet the active component that triggers
the production of cytokines in intravenous lipofection remains unknown.
Li et al. (16) recently showed that cationic lipidic vectors undergo
dynamic changes in their biophysical characteristics after exposure to
mouse serum. Serum initially causes aggregation of lipidic vectors
(16). Prolonged interactions with serum lead to vector disintegration
(16). Vector aggregates, due to their relatively large size, can be
efficiently taken up by circulating immune cells. Yet the fact that
liposomes also form aggregates in serum but are much weaker in inducing
cytokine production suggests that aggregates per se are not the major
component that is responsible for cytokine production. DNA formulated
in lipidic vectors appears to play an important role in inducing
cytokine production, although DNA alone is not active.
Recently, it has been shown (1, 12, 24, 26, 28, 33) that nucleic acids
including oligodeoxynucleotides and plasmid DNA can serve as a strong
immunogen if they are delivered to immune cells in an "intact"
form. Further studies showed that the functional motifs in nucleic
acids are the unmethylated CpG sequences. Interestingly, CpGs are
usually unmethylated in bacterial DNA, whereas in mammalian DNA,
~75% of the CpGs are methylated to 5'-methylcytosine (22). This structural difference between bacterial and mammalian DNAs is a
signal for the induction of innate immunity to microbial infections
(13, 31). An unmethylated CpG dinucleotide flanked by two 5'
purines (optimally a GpA) and two 3' pyrimidines (optimally a TpC
or TpT) has strong stimulatory effects on murine and human lymphocytes
in vitro and murine lymphocytes in vivo. These stimulatory effects
include triggering B-cell proliferation; resistance to apoptosis;
release of interleukin (IL)-6 and IL-12; natural killer cell secretion
of IFN- and increased lytic activity; and monocyte and/or
macrophage secretion of IFN-
/
, IL-6, IL-12,
granulocyte-macrophage colony-stimulating factor, chemokines, and
TNF-
(1, 12, 24, 26, 28, 33). Although these studies suggest that
CpG-enriched nucleic acids can be used to purposely stimulate immune
response in vaccination, they also point out a potential problem in the use of plasmid DNA in gene therapy. Indeed, the CpG-induced immune response inhibited gene expression in a dose-dependent manner when
plasmid DNA was delivered by lipid 67 via intratracheal administration (S. H. Cheng, personal communication). Intratracheal administration of
free plasmid DNA also induced the production of proinflammatory cytokines (5, 26), but the immune response was significantly enhanced
when lipid-DNA complexes were used (5). To determine whether CpG
sequences play an important role in inducing cytokine production in
intravenous lipofection, we investigated the effect of methylation on
cytokine production. As shown in Fig. 8, methylation of pCMVL resulted
in a significant decrease in the level of cytokines. The decrease in
cytokine production is not due to the lack of gene expression because
intravenous delivery of sonicated plasmid DNA (which is already
inactivated) by LPD could give rise to cytokine production at a level
similar to that when intact plasmid DNA was used. DNA alone is not
active in inducing cytokine production. This might be due to the fact
that free DNA is rapidly degraded in blood (16). Random degradation of
DNA by nucleases in the blood may result in a decrease in CpG contents.
This is in agreement with an in vitro study (12) in which DNase-treated
plasmid DNA is much weaker in inducing cytokine production compared
with intact plasmid DNA.
In summary, intravenous administration of cationic lipidic vectors is associated, in addition to expression of transgene, with production of cytokines. Cytokines play an important role in inactivation of gene expression. Production of cytokines is mainly due to the unmethylated CpG sequences in plasmid DNA. Thus decreasing the CpG-mediated immune response while not affecting gene expression may represent an important approach to improve cationic lipid-mediated gene transfer.
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ACKNOWLEDGEMENTS |
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The first two authors contributed equally to this work.
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FOOTNOTES |
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The work was supported by National Cancer Institute Grants CA-59327, CA-64654, and CA-71731 (to L. Huang); National Heart, Lung, and Blood Institute Grant HL-32154 (to B. Pitt); National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-44935; and a contract from Targeted Genetics Corporation (to L. Huang).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: L. Huang, Dept. of Pharmacology, Univ. of Pittsburgh School of Medicine, W1351 Biomedical Science Tower, Pittsburgh, PA 15261 (E-mail: leaf{at}prophet.pharm.pitt.edu).
Received 20 November 1998; accepted in final form 12 February 1999.
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