Cationic Lipid-mediated Gene Transfer Research Articles

Binding and uptake of cationic lipid:pDNA complexes by polarized airway epithelial cells.

To better understand the barriers associated with cationic lipid-mediated gene transfer to polarized epithelial cells, Fischer rat thyroid (FRT) cells and polarized normal human bronchial epithelial (NHBE) cells grown on filter supports at an air-liquid interface were used to study the binding and uptake of cationic lipid:plasmid DNA (pDNA) complexes. The efficiencies of binding and uptake of cationic lipid:pDNA complexes by these cell systems were monitored using fluorescence microscopy of fluorescently tagged lipid or pDNA probes. Fluorescent probe bound to the cell surface was differentiated from internalized probe by adding trypan blue, which quenched the fluorescence of bound but not internalized probes. For proliferating cells, binding and internalization of the cationic lipid:pDNA complexes were determined to be efficient. In contrast, little binding or internalization of the complexes was observed using polarized epithelial cells. However, after aspirating a small area of cells from the filter support, virtually all of the cells adjoining this newly formed edge bound and internalized the cationic lipid:pDNA complexes. To determine if their uptake in edge cells was related to the ability of the complexes to access the basolateral membranes of these cells, the binding and uptake of complexes was monitored in polarized NHBE cells that had been pretreated with EGTA or Ca2+-free media, strategies known to disrupt tight junctions. Cells treated in this manner bound and internalized cationic lipid:pDNA complexes efficiently and also expressed significant levels of transgene product. Control cells with intact tight junctions neither bound complexes nor expressed significant transgene product. These data confirm and extend earlier observations that the polarized apical membranes of airway epithelial cells are resistant to transfection by lipid:pDNA complexes. Further, in contrast to previous studies that have shown the entry step of complexes is not an important barrier for COS and HeLa cells, binding and entry of complexes in polarized NHBE cells appear to be rate limiting. These findings suggest that strategies designed to open the tight junctions of polarized epithelial cells may improve gene delivery to these cells for diseases such as cystic fibrosis (CF).

Efficiency of cationic lipid-mediated transfection of polarized and differentiated airway epithelial cells in vitro and in vivo.

Systematic analysis of a large number of different cationic lipids has led to the identification of novel structures (GL-67) and formulations of cationic lipid:plasmid DNA (pDNA) complexes that facilitate high levels of gene expression in lungs of mice. However, despite significant improvement in gene transfer activity, we show here that the efficiency of GL-67-mediated gene transduction of intact airway epithelia is still relatively low. Administration of GL-67:pCF1-CFTR (encoding the cystic fibrosis transmembrane conductance regulator) complexes into the nasal epithelium of cystic fibrosis (CF) transgenic mice resulted only in marginal correction of the ion transport defects. Measurements of nasal potential differences (PD) showed no correction of the sodium (Na+) transport defect, and only partial restitution of the chloride (Cl-) transport defect was achieved in a small proportion of the animals after perfusion of the nasal epithelium with the complexes. Furthermore, in contrast to results obtained following instillation of GL-67:pDNA complexes into the lungs of mice, perfusion of GL-67:pDNA into the nasal epithelium resulted only in a moderate enhancement of gene transduction activity relative to that attained with naked pDNA alone. To determine the basis for this low efficiency of transfection, a series of studies was conducted to identify some of the barriers governing cationic lipid-mediated gene transfer to the airway epithelium. We show here that the transfection activity of GL-67 was affected by the polarization, differentiation, and proliferative state of the cells. Diminished transfection activity was observed with nonmitotic, highly polarized and differentiated airway epithelial cells. This observed reduction in gene expression with nonmitotic cells was determined to be due in part to inefficient nuclear translocation of the pDNA from the cytoplasm. Together these data indicate that much improvement in the ability of cationic lipids to transfect polarized and differentiated airway epithelial cells is a necessary prerequisite for effective cationic lipid-mediated gene therapy of airway diseases such as CF.

Reversed-Phase High Performance Liquid Chromatographic Analysis of Cationic Lipid-Based Gene Transfer Agents

Cationic lipid-mediated gene transfer represents a promising approach for the treatment of a number of diseases. Since the successful introduction of DOTMA:DOPE (Lipofectin), a variety of cationic lipids have been developed for use in gene transfer. Some of the more active cationic lipid formulations, including GL-67:DOPE, DC-chol:DOPE, DMRIE:DOPE and DOTAP, have been used in human clinical trials. It is of critical importance to develop robust analytical methods for the determination of the chemical purity of these formulations. We report here efficient, sensitive, and reproducible reversed-phase HPLC methods for use in determining the chemical purity of cationic lipid formulations. GL-53:DOPE, GL-67:DOPE, DMRIE:DOPE, DC-chol:DOPE, GAP-DLRIE:DOPE, DOTMA:DOPE (Lipofectin), DDAB:DOPE (Lipofectace), DOSPA:DOPE (Lipofectamine), DOGS (Transfectam), and DOTAP were analyzed by HPLC on C8 or C18 bonded phase columns with aqueous/mixed organic mobile phases containing trifluoroacetic acid and with ELSD detection in the gradient elution mode. Baseline resolution of the components of the GL-53:DOPE formulation was achieved by optimization of the solvent system and gradient profile. Capacity factors (k') of the cationic lipids were greatly affected by the end-capping chemistry of the C18 bonded phases. The calibration curves for GL-53, DC-chol, DMRIE, and DOPE were determined in the range of 1.6–200.0 μg. The detection limits for these compounds were determined to be 0.4–1.6 μg

Phosphate-enhanced transfection of cationic lipid-complexed mRNA and plasmid DNA

Cationic lipid-mediated gene transfer has been shown to be a competent albeit inefficient mechanism of promoting cellular gene transfer. One way to improve the efficacy of cationic lipid-mediated transgene expression is to optimize conditions for complex formation between the lipids and nucleic acids. In this report we describe the beneficial effects of using phosphate buffer to precondition lipofectin (a 1:1 (w/w) mixture of N-[1-(2,3-dioleyloxy)propyl]- n, n, n-trimethylammonium chloride (DOTMA), and dioleoyl phosphatidylethanolamine (DOPE)) prior to complexing with plasmid DNA or mRNA. Under such optimized conditions we studied the kinetics of DNA- and RNA-mediated transgene expression in a human osteosarcoma cell line (HOS). Preincubation of lipofectin in phosphate buffer resulted in up to 26- and 56-fold increases in luciferase expression from plasmid DNA and mRNA, respectively. Addition of chloroquine (50 μM), which enhanced plasmid-mediated gene delivery 3-fold, was synergistic with phosphate resulting in an additional 46-fold increase in luciferase expression. The preincubation with phosphate shortened both the time required for cellular uptake and the time to achieve maximal transgene expression. Optimal transfection was achieved in the presence of 30–80 mM phosphate, at pH 5.6–6.8 under which the phosphate anion is divalent. The effect of phosphate anion was specific in that monovalent Cl - and acetate anions were not stimulatory. These results demonstrate that divalent phosphate anion plays a stimulatory role during complex formation and transfection when cationic lipids come in contact with negatively charged nucleic acids and cell membranes. These findings delineate specific conditions which dramatically enhance transfection efficiency for both DNA and mRNA, and provide an effective procedure for gene transfection studies.

Cationic lipid-mediated gene transfer: effect of serum on cellular uptake and intracellular fate of lipopolyamine/DNA complexes

Most of the cationic lipids used for gene transfer experiments drastically lose their efficiency in the presence of serum. We used a cationic lipid with a spermine head group and its fluorescent analog to study the cellular uptake and the intracellular fate of lipoplexes in the presence and absence of serum. We found that the amount of DNA and lipid taken up by the cells was not related to the efficacy of the gene transfer. When the lipofection was performed in the presence of serum, lipoplexes were contained within small intracellular vesicles. In the absence of serum, the vesicles were larger and heterogeneous in size and shape. By analysis of their size distribution, we showed that lipoplexes preformed in the absence of serum tended to aggregate. This aggregation was inhibited in the presence of serum. We used a carbonate formulation that led to the preformation of large particles: those large particles gave a high lipofection efficiency in the presence of serum and their intracellular distribution was identical to that observed in the absence of serum.

Cationic lipid-mediated gene transfer: analysis of cellular uptake and nuclear import of plasmid DNA.

Cationic lipids are widely used for gene transfer in vitro and show promise as vectors for in vivo gene therapy applications. However, there is limited understanding of the cellular mechanisms involved in nonviral gene transfer. We investigated two major steps that could be limiting barriers to cationic lipid-mediated gene transfer in vitro. We used a fluorescent plasmid to study the cellular uptake and the intracellular fate of lipoplexes during in vitro transfection of fibroblast cells and found that 100% of the cells take up lipoplexes. The intracellular staining observed with lipoplexes was clearly different from that obtained with endocytosed fluorescent dextran. This suggests that cells readily take up lipoplexes by a mechanism that could be different from endocytosis in our conditions. However, the escape of DNA from intracellular vesicles could be a major limiting barrier to gene transfer. Direct injection of plasmid DNA into the nucleus and cytoplasm of cells indicated that DNA traffic from the cytoplasm to the nucleus might be also an important limiting step.

A low rate of cell proliferation and reduced DNA uptake limit cationic lipid-mediated gene transfer to primary cultures of ciliated human airway epithelia.

Complexes of DNA and cationic lipid offer potential advantages for gene transfer to airway epithelia. However, we found that application of DNA-lipid (DMRIE-DOPE) complexes to primary cultures of human ciliated airway epithelia or explants of rabbit trachea generated only low levels of gene transfer. In contrast, when we applied the DNA-lipid to immature human epithelia shortly after seeding, transgene expression was substantially higher. We identified two barriers that limit gene transfer. First, uptake of the DNA-lipid complexes into airway cells across the apical membrane decreased rapidly with time after seeding and paralleled the decrease in transgene expression. Second, cell division decreased with time after seeding, and we found that cells in mitosis (labeled with BrdU) were much more likely to express transgene than BrdU-negative cells. These data suggest that the entry step across the apical membrane and the low rate of cell division are important barriers for cationic lipid-mediated gene transfer to airway epithelia. Attempts to modify these two processes may yield improvements in the efficiency of gene transfer to the airways in cystic fibrosis.

Effect of co-lipids in enhancing cationic lipid-mediated gene transfer in vitro and in vivo.

Complexes of DNA and cationic lipids are promising vectors for gene transfer. Most cationic lipid formulations contain both a cationic component and a neutral co-lipid. We found that the co-lipid could influence DNA uptake in COS-1 cells, but processes subsequent to uptake were even more important in determining gene expression. We compared dioleoylphosphatidylethanolamine (DOPE) and structural analogs of DOPE combined with cationic lipids and found that DNA uptake and transgene expression did not always correlate. Transgene expression was dependent on DNA uptake into the cell, on entry of DNA into the cytoplasm, and on release of DNA from the lipid complex. We found that some co-lipids had a greater effect on DNA uptake, whereas others had a greater effect on steps subsequent to entry. Based on those results, we tested the hypothesis that co-lipids conferring different properties could be combined to enhance gene transfer. The results showed that a combination of co-lipids had a synergistic effect on expression. We also found that structural analogs of DOPE were more effective than DOPE in enhancing gene transfer to mature human airway epithelia studied in vitro and to mouse lung studied in vivo. These data provide insight into the mechanism by which co-lipids influence cationic lipid-mediated gene transfer and show that optimization of the effects of co-lipids can enhance gene transfer both in vitro and in vivo.

Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung.

Studies have indicated that although abundant levels of transgene expression could be achieved in the lungs of mice instilled with cationic lipid:pDNA complexes, the efficiency of gene transfer is low. As a consequence, a relatively large amount of the complex will need to be administered to the human lungs to achieve therapeutic efficacy for indications such as cystic fibrosis. Because all cationic lipids exhibit some level of cytotoxicity in vitro, we assessed the safety profile of one such cationic lipid, GL-67, following administration into the lungs of BALB/c mice. Dose-dependent pulmonary inflammation was observed that was characterized by infiltrates of neutrophils, and, to a lesser extent, macrophages and lymphocytes. The lesions in the lung were multifocal in nature and were manifested primarily at the junction of the terminal bronchioles and alveolar ducts. The degree of inflammation abated with time and there were no apparent permanent fibrotic lesions, even in animals that were treated at the highest doses. Analysis of the individual components of the complex revealed that the pulmonary inflammation was primarily cationic lipid-mediated with a minor contribution from the neutral co-lipid DOPE. Associated with the lesions in the lungs were elevated levels of the pro-inflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma) that peaked at days 1-2 post-instillation but resolved to normal limits by day 14. Total cell counts, primarily of neutrophils, were also significantly elevated in the bronchoalveolar lavage fluids of GL-67:pDNA-treated mice between days 1 and 3 but returned to normal limits by day 14. No specific immune responses were detected against the cationic lipid or plasmid DNA in mice that had been either instilled or immunized with the individual components or complex, nor was there any evidence of complement activation. These studies indicate that a significant improvement in the potency of cationic lipid:pDNA formulations is desirable to minimize the toxicity associated with cationic lipids.

Cationic lipid-mediated delivery and expression of prepro-neuropeptide Y cDNA after intraventricular administration in rat: feasibility and limitations

The utility of in vivo lipofection for delivery and expression of a neuropeptide gene in the adult rat brain was explored. Prepro-neuropeptide Y (NPY) cDNA was cloned into the episomal eucaryotic expression vector pCEP4. This construct was complexed to lipofectamine or lipofectin. Complexed DNA was injected into the lateral ventricles of adult rats. Brains were removed for analysis following various time intervals. Polymerase chain reaction (PCR) reactions were designed for specific detection of endogenous and vector derived NPY sequence, respectively. PCR of DNA preparations from 5 major brain regions (frontal and parietal cortex, striatum, hypothalamus, brain stem) demonstrated presence of vector DNA up to 1 month (longest interval studied) in all brain regions. Reverse-transcription (RT-) PCR of DNase treated RNA-preparations from brain tissue demonstrated presence of both vector-derived and endogenous NPY mRNA in treated animals, while only endogenous mRNA was detected in controls. In situ hybridization histochemistry indicated scattered patches of vector uptake into tissue in the vicinity of the CSF compartment, but not into deeper located structures. Weight gain was not affected, indicating that the expression levels achieved may not be sufficient to play a functional role, and/or may need to be targeted to specific brain areas. These findings suggest a potential for cationic lipid mediated gene transfer in the brain as an experimental tool and as a possible future therapeutic principle, but also indicate the need for optimization of delivery strategies in order to achieve functionally relevant expression levels.

Cellular and Molecular Barriers to Gene Transfer by a Cationic Lipid

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.

Optimization of cationic lipid-mediated gene transfer to airway epithelia

The use of cationic lipids for gene transfer to airway epithelia has shown promise in in vitro and in vivo studies. However, previous studies have used a wide variety of different lipid preparations and different formulations. Few studies have been designed to optimize the variables involved in transfection, and none have been focused on airway epithelia. Therefore we examined variables that affect cationic lipid-mediated transfection of HeLa cells and of airway epithelial cells grown on permeable filter supports at the air-liquid interface. To quantitate expression of cDNA, we assayed expression of luciferase. We found that the ratio of DNA to lipid was an important variable that determined transfection efficiency. In both HeLa and airway epithelial cells, the optimum charge ratio of cationic lipid to anionic DNA was approximately 1.25, consistent with the notion that a positively charged complex facilitates interaction with the negatively charged cell membrane. After testing a series of readily available cationic lipids, we found that 1,2-dimyristyloxypropyl-N,N-dimethyl-hydroxyethyl ammonium bromide (DMRIE)/dioleoyl phosphatidylethandamine (DOPE) appeared to show good efficacy. The concentration of DNA and cationic lipid also played an important role: in HeLa cells the optimum concentration of cationic lipid was approximately 5 microM and in airway epithelial cells was approximately 15 microM.(ABSTRACT TRUNCATED AT 250 WORDS)