Lipid-mediated gene transfer into primary neurons using FuGene: comparison to C6 glioma cells and primary glia.

Gene transfer into cells of CNS origin is an important tool to counteract neurodegeneration by introducing, e.g., neuroprotective molecules. Although viral gene transfer reveals the highest gene transfer efficiency, liposome-mediated gene transfer seems to become an attractive alternative. In this study we investigated the lipid-mediated gene transfer into primary neurons in vitro by using the novel nonliposomal lipid FuGene and compared it to primary glia and malignant C6 glioma cells. FuGene-mediated gene transfer was useful to transfer the reporter gene beta-galactosidase into C6 glioma cells, primary glia, and primary neurons. Lipofection was highly dependent on the surface bottom and did not result in good efficiencies when using glass compared to plastic dishes. Comparing to a standard lipofection (1 x 8 h), lipofection on 3 consecutive days for 6 h each ("boosting") markedly increased the gene transfer efficiency in primary glia (up to sevenfold) and in primary neurons (up to sixfold). The use of endotoxin-free DNA only slightly increased the transfection efficiency. Immunohistochemistry demonstrated MAP-2-positive neurons (up to 1614 neurons/16-mm well; 2.4% of total neurons) as well as TH-positive neurons (up to 48 neurons/16-mm well; 12.7% of TH neurons) expressing beta-galactosidase. We conclude that FuGene-mediated gene transfer is an attractive alternative to introduce genes of interest into cells of glial and neuronal origin; however, this technique lacks sufficient gene transfer efficiency.

Enhancement of liposome-mediated gene transfer to human airway epithelial cells by replication-deficient adenovirus.

We hypothesized that replication-deficient adenovirus (Ad), when complexed with plasmid DNA (pl) and cationic liposomes (L), would enhance liposome-mediated gene transfer in cultured human airway epithelial cells. Pl/L/Ad complexes were formed using charge-charge interactions. A gel electrophoresis retardation assay showed plasmid DNA to be associated with the virus in a high-molecular-weight, low-mobility complex, the diameter of which was 300 to 350 nm. Compared to pl/L alone, pl/L/Ad enhanced luciferase expression on average by 1 log-fold in human airway epithelial cells which express either mutant or wild-type cystic fibrosis transmembrane conductance regulator (CFTR). Transgene expression was sustained at high levels for up to 7 days following transfection with pl/L/Ad. Using a heat-stable alkaline phosphatase reporter gene, we showed that a larger fraction of cells was transfected by pl/L/Ad compared to pl/L. Finally, cells were exposed to Ad for 0 to 24 hours prior to pl/L or exposed to pl/L prior to Ad. We found that enhancement was significantly greater using pl/L/Ad compared to the simultaneous addition of Ad with the pl/L complexes. In addition, when pl/L was added 4 to 24 hours prior to Ad, some enhancement was found, suggesting that plasmid DNA remained in a compartment in the cell for several hours and became available for transcription with the addition of Ad. When Ad was added prior to pl/L, enhancement was found suggesting that the effect of the virus on cell membranes may persist for up to 24 hours. We conclude that the tripartite pl/L/Ad complex significantly enhances liposome-mediated transgene expression for a prolonged period of time in human bronchial epithelial cells.

Periadventitial lacZ gene transfer to pig carotid arteries using a biodegradable collagen collar or a wrap of collagen sheet with adenoviruses and plasmid-liposome complexes.

BACKGROUND: Periadventitial gene therapy is a promising alternative for the treatment of stenosis, vessel wall thickening and other complications in vascular surgery. METHODS: We compared lacZ gene transfer efficiency of DOTMA: DOPE (1:1 w/w) plasmid/liposome complexes and adenoviruses in pig carotid arteries using perivascular delivery with either a collagen collar or a wrap of collagen sheet. Safety of the gene transfer was studied by clinical chemistry, tissue pathology and PCR analysis of lung, liver, kidney, spleen, skeletal muscle and gonads. RESULTS: Gene transfer efficiency using the periadventitial collar was fourfold higher than using the collagen wrap with adenovirus at 7 days (10.22 +/- 2.96 vs 2.78 +/- 1.28 positive cells/mm2; p = 0.18) and 4.3-fold at 14 days (13.46 +/- 3.49 vs 3.11 +/- 0.88 positive cells/mm2; p = 0.03). Gene transfer efficiency at 7 days with adenovirus was fivefold higher than with the plasmid/liposome complexes both using the collar (10.22 +/- 2.96 vs 2.07 +/- 0.95 positive cells/mm2; p = 0.01) and the collagen wrap (2.78 +/- 1.28 vs 0.45 +/- 0.35 positive cells/mm2; p = 0.03). No lacZ activity was detected in plasmid/liposome transfected arteries at 14 days. In spite of the local gene delivery methods a moderate systemic distribution of the transgene was detected in the major organs by PCR analysis. CONCLUSIONS: This study shows that: (i) adenovirus delivered with the periadventitial collar or the collagen wrap is well tolerated and may become an efficient new tool in vascular gene therapy, and (ii) gene transfer vector delivered in the periadventitial collar reaches the target tissue more efficiently than the vector in the collagen wrap.

Recent advances in liver-directed gene therapy: implications for the treatment of dyslipidemia.

Somatic gene therapy for the treatment of dyslipidemia is an area of active investigation. A substantial body of data indicates that the transfer of various lipid-lowering genes to the liver is an effective method of restoring normal plasma lipids in animal models of dyslipidemia. Most studies have used adenoviral vectors because of their excellent gene-transfer efficiency. However, the first and second-generation adenoviral vectors used in these experiments are highly toxic and are associated with substantial morbidity and mortality. This article reviews current data on the properties of two novel vectors, the adeno-associated virus and the helper-dependent adenovirus that is devoid of all protein-encoding genes. Each type of vector has its advantages and drawbacks. They appear to be the most promising vectors to date for liver-directed gene transfer in the treatment of dyslipidemia.

A novel, long-circulating, and functional liposomal formulation of antisense oligodeoxynucleotides targeted against MDR1.

The goal of this study was to develop a small, stable liposomal carrier for antisense oligodeoxynucleotides (asODN) that would have high trapping efficiencies and long circulation times in vivo. Traditional cationic liposomes aggregate to large complexes and, when injected intravenously, rapidly accumulate in the liver and lung. We produced charge-neutralized liposome-asODN particles by optimizing the charge interaction between a cationic lipid and negatively charged asODN, followed by a procedure in which a layer of neutral lipids coated the exterior of the cationic lipid-asODN particle. The coated cationic liposomes had an average diameter of 188 nm and entrapped 85-95% of the asODN. The biodistribution and pharmacokinetics of an 18-mer 125I-labeled phosphorothioate ODN formulated by this method were determined after tail vein injection in mice. The majority of the asODN was cleared from blood, with a half-life of >10 hours compared with <1 hour for free asODN. When coupled with an anti-CD19 targeted antibody, this formulation was also effective at delivering an MDR1 asODN to a multidrug-resistant human B-lymphoma cell line in vitro, decreasing the activity of P-glycoprotein. No inhibition was found for nontargeted formulations or for free asODN. A number of therapeutic opportunities exist for the use of small, stable, long-circulating, and targetable liposomal carriers such as this, with high trapping efficiencies for asODN.

Nuclear gene targeting using negatively charged liposomes.

Oligonucleotides are a very useful tool to control gene activity. Oligos work by complementary base-pairing with target sequences either in the nucleus or in the cytosol (Zelphati, O., Szoka, F.C., Jr., 1996. Liposomes as a carrier for intracellular delivery of antisense oligonucleotides: a real or magic bullet? J. Contr. Rel. 41, 99-119). In a new approach using chimeric oligonucleotides (Yoon, K., Cole Strauss, A., Kmiec, E.B., 1996. Targeted gene correction of episomal DNA in mammalian cells mediated by a chimeric RNA-DNA oligonucleotide. Proc. Natl. Acad. Sci. USA 93, 2071-2076) conversion of single base mutations with help of intranuclear repair mechanisms maybe an advantageous method to cure genetic diseases which are based on single point mutations. These chimeric oligonucleotides are constructed in a way that they form an intramolecular double strand of DNA and modified RNA-bases. We used a fluorescent labelled pure 68-mer DNA-analogue of a chimeric oligonucleotides to follow the intracellular fate of these kind of genetic material. The oligos were complexed with protamine sulfate and coated with three different liposomal formulations. The AVE-3 formulation shows enhanced properties compared to a classical neutral and negatively charged formulation. Nuclear localisation of oligos could only be observed with the AVE-3 formulation. Furthermore only the negatively charged liposome formulations interact with the protamine-complexed oligonucleotides.

Mannose receptor-mediated gene transfer into macrophages using novel mannosylated cationic liposomes.

A novel mannosylated cholesterol derivative, cholesten-5-yloxy-N-(4-((1-imino-2-beta-D-thiomannosyl -ethyl)amino)bu tyl) formamide (Man-C4-Chol), was synthesized in order to perform mannose receptor-mediated gene transfer with liposomes. Plasmid DNA encoding luciferase gene (pCMV-Luc) complexed with liposomes, consisting of a 6:4 mixture of Man-C4-Chol and dioleoylphosphatidylethanolamine (DOPE), showed higher transfection activity than that complexed with 3beta[N-(N', N'-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol)/DOPE(6:4) and N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)/DOPE(1:1) liposomes in mouse peritoneal macrophages. The presence of 20 mM mannose significantly inhibited the transfection efficiency of pCMV-Luc complexed with Man-C4-Chol/DC- Chol/DOPE(3:3:4) and Man-C4-Chol/DOPE(6:4) liposomes. High gene expression of pCMV-Luc was observed in the liver after intravenously injecting mice with Man-C4-Chol/DOPE(6:4) liposomes, whereas DC-Chol/DOPE(6:4) liposomes only showed marked expression in the lung. The gene expression with Man-C4-Chol/DOPE(6:4) liposome/ DNA complexes in the liver was observed preferentially in the non-parenchymal cells and was significantly reduced by predosing with mannosylated bovine serum albumin. The gene expression in the liver was greater following intraportal injection. These results suggest that plasmid DNA complexed with mannosylated liposomes exhibits high transfection activity due to recognition by mannose receptors both in vitro and in vivo. Gene Therapy (2000) 7, 292-299.

Anti-inflammatory gene therapy directed at the airway epithelium.

Cystic fibrosis (CF) is characterised by chronic airway inflammation. Pro-inflammatory mediators in the lung are regulated by the transcription factor nuclear factor kappa B (NFkappaB). We have assessed the effect of adenovirus and liposome-mediated overexpression of the NFkappaB inhibitor IkappaBalpha, as well as liposome-mediated transfection with oligonucleotides resembling NFkappaB consensus binding sites (decoys) in a cystic fibrosis airway epithelial cell line (CFTE). Electrophoretic mobility shift assays (EMSA) were used to assess NFkappaB activity and secretion of the pro-inflammatory cytokine interleukin-8 (IL-8) was measured by ELISA. At a MOI of 30, Ad-IkappaBalpha significantly decreased IL-8 secretion to 60% and 43% of control unstimulated and TNF-alpha stimulated cells, respectively. At this MOI, approximately 70% of cells are transduced. EMSA showed an approximately 50% decrease in NFkappaB activation. Liposome-mediated transfection of IkappaBalpha did not reduce IL-8 secretion, probably due to low transfection efficiency (approximately 5% of cells). Liposome-mediated transfection of CFTE cells with rhodamine-labeled decoy oligonucleotides indicated a transfection efficiency close to 100%. TNF-alpha stimulated IL-8 secretion was reduced by approximately 40% using this approach. EMSA confirmed a significant decrease of NFkappaB activation. Decoy oligonucleotides may be a promising approach for reduction of NFkappaB-mediated pulmonary inflammation. Gene Therapy (2000) 7, 306-313.

Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.

The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory. Gene Therapy (2000) 7, 401-407.

Catheter-mediated vascular endothelial growth factor gene transfer to human coronary arteries after angioplasty.

Blood vessels are among the easiest targets for gene therapy. However, no data are available about the safety and feasibility of intracoronary gene transfer in humans. We studied the safety and efficacy of catheter-mediated vascular endothelial growth factor (VEGF) plasmid/liposome (P/L) gene transfer in human coronary arteries after percutaneous translumenal coronary angioplasty (PTCA) in a randomized, double-blinded, placebo-controlled study. The optimized angioplasty/gene delivery method was previously shown to lead to detectable VEGF gene expression in human peripheral arteries as analyzed from amputated leg samples. Gene transfer to coronary arteries was done with a perfusion-infusion catheter, using 1000 microg of VEGF or beta-galactosidase plasmid complexed with 1000 microl of DOTMA:DOPE liposomes. Ten patients received VEGF P/L, three patients received beta-galactosidase P/L, and two patients received Ringer lactate. Gene transfer to coronary arteries was feasible and well tolerated. Except for a slight increase in serum C-reative protein in all study groups, no adverse effects or abnormalities in laboratory parameters were detected. No VEGF plasmid or recombinant VEGF protein was present in the systemic circulation after the gene transfer. In control angiography 6 months later, no differences were detected in the degree of coronary stenosis between treatment and control groups. We conclude that catheter-mediated intracoronary gene transfer performed after angioplasty is safe and well tolerated and potentially applicable for the prevention of restenosis and myocardial ischemia.

Improved lipid-mediated gene transfer in C6 glioma cells and primary glial cells using FuGene.

Gene therapy is a potent method to counteract neurodegeneration by introducing genetic information encoding neuroprotective factors. In this study cationic lipids were used to transfer DNA into C6 glioma cells and primary glial cells. When comparing the novel compound FuGene with other commercially-available lipids, it was found that FuGene markedly enhanced gene transfer of a beta-galactosidase reporter plasmid into C6 glioma cells. FuGene had several advantages compared to other lipids, such as a very low toxicity and the capability of transfection under serum conditions. When optimizing, a DNA-lipid ratio of 150 ng DNA/1 microl FuGene and a concentration of 3 microl FuGene/1 ml medium was found to be optimal. The incubation time peaked after 8 h and the expression time reached an optimum between 2 and 6 days. When cells were transfected on 3 consecutive days for 6 h each ('boosting'), the transfection efficiency was markedly enhanced in primary glial cells. When using endotoxin-free DNA the transfection efficiency could be enhanced up to 3 times. The optimal transfection efficiency in C6 glioma cells and in primary glial cells was found to be 16.3 +/- 0.3% and 5.1 +/- 0.37% of total cells, respectively. In conclusion this study shows that the novel compound FuGene has a very high potential to transfer DNA into cells of glial origin, and it might be an interesting canditate for ex vivo and in vivo gene therapeutic approaches.

Subcellular trafficking of the cytoplasmic expression system.

Cationic liposomes have provided many advantages over viral vector formulations; however, the problem of inefficient gene expression remains. This is due in part to the nuclear membrane, which limits DNA entry into the nucleus. Cytoplasmic expression systems using T7 RNA polymerase have been developed to express genes in the cytoplasm and avoid the need for nuclear import of DNA. Although these systems show improved transgene expression, little is known about how they function in transfected cells. Direct comparisons between a cytoplasmic and nuclear expression system were carried out with a 293 cell line stably expressing T7 RNA polymerase. A formulation for optimal reporter gene expression was developed and used in conjunction with a variety of subcellular trafficking inhibitors to study the process of DNA endocytosis. Transfected cells were also studied at different stages of the cell cycle to determine the dependence of each system on mitosis. These results showed that cytoplasmic and nuclear expression systems utilize similar endocytosis pathways to the point of endosomal release. Once DNA is released into the cytoplasm, the cytoplasmic expression system shows immediate expression that is proportional to the amount of DNA released. In contrast, DNA targeted for nuclear expression requires additional time for nuclear entry. The level of nuclear expression is also restricted by the limited amount of DNA that is imported into the nucleus. Finally, mitosis is required for effective nuclear expression but not for cytoplasmic expression. Therefore, the cytoplasmic expression system has considerable advantages over traditional nuclear expression systems and may be an effective method for transfecting nondividing cells. Efficient expression of genes delivered by nonviral vectors is hindered owing to poor nuclear transport of plasmid DNA. A potential solution to this problem would be to use a cytoplasmic expression system. Previous studies have shown that this method produces enhanced gene expression when compared with traditional nuclear expression systems; however, the actual mechanisms by which the cytoplasmic expression system works remains unknown. This article focuses on a direct comparison between cytoplasmic and nuclear expression in terms of optimal DNA delivery formulations, intracellular trafficking of DNA, and cell cycle dependence. These results indicate that the cytoplasmic expression system has two primary advantages over nuclear expression in that it does not rely on nuclear DNA transport or mitosis for efficient expression.

Synthesis of a single-tailed cationic lipid and investigation of its transfection.

Single-tailed cationic lipids were originally reported to have low transfection efficiency and high toxicity in plasmid delivery. We hypothesized that particular single-tailed cationic lipids may also function in plasmid transfection. To test this hypothesis, we synthesized a new cationic lipid-oleoyl ornithinate (OLON). To decrease cytotoxicity, we then introduced a potential biodegradable ester bond in the tail of lipid yielding 6-lauroxyhexyl ornithinate (LHON). The data demonstrated that the cytotoxicity of LHON was lower than that of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or OLON. To investigate the transfection activity of the new lipids and determine the cellular uptake of DNA/liposome complexes, we compared the transfection of liposomes produced from double-tailed 1',2'-dioleyl-sn-glycero-3'-succinyl-1, 6-hexanediol ornithine conjugate (DOGSHDO) with an ornithine headgroup, single-tailed OLON with an ornithine head group, double-tailed DOTAP with quaternary amine group, and single-tailed cetyltrimethylammonium bromide (CTAB) with a quaternary amine group. At the optimal ratios as defined in transfection experiments, OLON/DOPE had more than 10 times the transgene expression than other liposomes even though the DNA uptake was not necessarily greater. In the experiments comparing the release of DNA from DNA/liposome complexes by anionic substances, a greater fraction of DNA was released from DNA/OLON/DOPE complexes than that from DNA/DOTAP/DOPE complexes.

Efficient inducation of local and systemic antitumor immune response by liposome-mediated intratumoral co-transfer of interleukin-2 gene and interleukin-6 gene.

Interleukin 2 (IL-2) expressing plasmid and interleukin 6 (IL-6)-expressing plasmid were encapsulated in liposome and administrated intratumoraly into tumor-bearing mice 4 days after subcutaneous inoculation of B16F10 melanoma cells. The results showed that treatment of tumor-bearing mice with IL-2 gene or IL-6 gene transfer inhibited the growth of subcutaneous tumor and prolonged the survival of tumor-bearing mice significantly when compared with the treatment of PBS or control gene transfer mediated by liposome (P < 0.01). Combined transfer of IL-2 gene and IL-6 gene was found to elicit inhibitory effects on the growth of B16F10 tumor more significantly and prolonged the survival period of tumor-bearing mice more obviously. We investigated the local immunity in tumor microenvironment and found that IL-2 and IL-6 gene transfer could significantly increase the expression of lymphocyte function-associated antigen-1 on tumor infiltrating lymphocytes (TIL) and MHC-I molecule on tumor cells freshly isolated from the tumor mass. The NK and CTL activity of TIL increased markedly after the combined transfer of these two cytokine genes. We also observed the systemic antitumor immune response in the tumor-bearing mice and demonstrated that NK and CTL activity of splenocytes and the production of IL-2, tumor necrosis factor and interferon-gamma from splenocytes increased obviously in mice after the combined transfer of IL-2 and IL-6 gene. In conclusion, local and systemic antitumor immunity of the tumor-bearing host could be induced efficiently after the combined gene transfer. The enhanced specific and non-specific antitumor immunity might be responsible for the more potent antitumor effects of the combined gene therapy.

Enrichment of transiently transfected mesangial cells by cell sorting after cotransfection with GFP.

Early passage mesangial cells, like many other nonimmortalized cultured cells, can be difficult to transfect. We devised a simple method to improve the efficiency of transient protein expression under the transcriptional control of promoters in conventional plasmid vectors in rat mesangial cells. We used a vector encoding modified green fluorescent protein (GFP) and sterile fluorescence-activated cell sorting (FACS) to select a population consisting of >90% GFP-expressing cells from passaged nonimmortalized cultures transfected at much lower efficiency. Only 10% transfection efficiency was noted with a beta-galactosidase expression vector alone, but cotransfection with GFP followed by FACS and replating of GFP+ cells yielded greater than fivefold enrichment of cells with detectable beta-galactosidase activity. To demonstrate the expression of a properly oriented and processed membrane protein, we cotransfected GFP with a natriuretic peptide clearance receptor (NPR-C) expression vector. Plasmid-dependent cell surface NPR-C density was enhanced by 89% after FACS, though expression remained lower in selected mesangial cells than in the CHO cell line transfected with the same vector. We conclude that cotransfection of rat mesangial cells with GFP, followed by FACS, results in improvement in transient transfection efficiencies to levels that should suffice for many applications.

Successful transfection of lymphocytes by ternary lipoplexes.

Transgene expression in lymphoid cells may be useful for modulating immune responses in, and gene therapy of, cancer and AIDS. Although cationic liposome-DNA complexes (lipoplexes) present advantages over viral vectors, they have low transfection efficiency, unfavorable features for intravenous administration, and lack of target cell specificity. The use of a targeting ligand (transferrin), or an endosome-disrupting peptide, in ternary complexes with liposomes and a luciferase plasmid, significantly promoted transgene expression in several T- and B-lymphocytic cell lines. The highest levels of luciferase activity were obtained at a lipid/DNA (+/-) charge ratio of 1/1, where the ternary complexes were net negatively charged. The use of such negatively charged ternary complexes may alleviate some of the drawbacks of highly positively charged plain lipoplexes for gene delivery.

New biocompatible cationic amphiphiles derivative from glycine betaine: a novel family of efficient nonviral gene transfer agents.

With the aim of developing new efficient agents for transfecting of eukaryotic cells we have designed and synthesized a novel family of cationic lipid vectors derived from glycine betaine. In this study we present three novel molecules differing by the length of their aliphatic chains (R=12,R=14,R=16). The lyotropic properties of these cationic lipids have been determined, and their transfection efficiency on different cell lines evaluated, using a luminescent assay. Two of these compounds, GB14 and GB12 are efficient in vitro experiments. Cytoxicity evaluation of these new molecules showed promising results with a low cytotoxicity, especially when co-lipids were included in the formulation. These compounds represent a new family of gene transfer vectors which display good transfection efficiency and low toxicity, possibly due to the natural properties of glycine betaine. These compounds have great potential for the future development of in vivo gene transfer protocols.

Characterization and in vivo testing of a heterogeneous cationic lipid-DNA formulation.

PURPOSE: To identify characteristics of lipid-DNA complexes that correlate with in vivo expression data. METHODS: DOTIM:cholesterol liposomes (1:1 mole ratio) and DNA expressing chloramphenicol acetyl transferase (CAT) were complexed at a 4.2:1 mass ratio (cationic lipid:DNA). Complexes were fractionated by density gradient centrifugation. analyzed for particle size and zeta potential and quantitated using HPLC methods. The unfractionated complexes, "purified" fractions of the complexes, and purified complexes supplemented with liposomes were administered to mice by intravenous injection (i.v.) and intratracheal instillation (i.t.) and their ability to express gene product was assessed. RESULTS: Centrifugation separated two distinct populations within complexes one consisting of free liposomes and the other of lipid complexed with DNA. The vesicle diameter and zeta potential among separated fractions varied from 113 to 354 nm. and + 24 to + 34 mV respectively. Re-centrifugation of the 'purified' fractions containing the lipid-DNA population produced a single band. CAT expression in lung tissue 24 hr post-i.v. was observed with the unfractionated complex, but not the purified form. Some activity was 'restored' with the liposome-supplemented complexes. In contrast, the same series of complexes administered by i.t. resulted in no significant difference in lung expression (p=0.16 ANOVA). CONCLUSIONS: An uncomplexed liposome population exists within DOTIM:cholesterol-DNA complexes that influences the expression of complexes administered i.v. but not i.t..