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.

MATERIALS AND METHODS

Cells and Culture

Reagents

Plasmids and Vaccinia Virus

Cationic Lipids

Vaccinia Virus

Ethidium-labeled DNA

Gold-labeled DNA

Markers of Endocytosis

Transfection

Assays

Luciferase Activity

X-Gal Staining

FACS

Dot Blot

Microscopy

Fluorescent Microscopy

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 HO and, following removal of the wells, mounted in Gel/Mount (Biomeda Corp., Foster City, CA).

Electron Microscopy

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.

Studies in Oocytes

RESULTS AND DISCUSSION

Formation of the Cationic Lipid-DNA Complex

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.

Entry of Cationic Lipid-DNA Complex into Cells

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.

Mechanism of Entry into the Cell

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.

Intracellular Disposition of Lipid-DNA Complexes

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).

Percentage of Cells with Cytoplasmic DNA

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.

Entry of DNA into the Nucleus and Nuclear Transcription

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.

Summary of Barriers to Cationic Lipid-mediated Transfection

Formation of the Lipid-DNA Complex

Entry of DNA into the Cell

Escape of DNA from the Endosomes

Dissociation of the DNA from Lipid

Entry of the DNA into the Nucleus

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.

FOOTNOTES

*

This work was supported by the National Institutes of Health and the Howard Hughes Medical Institute. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§

Parker B. Francis Fellow.

¶

Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Howard Hughes Medical Institute, University of Iowa College of Medicine, 500 EMRB, Iowa City, IA 52242. Tel.: 319-335-7619; Fax: 319-335-7623.

The abbreviations used are: 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); MEM, minimal essential medium; DMEM, Dulbecco's modified Eagle's medium; CMV, cytomegalovirus; RSV, Rous sarcoma virus; NBD, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole; X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside; PBS, phosphate-buffered saline; FACS, fluorescence-activated cell sorting; TEM, transmission electron microscopy; MBS, modified Barth's solution; DOTMA, N-1-[2,3-bis(oleoyloxy)]propyl]-N,N,N-trimethylammonium chloride.

J. Zabner, A. J. Fasbender, T. Moninger, K. A. Poellinger, and M. J. Welsh, unpublished observation.

ACKNOWLEDGEMENTS

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