(Received for publication, February 21, 1995; and in revised form, June 2, 1995)
From the
Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE)) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.
Gene transfer could represent an important advance in the treatment of both genetic and acquired diseases. Thus, there has been increasing attention focused on the development of gene transfer vectors. Viral vectors, such as recombinant adenovirus vectors, have a number of advantages for gene transfer, including their efficiency and their wide range of cell targets(1, 2) . But they also have a number of disadvantages, including the fact that they can generate several types of immune response, they often contain viral genes which could be transcribed, and there is a possibility of recombination or complementation. As a result of such limitations, there has been substantial effort focused on nonviral vectors, particularly the use of cationic lipids(3, 4, 5, 6, 7, 8, 9) . Cationic lipids are commercially available and are widely used in the research laboratory. For gene therapy applications, cationic lipids are currently under investigation as transfer vectors for treatments focused on melanoma (10) and cystic fibrosis(11, 12, 13, 14) .
Despite their availability, wide use, and potential application as vectors for gene therapy, there is limited understanding of the cellular and molecular mechanisms involved in cationic lipid-mediated gene transfer. As a result, wide variations in formulations and protocols have been described. For example, published reports of gene transfer to airway epithelia have used lipid to DNA charge ratios of 0.03 to 3 (net positive charge from the cationic component of the lipid divided by the net negative charge of the DNA) with the same or related compounds(13, 14, 15, 16, 17, 18, 19) . Evaluation of cationic lipids usually involves comparison of different lipids and different formulations using expression of a transgene as the end point. However, such an approach is an empiric one that may provide little understanding of intermediate steps involved in transfection. Without knowledge of the cellular mechanisms of gene transfer and the limiting barriers involved, it will be difficult to take a rational approach to develop improved methods of gene transfer and it is difficult to test specific hypotheses related to cellular and molecular mechanisms.
The goal of this work was to evaluate some of
the cellular mechanisms involved in cationic lipid-mediated gene
transfer and to identify the steps that may impair transfer. As a
starting point, we used DMRIE/DOPE ()(a 1:1 mixture of N-[1(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)
ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE))
as a model cationic lipid. In a systematic structure-activity analysis
of a large number of lipids, Felgner and colleagues (4) recently identified DMRIE/DOPE as a promising vector for
gene transfer. In a comparison of several lipids, we also found
DMRIE/DOPE to be effective and have optimized a series of factors and
conditions required for efficient gene transfer to HeLa cells and to
canine airway epithelium(12) . In addition, protocols approved
by the National Institutes of Health Recombinant DNA Advisory Committee
propose to use DMRIE/DOPE as the vector for gene transfer to humans
with cystic fibrosis(13) , melanoma (20) , metastatic
renal cell carcinoma(21) , and hepatic metastases of colorectal
carcinoma(22) . Our results identify a number of steps at which
lipid-mediated transfection is inefficient. The identification of these
steps provides the opportunity to further improve the process, thereby
increasing its utility and application.
To study endocytosis of other markers, COS-1 cells were
cultured on collagen-coated four-well (2 cm each) glass
slides. Following a DMEM wash, 500 µl of each fluorescent probe
solution was placed on the cells for 4-6 h. The cultures were
then washed in DMEM, covered with COS media, and returned to the
incubator. At 24 h. the cells were washed in PBS, pH 7.4, and fixed in
2% freshly prepared formaldehyde in PBS for 15 min. The slides were
then rinsed three times in PBS and once in double-distilled
H
O and, following removal of the wells, mounted in
Gel/Mount (Biomeda Corp., Foster City, CA).
To follow the cellular entry and fate of DNA, COS cells were transfected with gold-labeled DNA complexed with DMRIE/DOPE at a 1:5 (w/w) ratio. Cells were fixed at various times in 2.5% gluteraldehyde and processed using standard EM procedures. Briefly, the samples were post-fixed in 1% osmium tetroxide, followed by 2.5% aqueous uranyl acetate, and then dehydrated in a graded series of ethanol washes. Thin sections (70 nm) of the Eponate 12-embedded specimen were placed on 135-mesh hexagonal copper grids and stained with uranyl acetate and Reynold's lead citrate.
To identify
lysosomes, we localized acid phosphatase(47) . After treatment
with the lipid-DNA-gold complex, cells were fixed in 2% gluteraldehyde
in 0.1 M cacodylate buffer, pH 7.2, at 4 °C for 1 h.
Cultures were then rinsed three times for 10 min in 0.1 M cacodylate buffer at room temperature, rinsed three times for 10
min in Tris-maleic buffer, pH 5.0, at 37 °C, and incubated in
reaction solution consisting of 0.25% sodium -glycerophosphate and
0.08% lead nitrate in Tris-maleic buffer for 1 h at 37 °C. Cells
were rinsed in Tris-maleic buffer and cacodylate buffer as above,
post-fixed in 2% osmium tetroxide for 1 h, and processed for TEM as
described previously.
Although the preparation had been optimized for transfection, we were surprised to find a very heterogeneous population of complexes. Fig.1A shows free DNA that has not been complexed with lipid, and Fig. 1(B-F) show examples of the type of lipid-DNA particles we observed. In some cases the DNA appeared to be compacted into relatively dense particles, but as shown in Fig.1B, compacted and free DNA could be observed in the same field. Often when dense aggregates were observed, DNA also appeared to extend from the complex, forming looped structures (Fig.1E). In some cases the DNA appeared to be free (Fig. 1D), and in other cases it may have been coated with lipid to form an extended structure (Fig.1E). In some cases, relatively large aggregates appeared to form (Fig.1C), and less frequently we saw strands of complexes (Fig.1F). The complexes were quite heterogeneous, although the most frequently observed complexes resembled those in Fig.1(B and E). In all cases the complexes appeared to be at least 100 nm or larger, at least in one dimension.
Figure 1: Electron photomicrographs of lipid-DNA complexes. Lipid-DNA complexes were prepared at a ratio of 5:1 (w/w), and the methods used for electron microscopy are described under ``Materials and Methods.'' PanelA shows appearance of plasmid DNA without lipid. Panels B-F show examples of the various types of complexes that were observed. In panelB the openarrow shows uncomplexed plasmid and the solidarrow shows plasmid complexed with lipid. Bar indicates 100 nm.
Gershon and colleagues (31) have also imaged lipid-DNA complexes formed from calf thymus DNA or plasmid DNA complexed with an equimolar mixture of N-1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammonium chloride (DOTMA) and phosphatidylethanolamine. At a lipid-DNA charge ratio of 1.0, they showed an electron photomicrograph of a rod-like complex approximately 700 nm long, which most closely resembled the structures indicated by solidarrows in Fig.1B of the present study. However, there was no indication of the substantial heterogeneity that we observed. Besides the difference in lipid, one additional difference between our study and theirs is that they used the Kleinschmidt method(30) , whereas we used a much different method to place the sample on the grid.
At present we do not know whether one or all of the various forms of complex shown in Fig.1is the most efficient transfection particle. However, methods designed to produce homogeneous complexes and to identify the complex(s) that is most effective at transfection could provide an important advance in improving gene transfer.
Figure 2: Effect of incubation time on DMRIE/DOPE-mediated DNA entry into COS cells. Data are histograms of relative fluorescence intensity versus number of cells following increasing duration of exposure to DMRIE/DOPE and 1 µg of ethidium-labeled DNA (5:1, w/w ratio). Uptake of complexed DNA was evaluated by FACS as described under ``Materials and Methods.'' In each panel we plot two histograms. One histogram (control), showing cells that were exposed to DNA alone for 24 h, is repeated in all 6 panels. The second histogram in each panel was from cells incubated with the lipid-DNA complex for the indicated times. The histogram labeled 0min was from cells not exposed to DNA. The percentage of highly fluorescent cells was calculated by subtracting the control histogram (cells exposed to DNA alone) from the experimental histogram. Less than 5% of the cells were highly fluorescent at 5 and 30 min. The percentage of highly fluorescent cells at 1 h was 36.3%, at 6 h was 68.4%, and at 24 h was 72.3%. These values are likely underestimates of the actual percentage of cells that contain labeled DNA, because the figure shows that the entire histogram for treated cells shifted position at late time points.
To further evaluate
efficiency of uptake, we used three different cell types: COS, HeLa,
and C127. Fig.3shows that more of the COS and HeLa cells took
up ethidium-labeled DNA than did C127 cells, suggesting cell
type-dependent variability in lipid-DNA uptake. To learn whether the
lipid-DNA uptake by different cells paralleled expression of transgene,
we measured the percentage of cells showing nuclear localized
-galactosidase activity after transfection with pCMV-
Gal and
we measured luciferase activity after transfection with pRSV-Luc. Fig.3shows that both measures of expression paralleled the
uptake of DNA; COS and HeLa cells showed more expression than did C127
cells. These data are consistent with previous observations that the
efficiency of cationic lipid-mediated transfection varies for different
cell types(5, 7) . More importantly, the correlation
between the percentage of cells taking up DNA and the percentage of
cells expressing transgene indicates that in some cells lipid-DNA
uptake may be an important barrier to transfection. However, they also
suggest that additional barriers to transfection may be responsible for
differences in efficiency observed with different cell types.
Figure 3: DNA uptake and expression in COS, HeLa, and C127 cells. Uptake was evaluated as described in legend of Fig.2. A, the percentage of highly fluorescent cells was calculated by subtraction of the control histogram (DNA alone) from the experimental histogram. B, the percentage of cells transfected was evaluated by X-gal staining. C, expression of luciferase was evaluated by measurement of total luciferase activity. Cells were transfected and assays performed as described under ``Materials and Methods.'' COS cells (openbars), HeLa cells (cross-hatched bars), and C127 cells (graybars) in 35-mm dishes were transfected with 0.5 µg of plasmid and DOPE/DMRIE at a 5:1 lipid-DNA (w/w) ratio in 1.5 ml of Eagle's MEM. DNA uptake was measured 6 h after exposure to lipid-DNA complex. X-Gal staining and luciferase activity were measured 48 h after transfection. Data are means ± S.E. from three to six experiments. Asterisks indicate values for C127 cells are significantly different from those for COS or HeLa cells (p < 0.01).
The
fact that a large percentage of cells took up some DNA seemed
encouraging. To more accurately assess the efficiency of the uptake
step, we measured the amount of DNA that was in the cells. To do this
we exposed cells to the lipid-DNA complex for varying intervals of
time, then removed the complex by rinsing and measured the amount of
intracellular DNA by dot blot with a probe to the plasmid DNA. Fig.4shows the results in COS cells. At 5 min the amount of DNA
in the cells was very small, but the amount increased progressively
with time. These results are consistent with our data using
ethidium-labeled DNA and suggest that covalent modification with
ethidium monoazide did not substantially alter the ability of DNA to
enter the cell. The dot blots show that after 6 h of exposure the cells
had taken up approximately 60% of the DNA that was added (n = 6). These data are consistent with the previous
observation that when NIH 3T3 cells were exposed to
DOTMA/DOPERNA, 20-30% of the RNA became
RNase-resistant(32) . (However, the ability of lipids to
protect DNA from the activity of DNase (31) makes
interpretation of those results less clear.) By standardizing the dot
blots to known amounts of DNA, we estimated the absolute amount of DNA
that entered the cells. At the start of the experiment we added 2
µg of DNA to COS cells, and 6 h later we found that the cells had
taken up 1.2 ± 0.1 µg (n = 3). We calculate
that on average each cell took up 2.95
10
plasmids.
It is likely that a large percentage of the cells contained a very
large number of plasmids. However, our expression data obtained under
similar conditions showed that less than 50% of the cells expressed the
transgene. Thus, steps subsequent to uptake may be important
impediments to transfection.
Figure 4: Dot blot of plasmid DNA in cell extract of COS cells. Cells in 35-mm dishes were transfected with DMRIE/DOPE and 2 µg pRSV-Luc (5:1, w/w ratio). At the indicated times cells were removed for analysis as described under ``Materials and Methods.'' For the 24-h time point, cells were exposed to the lipid-DNA complex for 6 h in serum-free media and then an additional 18 h with serum-containing media. Figure shows autoradiogram of representative results. Cells treated with DNA alone for 24 h were used as a negative control. Figure also shows the dilution series of DNA in the bottom row. Similar results were obtained in two other experiments.
Figure 5:
Electron photomicrographs of COS cells
transfected with gold-labeled DNA complexed with lipid. Cells were
exposed to DMRIE/DOPEDNA complexes and then removed for electron
microscopy at the following times: panelA, 5 min; panelB, 30 min; panelC, 1 h; panelD, 6 h; panelE, 24 h; panelF, 24 h. Cells transfected with plasmid that
had not been labeled with gold are shown in panelF. Bar indicates 100 nm. Gold particles were 10
nm.
Uptake of complexes predominantly by
endocytosis is not what we had expected. It has often been assumed that
cationic lipid-mediated transfection results from fusion of the
positively charged complex with the plasma membrane resulting in direct
entry of the lipid into the cytoplasm(3, 8) . This
notion was based on the observations that (a) lipid-DNA
complexes containing fluorescently labeled DOPE seemed to stain the
cell surface (3) and (b) positively charged DOTMA/DOPE
liposomes fuse with negatively charged liposomes composed of
phosphatidylserine and phosphatidylethanolamine or
phosphatidylcholine(33) . However, several observations have
suggested that endocytosis may be involved. The ability of chloroquine
to enhance cationic lipid-mediated transfection in some cases has been
interpreted to suggest that this agent, which increases endosome pH and
prevents endosome-lysosome fusion, may aid escape of the complex from
the endosome(4, 6, 34) . Interestingly,
chloroquine inhibited DMRIE/DOPE transfection of COS cells(4) .
In addition, electron photomicrographs of a lipopoly (L-lysine)-DNA complex suggested that the complex was present
in endosomes(34) . Although our data do not allow us to exclude
the possibility that some of the DNA may have entered the cytoplasm
directly or that a different cationic lipid might produce a different
mechanism of entry, these results suggest that DMRIE/DOPEDNA
complexes enter COS and HeLa cells primarily through endocytosis.
Figure 6:
Fluorescence microscopic images of labeled
DNA and lipid after addition to COS cells. Photomicrograms are confocal
images superimposed on transmitted light image. DNA was labeled with
ethidium monoazide in panelA, complexes that include
isatoic ester-labeled DMRIE are shown in panelB, and
complexes which contained NBD-labeled DOPE are shown in panelC. DMRIE/DOPEDNA complexes were generated with the
labeled components at a lipid-DNA ratio of 5:1 (w/w). PanelsD and E show cells exposed to Texas Red-labeled
dextran and transferrin, respectively. Cells were exposed to lipid-DNA
complex, dextran, and transferrin for 6 h. Then the media was replaced
and cells incubated an additional 18 h before they were studied. In all
panels the fluorescence is shown as orange. N indicates nucleus. Bar indicates 20
µm.
We also used electron microscopy and gold-labeled DNA to assess the fate of lipid-DNA complexes 24 h after application to the cells. Fig.7(A-C) shows that the gold-labeled DNA remained in vesicles that were often found in a perinuclear location. Moreover, the vesicles were often very large, much larger than anything we observed at 1-6 h (Fig.5). This result is consistent with the appearance of discrete areas of perinuclear fluorescence observed with fluorescently labeled DNA and lipid (Fig.6, A-C). These results suggest that the lipid-DNA complex was endocytosed and moved toward the nucleus where the endosomes fused, and coalesced into large membrane-bound vesicles. Of note, all of the gold-labeled DNA was observed within the membrane-bound vesicular complexes; none was observed free in the cytoplasm or in the nucleus.
Figure 7: Electron photomicrograph of COS cells transfected with gold-labeled plasmid. Cells were exposed to a complex of gold-labeled plasmid and DMRIE/DOPE as described in legend to Fig.6. The lipid-DNA complex was removed by washing at 6 h, and cells were studied 24 h after the start of transfection. Panels A-C show examples of large membrane bound complexes. PanelD shows a higher magnification of panelC, and panelE is a higher magnification of panelA. Bars indicate 100 nm.
The appearance of the lipid-DNA was interesting in that it often developed a highly ordered pattern. Fig.7D shows a frequently observed regular lamellar pattern with a periodicity of approximately 3.2-4.5 nm. Fig.7E shows an example of what may represent this pattern in cross-section. The appearance is one of a series of regularly packed tubules. The lumen of the tubule was approximately 6.5 nm in diameter, and the center-to-center distance between tubules was approximately 17.5 nm. One way to explain the regular appearance would be to assume that a strand of DNA is surrounded by a bilayer or in some cases a tubular monolayer of lipid. Such arrangements could give the regularly shaped appearance described above and could account for the interaction of lipid and DNA.
We considered the possibility that the lipid-DNA complex is contained within lysosomes. To identify lysosomes, we used acid phosphatase enzyme-cytochemistry. Fig.8shows an example of an electron photomicrograph taken 24 h after application of the lipid-DNA complex. The figure shows that the gold-labeled DNA and the reaction product were present in different cellular compartments. This result suggests that the lipid-DNA was present in endosomes that did not fuse with the lysosomes. It is also possible that the presence of the lipid-DNA complex prevented fusion.
Figure 8: Electron photomicrograph of COS cells transfected with gold-labeled plasmid in which lysosomes are identified by acid phosphatase enzyme cytochemistry. Reaction product identifying lysosome is indicated by arrow. Bar indicates 100 nm.
These results indicate that most of the lipid-DNA complex is endocytosed and retained in the perinuclear area. However, because treatment of cells with lipid-DNA complex can lead to transgene expression, at least some of the DNA must escape from the endosomal compartment. Our inability to detect gold-labeled DNA free in the cytoplasm or nucleus might reflect the fact that not all of the DNA was labeled and some unlabeled DNA was able to escape from endosomes; it could be that labeling with gold prevented DNA from escaping from the endosome, or it could reflect the fact that very little DNA escaped from the endosome and the sensitivity of electron microscopic detection is not high enough to detect it. The fact that we are led to similar conclusions with results with three different techniques (light microscopic evaluation of fluorescently labeled DNA and fluorescently labeled lipid, EM evaluation of gold-labeled DNA, and quantitation of DNA uptake by dot blot) serves to validate the methods and strengthen the conclusions. Thus, escape of DNA from endosomes is an important barrier to transfection.
It is interesting that escape of DNA from
the endosome is also a major barrier for DNA delivery via
transferrin-coupled to DNA-polylysine complexes(37) . In that
system, addition of adenovirus to enhance escape from the endosomal
compartment improved transfection. Likewise with cationic
lipid-mediated DNA transfer, treatment of cells with adenovirus (200
plaque-forming units/cell) increased the efficiency of transfection
2-7-fold(38) . We have also observed that addition of 200
infectious units of adenovirus to the lipid-DNA complex increased
expression 4-fold, but it also increased expression when added to
plasmid without lipid. ()With only this modest increase in
expression, the qualitative methods we have used are not able to
identify the mechanisms by which adenovirus enhanced expression. It may
be that adenovirus enhanced escape from the endosomes, but other
mechanisms are possible, including binding of lipid-DNA complex to the
adenovirus or enhancement of DNA entry into the nucleus (see below).
Fig.9shows that even when 0.01 µg of
pTM-Gal was transfected with DMRIE/DOPE and cytoplasmic
transcription was driven by recombinant vaccinia virus, most cells were
positive for X-gal staining. By comparison, with 100-fold more of a
plasmid (pCMV-
Gal) that required nuclear delivery, only 10% of the
cells were positive. We considered the possibility that vaccinia virus
infection might have disrupted the endosomes, thereby releasing DNA
into the cytoplasm or making it available for transfer to the nucleus.
However, vaccinia virus infection did not increase expression from
cells transfected with pCMV-LacZ (not shown). Moreover, evaluation of
the cells by electron microscopy 16 h after the transfection/infection
procedure revealed gold labeled DNA complexed to lipid in intact
vesicles and endosomes, in addition to numerous intracellular viral
particles (Fig.10). After virus infection, we observed no free
gold particles in the cytoplasm.
Figure 9:
Percentage of X-gal-positive cells
following transfection with varying amounts of DNA. All transfections
used a DMRIE/DOPE:DNA ratio of 5:1 (w/w). Transfection was performed
for 6 h, the media was replaced, and 10 h later cells were stained with
X-gal reagent. Openbars indicate cells transfected
with pTM-Gal plus vTF7-3 (m.o.i. of 5). Solidbars represent cells that were transfected with pCMV-
Gal. Hatchedbar indicates cells infected with a
recombinant vaccinia virus expressing
-galactosidase, vTF7-LacZ,
plus vTF7-3 (both at m.o.i. of 5).
Figure 10: Electron photomicrograph of COS cells treated with lipid-DNA plus recombinant vaccinia virus. Cells were treated with 1 µg DNA at a lipid-DNA (w/w) ratio of 5:1 and 1 h before recombinant vaccinia virus (vTF7-3, m.o.i. of 5). Cells were prepared 16 h later. Closedarrow indicates an example of a vaccinia virus; openarrow shows the lipid-DNA complex in a perinuclear vesicle. N indicates nucleus. Note that vaccinia virus replicates in these cells. Disruption of intracellular membranes was not observed. Bar indicates 100 nm.
These data indicate that after transfection most cells contain at least some DNA in the cytoplasm. This conclusion is consistent with the observation of Gao and Huang(39) , who delivered T7 RNA polymerase to cells along with cationic lipid and plasmid containing a T7 promoter driving CAT expression. They found that total transgene expression was greater than that observed with a plasmid that required nuclear expression; however, they made no assessment of the percentage of cells transfected. These data suggest that one of the most important barriers to transfection may be movement of DNA from the cytoplasm to the nucleus.
Figure 11: Secreted alkaline phosphatase production from oocytes injected with DNA and lipid-DNA complexes. Nuclear or cytoplasmic injections of oocytes were performed and alkaline phosphatase activity in the media was measured as described under ``Materials and Methods.'' PanelA shows alkaline phosphatase activity following nuclear or cytoplasmic injections. PanelsB and C show alkaline phosphatase production after injection of DNA alone or DNA complexed with lipid at the indicated lipid-DNA ratios into the cytoplasm or nucleus, respectively. The total amount of DNA injected was constant for all conditions. Data are mean ± S.E., n = 4-12 for each condition.
This result indicates that DNA traffic from the cytoplasm to the nucleus is an inefficient process. This conclusion is consistent with Capecchi's observation (40) that injection of plasmid into the nucleus of a mouse cell line led to protein expression in over 50% of cells, whereas injection into the cytoplasm led to expression in <0.01% of cells. The fact that we observed transfection in mammalian cells treated with lipid-DNA, but not the oocyte may be in part due to the fact that the mammalian cells are dividing whereas the oocyte is stationary. In contrast to our data, an 18-bp oligonucleotide delivered to endothelial cells with DOTMA/DOPE accumulated in the nucleus(9) . Moreover, when a 28-bp oligonucleotide was injected into the cytoplasm of CF-1 cells or fibroblasts, the DNA rapidly and preferentially accumulated in the nucleus(41) . The reason for the difference between our results using a plasmid and the results with oligonucleotides most likely relate to the size of the DNA. Oligonucleotides may readily pass through nuclear pores, which have a diffusion limit of approximately 40,000 Da(42) , whereas the much larger plasmid would not.
Because Bennett et al.(9) suggested that cationic lipids may alter the intracellular distribution of oligonucleotides by increasing delivery to the nucleus, we asked whether the lipid-DNA complex might improve nuclear targeting of the plasmid and thereby increase expression. However, when we injected the complex into the cytoplasm at varying lipid-DNA ratios, alkaline phosphatase production was not substantially increased (Fig.11B), suggesting that complexing plasmid with lipid did not improve transport to the nucleus.
We also asked whether complexing the DNA with the lipid would impair transcription when the complex is injected into the nucleus. Fig.11C shows that when we used a lipid-DNA ratio of 5:1 (w/w), which was optimal for transfection, the production of secreted alkaline phosphatase was inhibited compared to injection of DNA alone. In contrast, when we used lipid-DNA (w/w) ratio of 1:1, which is suboptimal for transfection and in which there is much more uncomplexed DNA, the cells produced alkaline phosphatase. This result suggests that complexing DNA with lipid prevents expression of the encoded protein, probably because the DNA does not dissociate from the lipid and is not available for transcription. This observation is consistent with the finding that DNA complexed with cationic lipid is protected from DNase in vitro(31) . Thus, dissociation of DNA from the lipid complex would appear to be another important variable limiting gene transfer and expression.
In the presence of these barriers, each of which can provide a major limitation, how is it that cationic lipids can mediate gene transfer and expression? Our data suggest that cationic lipid-mediated transfection is a rather inefficient process that proceeds through a mass action effect(44, 45) . A large amount of DNA is delivered to the cell, a small percentage of that is released from the endosomes, and a small percentage of that makes its way from the cytoplasm to the nucleus where it is transcribed. The inefficiency of each step suggests that specific attention must be paid to developing ways to overcome each of the different barriers. Although we know of no specific toxicity associated with delivery of a large amount of DNA to a cell, delivery of large amounts of lipid does have toxicity. Focusing attention on each of the barriers to gene transfer may allow a decrease in the amount of lipid required and hence reduce toxicity. If efficiency can be improved and lipid toxicity minimized, cationic lipids could be attractive vectors for diseases in which repeated administration is required.
In considering the various barriers encountered with lipid-mediated gene transfer, it is interesting to remember that viruses, such as adenovirus, have solved many of these problems. They bind to specific receptors for cell uptake, they have mechanisms for release of viral DNA from the endosome, and they have mechanisms to target the DNA to the nucleus(46) . Perhaps a better understanding of the mechanisms used by a variety of viruses will allow us to adapt some of the advantages and features of viral systems and yet avoid their disadvantages in designing better nonviral vector-mediated gene transfer techniques.
Our data suggest that there is much opportunity for improving cationic lipid-mediated gene transfer and that as the process is improved, it could be used successfully in an even larger number of applications.