Developing non-viral DNA delivery systems for cancer and infectious disease.

Efforts to deliver therapeutic genes are frequently rebuffed by the body's adaptive immune response against viral delivery vectors. Attempts to circumvent this problem using non-viral delivery systems have encountered problems with transient expression and inflammatory responses induced by reaction of the innate immune system reacting against bacterial DNA. However, within the past decade, these barriers to non-viral DNA delivery have been recognized as potential allies in the development of novel vaccines for cancer and infectious disease. This review summarizes preclinical and current clinical studies testing the formulation, delivery route and adjuvant options in the development of novel DNA-based vaccines.

Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

Aspartoacylase gene transfer to the mammalian central nervous system with therapeutic implications for Canavan disease.

With the ultimate goal of developing safe and effective in vivo gene therapy for the treatment of Canavan disease and other neurological disorders, we developed a non-viral lipid-entrapped, polycation-condensed delivery system (LPD) for central nervous system gene transfer, in conjunction with adeno-associated virus (AAV)-based plasmids containing recombinant aspartoacylase (ASPA). The gene delivery system was tested in healthy rodents and primates, before proceeding to preliminary studies in 2 children with Canavan disease. Toxicity and expression testing was first carried out in human 293 cells, which demonstrated effective transduction of cells and high levels of functional ASPA activity. We performed in vivo toxicity and expression testing of LPD/pAAVaspa and LPD/pAAVlac in rodents, which demonstrated widespread gene expression for more than 10 months after intraventricular delivery, and local expression in deep brain nuclei and white matter tracts for more than 6 months after intraparenchymal injections, with no significant adverse effects. We also performed intraventricular delivery of LPD/pAAVaspa to 2 cynomologous monkeys, with 2 additional monkeys receiving LPD and saline controls. None of the monkeys demonstrated significant adverse effects, and at 1 month the 2 LPD/pAAVaspa monkeys were positive for human ASPA transcript by reverse transcriptase polymerase chain reaction of brain tissue punches. Finally, we performed the first in vivo gene transfer study for a human neurodegenerative disease in 2 children with Canavan disease to assess the in vivo toxicity and efficacy of ASPA gene delivery. Our results suggest that LPD/pAAVaspa is well tolerated in human subjects and is associated with biochemical, radiological, and clinical changes.

The effects of jet nebulisation on cationic liposome-mediated gene transfer in vitro.

Nebulisation is currently the most acceptable and practical delivery system for repeated applications of gene therapy to the lower airways of cystic fibrosis (CF) patients. We have assessed whether this route of administration offers other benefits with regard to respiratory gene transfer. A standard jet nebuliser (Acorn System 22, Medicaid) was used to transfer the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE to three epithelial cell lines in vitro, two non-CF and one CF, using a novel collection system. In all three cell lines, nebulisation resulted in significantly (P < 0.05) improved transfection efficiency compared with instillation. At a constant DNA: liposome ratio of 1:5 (wt:wt), transfection efficiency was inversely related to increasing concentrations of DNA-liposomes before nebulisation. This effect was not related to the amount of DNA delivered and measurements of both zeta potential and mean aerodynamic particle size before and after nebulisation did not show concentration-related differences. The increased transfection efficiency did not relate either to the physical consequences of the nebulisation processes nor the effects of nebulisation on the complexes before instillation. Significantly increased transfection efficiency was seen following nebulisation with 95% O2/5% CO2 in comparison with 21% O2/78% N2 (air); this did not relate to changes in either the pH or temperature of the solution bathing the cells. The data confirm that nebulisation is appropriate for gene delivery to the lower airways in clinical practice and points to factors that may optimise gene transfer efficiency.

The effect of mucolytic agents on gene transfer across a CF sputum barrier in vitro.

Trials of gene transfer for cystic fibrosis (CF) are currently underway. However, direct application to the airways may be impeded by the presence of airway secretions. We have therefore assessed the effect of CF sputum on the expression of the reporter gene beta-galactosidase complexed with the cationic liposome DC-Chol/DOPE in a number of cell lines in vitro. Transfection was markedly inhibited in the presence of sputum; the effect was concentration dependent and was only partially ameliorated by removal of sputum with phosphate-buffered saline (PBS) washing before gene transfer. However, treatment of the sputum-covered cells with recombinant human DNase (rhDNase, 50 micrograms/ml) but not with N-acetylcysteine, Nacystelyn, lysine (all 20 mM) or recombinant alginase (0.5 U/ml) significantly (P < 0.005) improved gene transfer. Adenovirus-mediated gene transfer efficiency in the presence of sputum was similarly inhibited, and again, treatment with rhDNase before transfection significantly improved gene transfer (P < 0.005). Transfection of Cos 7 cells in the presence of exogenous genomic DNA alone demonstrated similar inhibition to that observed with sputum and was also ameliorated by pre-treatment of DNA-covered cells with rhDNase. In a separate series of experiments performed in the absence of added sputum or genomic DNA, increasing concentrations of rhDNase resulted in a concentration-related decline in transfection efficiency. However, even at the highest concentration (500 micrograms/ml of rhDNase), transfection efficiency remained more than 50% of control. Thus, pre-treatment of CF airways with rhDNase may be appropriate before liposome or adenovirus-mediated gene therapy.

DC-Chol liposome-mediated gene transfer in rat spinal cord.

We examined the potential of non-viral vector-mediated gene transfection in the rat spinal cord. Reporter gene (beta-gal) or brain-derived neurotrophic factor (BDNF) cDNA containing a pCMV promoter complexed with DC-Chol liposomes was injected into the intact rat spinal cord gray matter. RT-PCR confirmed the increased expression of BDNF mRNA in the injection areas. X-gal staining demonstrated the localized expression of beta-gal reporter genes. No overt tissue damage caused by DC-Chol liposome/DNA complex injections was detected. These results suggest that cationic liposome-mediated delivery can be a practical method for gene transfer in spinal cord.

CFTR gene transfer reduces the binding of Pseudomonas aeruginosa to cystic fibrosis respiratory epithelium.

Much of the morbidity and mortality seen in cystic fibrosis (CF) is related to chronic infection of the respiratory tract with Pseudomonas aeruginosa. Some studies have attributed the strong relationship between CF and Pseudomonas colonization to the presence of increased numbers of specific cell-surface receptors, although other work suggests that this relates to the presence of mucus. Several groups are now assessing the use of gene transfer as a novel form of treatment for CF. We have examined whether P. aeruginosa binding to freshly obtained CF respiratory epithelial cells is increased, and have studied the effects of transfer of the CF transmembrane conductance regulator (CFTR) gene on this attachment. Binding of P. aeruginosa to noncultured nasal epithelial cells from both CF patients (n = 31) and healthy controls (n = 15) was studied with scanning electron microscopy. Binding was also assessed for CF cells following transfection with CFTR/liposome complexes. Epifluorescence microscopy was used to assess the effects of gene transfer on chloride fluxes. Adherence of P. aeruginosa directly to the cell surface of CF airway epithelium was significantly (P < 0.001) increased over that in non-CF controls. Liposome-mediated CFTR gene transfer resulted in a significant (P < 0.01) reduction in the numbers of bacteria bound to ciliated epithelial cells. Fluorescence microscopy confirmed correction of the basic chloride defect. Thus, in CF, the absence of normal CFTR results in increased binding of P. aeruginosa to respiratory epithelial cells. This abnormality can be corrected in vitro by restoration of CFTR function. This has important implications both for the pathogenesis of CF and for the future application and assessment of gene therapy for this disease.

A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis.

Cystic fibrosis (CF) is a common, serious, inherited disease. The major cause of mortality in CF is lung disease, due to the failure of airway epithelial cells to express a functional product of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A potential treatment for CF lung disease is the expression of CFTR in the airways following gene transfer. We have undertaken a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken 7 days after dosing were normal. No significant changes in clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients (15 days after dosing in one patient). Fluorescence microscopy demonstrated CFTR function ex vivo. In cells from nasal brushings. In total, evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients. These results provide proof of concept for liposome-mediated CF gene transfer.

Chloride secretion in the trachea of null cystic fibrosis mice: the effects of transfection with pTrial10-CFTR2.

1. An improved novel plasmid backbone, pTrial10, has been developed. We have used this vector to deliver the cDNA for the cystic fibrosis transmembrane conductance regulator (CFTR) to cells, both in vitro and in vivo, complexed with cationic liposomes. 2. Human 293 kidney epithelial cells (HEK 293) showed expression of an immunoprecipitable 165 kDa protein corresponding to CFTR when transfected in vitro with pTrial10-CFTR2, but not when the vector pTrial10 was used. 3. HEK 293 cells transfected with pTrial10-CFTR2, but not pTrial10, demonstrated a cAMP-dependent anion conductance, measured by fluorescence microscopy using a halide-sensitive probe, SPQ. 4. The CFTR-dependent, cAMP-sensitive chloride secretory response in murine tracheal epithelium could be measured if the calcium-dependent chloride secretory process was first maximally stimulated with a mixture of the Ca(2+)-ATPase inhibitor, TBHQ, and the calcium ionophore, A23187. With these conditions wild-type and CF-null (transgenic animals in which the cystic fibrosis (CF) gene has been disrupted so that no CFTR is produced) murine tracheas could be distinguished. The difference between the current elicited by forskolin in wild-type and CF tracheas was highly significantly different (P < 0.001), giving a CFTR-dependent current of 11.2 microA cm-2. 5. Transfection of the airways with pTrial10-CFTR2, but not pTrial10, significantly (P < 0.01) increased the CFTR-dependent chloride secretory current in CF tracheas. The degree of correction was greater when intra-tracheal installation rather than nasal insufflation was used to deliver the plasmids.

The tumor suppression activity of E1A in HER-2/neu-overexpressing breast cancer.

The HER-2/neu proto-oncogene is frequently amplified or overexpressed in human breast and ovarian cancers, and is significantly correlated with shorter survival. We have previously reported that the adenovirus type 5 early region 1A (E1A) gene product can repress HER-2/neu overexpression by repressing HER-2/neu promoter activity, and suppress the tumorigenic potential of HER-2/neu-overexpressing ovarian cancer cells. To examine E1A tumor suppressor function in breast cancer, we transduced E1A in vitro by adenovirus into both HER-2/neu-overexpressing and low expressing human breast cancer cell lines. In HER-2/neu-overexpressing cells, E1A greatly inhibited tumor cell growth in vitro. However, in HER-2/neu low expressing cancer cell lines, E1A had no significant effect on cell growth in culture medium. To test the therapeutic efficacy of E1A, we used both adenovirus-mediated and cationic liposome-mediated E1A gene delivery systems in an orthotopic breast cancer animal model. An advanced breast cancer model was established by inoculation of HER-2/neu-overexpressing human breast cancer cells in mammary fat pad and treated by local injections of either replication-deficient adenovirus expressing E1A, Ad.E1A(+) or a liposome-E1A DNA complex. As controls, mice bearing tumors were also treated with Ad.E1A(-) which is virtually the same adenovirus as Ad.E1A(+) except that E1A is deleted, a liposome-E1A frame-shift mutant DNA complex, or just PBS. In mice bearing a HER-2/neu-overexpressing breast cancer cell line, E1A delivered either by adenovirus or liposome significantly inhibited tumor growth and prolonged mouse survival compared with the controls. In fact, 60-80% of E1A-treated mice lived longer than 2 years versus only 0-20% of control mice (P<0.05). Western blot analysis showed that E1A protein was expressed in tumor tissue and immunohistochemical analysis showed that HER-2/neu p185 protein expression was suppressed. Taken together, our results indicated that both adenovirus and cationic liposome delivery systems were effective in transfering E1A gene for tumor suppression in a HER-2/neu-overexpressing breast cancer model.

Protamine sulfate enhances lipid-mediated gene transfer.

A polycationic peptide, protamine sulfate, USP, has been shown to be able to condense plasmid DNA efficiently for delivery into several different types of cells in vitro by several different types of cationic liposomes. The monovalent cationic liposomal formulations (DC-Chol and lipofectin) exhibited increased transfection activities comparable to that seen with the multivalent cationic liposome formulation, lipofectamine. This suggests that lipofectamine's superior in vitro activity arises from its ability to condense DNA efficiently and that protamine's primary role is that of a condensation agent, although it also possesses several amino acid sequences resembling that of a nuclear localization signal. While the use of polycations to condense DNA has been previously reported, the of protamine sulfate, USP as a condensation agent was found to be superior to poly-L-lysine as well as to various other types of protamine. These differences among various salt forms of protamine appear to be attributable to structural differences between the protamines and not due to differences in the net charge of the molecule. The appearance of lysine residues within the protamine molecule correlate with a reduction in binding affinity to plasmid DNA as well as an observed loss in transfection enhancing activity. This finding sheds light on the structural requirements of condensation agents for use in gene transfer protocols. Furthermore, protamine sulfate, USP is an FDA-approved compound with a documented safety profile and could be readily used as an adjuvant to a human gene therapy protocol.

The effect of cationic liposome pretreatment and centrifugation on retrovirus-mediated gene transfer.

Pretreatment of retroviral supernatants with the cationic liposomes DOTMA-DOPE (Lipofectin), DC-Chol-DOPE and DOSPA-DOPE (Lipofectamine) was found to enhance static transductions of TF-1 target cells. The relative effectiveness at increasing transduction efficiencies (TE) was: DOSPA > DC-Chol > DOTMA, resulting in average increases over nontreated controls of 11.9-, 6.2- and 1.2-fold, respectively. This pretreatment was found to be synergistic when combined with centrifugation, having the same order of effectiveness, and resulting in 57-, 35- and 27-fold increases over nontreated controls. For Lipofectamine and DC-Chol-DOPE liposomes, the combined approach yielded 2.2- and 1.3-fold increases over untreated centrifuged samples. Individual colonies picked from colony-forming unit granulocyte-macrophage assays of infected CD34+ cells were screened for the presence of the transgene by polymerase chain reaction (PCR). Colonies from cells infected using centrifugation were positive 27% of the time, while the combined approach had positive colonies 31 and 50% of the time for DC-Chol and Lipofectamine, respectively. The addition of protamine sulfate to the liposome-supernatant mixture during pretreatment was found to be inhibitory. With increasing centrifugal force, the TE of cells infected with Lipofectamine pretreated and untreated supernatants increased proportionally. However, the TE of the cells infected with the pretreated supernatants was significantly higher than the TE of the cells infected with untreated supernatants at all points examined. The increase in TE associated with liposomal pretreatment of retroviral supernatants was not shown to be attributed to a nonreceptor-mediated pathway for viral entry into the cell.

Cationic liposome-mediated gene transfer to tumor cells in vitro and in vivo.

Development of safe and effective technology for delivering functional DNA into cells in an intact organism is crucial to broad applications of gene therapy to human disease. Both viral and nonviral vectors have been developed. Of the technologies currently being studied, liposomal delivery system is particularly attractive. Cationic liposome-mediated gene transfection (lipofection), a relatively new technique pioneered by Felgner and coworkers (1), was highly efficient for transfecting cells in culture. The liposomes were composed of an equimolar mixture of a synthetic cationic lipid N-[1-(2,3,-dioleyloxy)propyl]-N,N,N,-trimethylammonium chloride (DOTMA) and a helper lipid dioleoyl-phosphatidylethanolamine (DOPE) Fig. 1). The DOTMA/DOPE mixture (Lipofectin) forms complexes with DNA by charge interaction upon mixing at room temperature. Other catronic lipids are DOTAP, LipofectAMINE, Lipofectam, and DC-chol. The DOTAP is a diester analog of DOTMA and commercially available. LipofectAMINE and Lipofectam are polycationic lipids with a spermine head group that show increased frequency and activity of eukaryotic cell transfection (2,3). 3ß-[N-(N',N'-dimethyaminoaminoethane) carbamoyl] cholesterol (DC-chol) (Fig. 1), a cationic cholesterol derivative, was introduced by Gao and Huang (4) and is routinely used in our laboratory. The DC-chol is now commercially available but can be easily synthesized with a single-step reaction from N,N-dimethylethylenediamine and cholesterol chloroformate (4), and improves the efficiency of transfection with minimal toxicity.Liposomes prepared with DC-chol and DOPE (3∶2 molar ratio) are stable at 4°C for at least 1 yr (unpublished data).

In vivo cytokine gene therapy of human tumor xenografts in SCID mice by liposome-mediated DNA delivery.

The human interleukin-2 (IL-2) gene was successfully delivered into established human tumor xenografts in SCID (severe combined immunodeficient) mice by cationic liposome-mediated DNA delivery. A bicistronic mammalian expression vector containing a reporter gene (beta-galactosidase) and human IL-2 cDNA was complexed with either lipofectin or DC-cholesterol liposomes and transferred to tumor xenografts by direct intratumoral injection. Transfection of tumors was confirmed by staining of tumor sections for beta-galactosidase activity and by reverse transcription-polymerase chain reaction (RT-PCR) for the presence of IL-2 mRNA. Growth suppression of tumor xenografts was observed in animals injected with plasmid-liposome complexes but not in animals that received liposomes or naked plasmid only. Complete tumor regression, mediated by the mouse natural killer cells, was observed in 50-80% of the mice treated with the plasmid containing the IL-2 cDNA. The effectiveness of the treatment was dependent on the transfection efficiency and the tumor size at the start of therapy. An initial IL-2 independent suppression of tumor growth was also observed with a plasmid carrying only the beta-galactosidase gene but this effect was temporary and did not lead to tumor regression. These results establish that human tumor xenografts growing in SCID mice can be transfected in vivo by liposome mediated gene delivery and that both IL-2-dependent and IL-2-independent factors may contribute to the tumor suppression observed here.

Mutant SV40 large T antigen as a therapeutic agent for HER-2/neu-overexpressing ovarian cancer.

The HER-2/neu gene is frequently amplified and/or its protein product, p185, is overexpressed in a number of human cancers. Overexpression of p185 correlates with poor prognosis and low survival rates in ovarian cancer patients. We previously found that the K1 mutant of SV40 large T antigen inhibits rat neu promoter and suppresses mutation-activated rat neu transformation in mouse fibroblasts. We show here that K1 also inhibits human HER-2/neu promoter in human ovarian cancer cells. To investigate whether K1 can suppress HER-2/neu transformation and thus is a potential therapeutic agent, we used an orthotopic ovarian cancer model in which mice were injected intraperitoneally with HER-2/neu-overexpressing human ovarian cancer cells to induce tumor development. The tumor-bearing mice were then treated with K1-liposome complex weekly. We found that liposome-mediated K1 gene transfer decreased the p185 protein level by K1 expression in these cancer cells and significantly prolonged mice survival; about 40% of these treated mice were alive for more than 1 year without any tumor development. On the other hand, the animals from control groups that did not receive this gene therapy all developed tumors and died within 7 months. The results indicate that liposome-mediated K1 gene transfer is able to suppress tumor development from HER-2/neu-overexpressing ovarian cancer cells in mice.

Quantitative fluorescence measurements of chloride secretion in native airway epithelium from CF and non-CF subjects.

Functional assessment of the efficacy of CFTR gene transfer protocols in humans has previously involved measurement of in vivo potential difference. We have studied whether freshly obtained airway epithelial cells may provide suitable tissue for studies of in vivo gene transfer using fluorescent digital imaging microscopy. Nasal epithelial cells from non-cystic fibrosis subjects (n = 6) and from cystic fibrosis (CF) patients (delta F508: delta F508, n = 5) were obtained by brushing and loaded with 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ). Addition of the cAMP-agonists forskolin (20 microM) and 3-isobutyl-1-methylxanthine (IBMX, 100 microM) produced an increased efflux of iodide from the cells which was significantly (P < 0.05) greater in non-CF than in CF cells. Efflux following addition of the calcium ionophore, ionomycin (100 microM) was similar in both non-CF and CF cells. Liposome-mediated transfection of CF nasal epithelial cells in vitro with CFTR-cDNA restored the cAMP-stimulated efflux to non-CF values. Bronchial epithelial cells from non-CF subjects showed responses to forskolin and ionomycin that were not different to those in non-CF nasal epithelia. These data demonstrate that the assay provides a useful method for assessing correction of abnormal ion transport in non-cultured CF epithelium and is likely to provide a further assay for assessment of in vivo gene transfer efficiency in protocols of gene therapy for CF.

In vitro liposome-mediated DNA transfection of epithelial cell lines using the cationic liposome DC-Chol/DOPE.

Clinical trials using cationic liposome-mediated DNA transfer have now been initiated for several disorders including cystic fibrosis. Previous studies have shown that the level of gene expression achieved may be dependent on the formulation of the DNA-liposome complex and the cell type transfected. We have investigated, in vitro, the effect of parameters such as DNA:liposome ratio, dose and concentration on the level of transgene expression in epithelial cell lines using the cationic liposome DC-Chol/1,2-dioleoyl phosphatidylethanolamine (DOPE). A narrow range of conditions was found to produce maximal level of transgene expression within a particular cell line, as detected using the reporter molecule beta-galactosidase (beta-gal). beta-Gal expression was significantly enhanced by formulation of the DNA-DC-Chol/DOPE complexes in physiological solution at pH 9.0. Under standard in vitro transfection conditions, increased incubation time of the DNA-liposome complexes with cells resulted in increased transgene expression. In contrast, at relatively high DNA and liposome dose and concentrations, beta-gal activity was maximal after only 1 h of incubation, with a subsequent decrease in expression with time. The maximum level of expression that could be produced using fully optimised transfection conditions, however, was still highly dependent on each cell type analysed. Correlation of these findings with similar studies in vivo are now critical to determine the optimal formulation of DNA-liposome complexes for clinical application.

Liposome-mediated in vivo E1A gene transfer suppressed dissemination of ovarian cancer cells that overexpress HER-2/neu.

The HER-2/neu proto-oncogene is frequently amplified or overexpressed in many different types of human cancers, a phenomenon that has been shown to correlate with shorter survival time and lower survival rate in ovarian cancer patients. We previously reported that increased HER-2/neu expression led to more severe malignancy and increased metastatic potential in animal models and that the adenovirus 5 E1A gene repressed HER-2/neu gene expression at transcriptional level and was able to suppress tumor growth when stably transfected into human ovarian cancer SKOV-3 cells which overexpress HER-2/neu. To investigate whether the E1A gene may be used as a therapeutic agent for HER-2/neu-overexpressing human cancers in living hosts, we first developed tumor-bearing mice by injecting SKOV-3 cells that overexpress HER-2/neu intraperitonealy into female nu/nu mice. Five days later, we used cationic liposomes to directly deliver the E1A gene into adenocarcinomas that developed in the peritoneal cavity and on the mesentery of the mice that received the SKOV-3 cell injection. We found that liposome-mediated E1A gene transfer significantly inhibited growth and dissemination of ovarian cancer cells that overexpress HER-2/neu in the treated mice; about 70% of these mice survived at least 365 days, whereas all the control mice that did not receive the gene therapy developed severe tumor symptoms and died within 160 days. The results suggest that liposome-mediated E1A gene transfer may serve as an effective therapy for human ovarian cancers that overexpress HER-2/neu by directly targeting the HER-2/neu oncogene.

HER-2/neu-targeting gene therapy--a review.

The HER-2/neu (also named c-erbB-2) oncogene is known to be overexpressed in many human cancers, including breast, ovarian, lung, gastric and oral cancers. In animal models, HER-2/neu overexpression was shown to enhance malignancy and metastasis phenotypes. Repression of HER-2/neu overexpression suppresses the malignant phenotypes of HER-2/neu-overexpressing cancer cells, suggesting that HER-2/neu may serve as an excellent target for developing anti-cancer agents. We have previously shown that the adenovirus-5 (Ad5) E1a gene products and the SV40 large T antigen (large T) inhibit transcription of the HER-2/neu promoter and accordingly suppresses transformation induced by HER-2/neu. In this review, we summarize our recent findings on using cationic liposomes or an Ad vector to deliver E1a or large T into tumor-bearing mice. Our results indicate that both cationic liposomes or an Ad vector can efficiently deliver E1a or large T into tumor cells in mice, and this results in suppression of tumor growth and longer survival of the mice.

New structures in complex formation between DNA and cationic liposomes visualized by freeze-fracture electron microscopy.

Structures formed during interaction of cationic liposomes and plasmid DNA were studied by freeze-fracture electron microscopy and their morphology was found to be dependent on incubation time and DNA concentration. These structures were formed with liposomes composed of DC-Chol and DOPE after 30 min incubation at DNA:lipid concentrations encompassing maximal transfection activity. They resembled liposome complexes (meatballs) and additionally bilayer-covered DNA tubules (spaghetti), whereby the DNA-tubules were found to be connected to the liposome complexes as well as occurring free in the suspension. At later times and higher DNA-to-liposome ratios the complexes grow larger while their membranes become discontinuous, allowing the self-encapsulation of the DNA. The relative transfection potency of the various morphologically distinct structures is discussed.