Liposome Method Research Articles

Phosphorothioate oligonucleotides induction into experimental choroidal neovascularization by HVJ-liposome system

Purpose. The purpose of this study was to determine whether the inactivated hemagglutinating virus of Japan (HVJ)-liposome method can induce phosphorothioate oligonucleotides effectively into an experimentally-induced choroidal neovascularization of rats. We also examined whether anti-sense phosphorothioate oligonucleotides against VEGF could be induced into choroidal neovascularization as a therapeutic agent by the HVJ-liposome method. Methods. The experiments were conducted on a rat model of choroidal neovascularization. FITC-labeled phosphorothioate oligonucleotides were coencapsulated in liposomes. The liposomes were coated with the envelope of inactivated HVJ and injected into the vitreous cavity following photocoagulation of pigmented rat eyes. The eyes were removed following injection, fixed, frozen and cut into thin sections. Induction of oligonucleotides was observed under a laser confocal scanning microscope for fluorescence and the development of choroidal neovascularization was evaluated histopathologically. Results. Phosphorothioate oligonucleotides were effectively induced into ganglion cells and into the cells of the choroidal neovascularization induced by laser photocoagulation. Highly effective induction of oligos was observed 3 to 14 days after intravitreal injection of HVJ-liposomes after which the level decreased. Antisense oligonucleotides against VEGF were induced specifically into cells in the choroidal neovascularization, however neovascularization was still observed. Conclusions. Phosphorothioate oligonucleotides can be effectively induced into ganglion cells, and specifically into cells in choroidal neovascularization. Although antisense oligonucleotides against VEGF failed to prevent choroidal neovascularization, the HVJ-liposome method provided a highly effective means of inducing antisense oligos for in vivo antisense therapy.

Cytokines and glomerulosclerosis.

Glomerulonephritis (GN), diabetic nephropathy, and uli. The expression efficiency into the glomeruli was hypertension are the major causes of chronic renal approximately 35%. Over-expression of TGF-b caused failure that finally require renal replacement therapy. extracellular matrix accumulation with a mild increase Different aetiological factors lead to renal injury, howin glomerular cell numbers 5 to 7 days after transfection ever, these diseases share the common histological [6 ]. Over-expression of TGF-b also altered the phenopathway of a pathological accumulation of extracellutype of mesangial cells since type I and type III lar matrix in the glomeruli. These features are clinically collagens were expressed in the mesangial area. referred to as glomerulosclerosis. Pathophysiological TGF-b actions have now been TGF-b plays an important role in regulating tissue demonstrated. The discovery that blocking TGF-b repair and remodelling following injury [1,2]. One of with either neutralizing antibodies [7] or the proteothe most important biological actions of TGF-b is the glycan decorin [8] stopped extracellular matrix accuregulation of extracellular matrix accumulation. TGFmulation offers the potential of developing agents that b stimulates the synthesis of individual matrix componmodulate or antagonize TGF-b as anti-fibrotic drugs. ents including proteoglycans, collagens, and glycoproWe have also reported that inhibition of TGF-b teins [3]. TGF-b also inhibits matrix degradation by gene expression by antisense oligonucleotides could decreasing the synthesis of proteases and increasing suppress the development of experimental GN [9]. the synthesis of protease inhibitors [4]. Finally, TGFFurthermore, continuous delivery of decorin, which b modulates the expression of integrin receptors and was accomplished by in vivo gene transfer into the alters their relative proportions on the cell surface in skeletal muscle, also inhibited the extracellular matrix a manner that could facilitate adhesion to the matrix expansion in experimental GN [10]. This evidence [5]. All of these actions leads to increased deposition strongly suggests the hypothesis that the inhibition of and accumulation of extracellular matrix surrounding over-expressed TGF-b can ameliorate the progression the cells in a tissue. All of these events have largely of renal diseases. been demonstrated in vitro in cultured cells. In an Recently it has been shown that TGF-b signals are experimental model of GN, TGF-b has also been transduced by contacting two transmembrane serine/ shown to be responsible for the accumulation of the threonine kinases known as type I and type II receptors pathological matrix in the glomeruli following simultaneously [11]. The type II receptor recognizes immunological injury. That is, all three actions of the active TGF-b ligand, whereas the type I receptor TGF-b actions on the extracellular matrix; (1) does not. Thus, TGF-b binds directly to the type II increased synthesis; (2) decreased degradation; and receptor, which is a constitutively active kinase. The (3) modulation of integrin receptors, have now been TGF-b binding type II receptor is then recognized by demonstrated to be involved in matrix deposition the type I receptor which is recruited into the complex in GN. and becomes phosphorylated by the type II receptor. It was not known, however, whether or not the overBoth receptors are required for TGF-b action in mamproduction of a single growth factor in the glomerulus malian cells [12] because mutations in either receptor resulted in glomerulosclerosis. Therefore, the haemagtype disrupts signalling in each case. Based on the glutinating virus of Japan (HVJ) liposome method differences between their ligand-binding properties, the was applied to create a new animal model expressing type II receptor acts upstream of the type I receptor, the single growth factor selectively in the glomerulus. and so these components may be thought of as primary The expression vectors carrying cDNA for TGF-b receptor and transducer, respectively [12]. These results were generated and introduced into the kidney by the motivated us to produce an inactive type II receptor HVJ-liposome method. Consequently, the selective construct to impede the activities of TGF-b. However, overexpression of TGF-b was observed in the glomerthe soluble type II receptor was reported to have about a 10-fold less binding affinity for TGF-b than does the Correspondence and offprint requests to: Enyu Imai, First Department cell-surface type II receptor [13]. This may partly of Medicine, Osaka University School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. depend on the fact that the soluble type II receptor is

Gene transfer to vein graft wall by HVJ–liposome method: time course and localization of gene expression

Background. A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ–liposome method. Methods. The HVJ–liposome complex containing either β-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate–labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries. Results. In experiment 1, all grafts incubated with β-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate–labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome–treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ–liposome treatment. Conclusions. These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ–liposome method and suggest the possibility of its clinical application to prevent vein graft failure.

Neutralization of aspartate residues in the murine inwardly rectifying K+ channel IRK1 affects the substate behaviour in Mg2+ block.

1. To investigate the molecular basis of the sublevels induced in the outward current during block by intracellular Mg2+, single-channel currents through inwardly rectifying K+ (IRK1) channels were studied. 2. cDNA encoding a functional murine IRK1 channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 3. Intracellular Mg2+ at micromolar concentrations induced sublevels in the outward current at one-third and two-thirds of the unitary amplitude seen in wild-type channels. Replacing Asp 172 with Asn (D172N) and Gln (D172Q) abolished these sublevels, i.e. the channel showed only the fully open and fully blocked states. 4. Both mutations reduced the Mg2+ sensitivity of the channel at 2 microM Mg2+. However, the Mg2+ sensitivity did not differ significantly at higher concentrations (10 microM) and voltages (+70 mV). 5. Channels expressed from D172E showed the sublevels, indicating that a negative charge is indispensable to the substate behaviour. 6. Channels from tandem tetramers of IRK1 with one and two D172N mutant subunits mainly showed sublevels with two-thirds amplitude, while those from tetramers with three D172N mutant subunits showed no sublevels. 7. These findings suggest that differences in Mg2+ binding patterns lead to different conductive states in a single-barrelled channel.

Transcription factor decoy for nuclear factor-kappaB inhibits tumor necrosis factor-alpha-induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.

Several cytokines and adhesion molecules released from endothelium play an important role in inflammation, immune responses, and probably atherogenesis. To determine whether the transcription factor nuclear factor-kappaB mediated expression of these genes involved in the inflammatory response of endothelial cells to tumor necrosis factor-alpha, by using transcription factor decoy oligodeoxynucleotides. We first transfected fluorescein isothiocyanate (FITC)-labeled double-stranded oligodeoxynucleotides into endothelial cells by a cationic liposome-mediated method of gene transfer. We then confirmed that the decoy oligodeoxynucleotides could block binding of nuclear factor-kappaB to its specific cis element effectively. In addition, we transfected the reporter gene chloramphenicol acetyltransferase driven by three repeated nuclear factor-kappaB binding sequences in the promoter and enhancer region. FITC-labeled oligodeoxynucleotides were detected in the nuclei of approximately 70% of the total cells. Tumor necrosis factor--stimulated expression of chloramphenicol acetyltransferase was partially inhibited by transfection of nuclear factor-kappaB decoy oligodeoxynucleotides, but not by transfection of scrambled oligodeoxynucleotides. Also nuclear factor-kappaB decoy oligodeoxynucleotides but not scrambled oligodeoxynucleotides inhibited tumor necrosis factor-induced expression of interleukin-6 and intracellular adhesion molecule-1 both at the messenger RNA and at protein level (assessed by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay). Our results demonstrate that nuclear factor-kappaB decoy oligodeoxynucleotides transfected by cationic liposome method inhibited tumor necrosis factor--induced expression of interleukin-6 and intracellular adhesion molecule-1 in endothelial cells.

Establishment of a Mouse Sertoli Cell Line Producing Rat Androgen-binding Protein (ABP)

The ultimate goal of this study was to compare the fate of rat testicular germ cells cocultured with mouse Sertoli cells that either do or do not produce rat androgen-binding protein (ABP). As a first step, we stably transfected a rat ABP expression construct into an immortalized mouse Sertoli cell line (TM4), which does not produce ABP when growing on plastic without hormones. The transfection of the pRc/CMV- rat ABP cDNA expression vector containing a neomycin resistance gene was made by either the liposome method (Dotap) or by polyethyleneimine transfection (PEI) into TM4 cell cultures. Neomycin-resistant clones were selected by adding Geneticin to the culture medium for 3 weeks. Analysis of over 25 clones revealed the presence of recombinant rat ABP when cell extracts and culture media were probed with a rabbit polyclonal antibody raised against rat testicular ABP, indicating the translation and secretion of a protein similar to rat testicular ABP. Transfected TM4 cells maintain the secretion of rat ABP for more than 40 days, with immunopositive rat ABP localized within cytoplasmic granules in the Golgi region and along cytoplasmic processes in TM4 transfected with either vector. Electron microscopic study revealed a higher development of cytoplasmic organelles involved in protein secretion.

Heterogeneous induction of apoptosis in colon cancer cells by wild-type p53 gene transfection.

To examine the effects of wild-type (wt)-p53 gene transfer on cancer cell growth and apoptosis induction, we transduced human wt-p53 cDNA into three colon cancer cell lines either with or without a mutation of the p53 gene using the HVJ-cationic liposome method. Wt-p53 gene transfer, thus, induced an apparent growth arrest in all cell lines, but its enhancement of the apoptotic rate varied (from about 4 to 70 times). The simultaneous doxorubicin treatment was able to enhance growth arrest and the apoptosis induction rate. These findings suggest that wt-p53 gene transfer using HVJ-cationic liposomes seems to be a potentially effective therapeutic strategy, however wt-p53 gene transfer still appears to be more effective in combination with other cytotoxic treatments.

Prevention of Hypoxic Liver Cell Necrosis byin VivoHumanbcl-2Gene Transfection

Prevention of hypoxic cell death is a key to successful liver transplantation. We developed a new method for preventing liver hypoxic cell death by introducing an anti-cell death gene directly into rat livers. When the humanbcl-2gene (hbcl-2) was directly transfected into rat livers together with non-histone chromosomal protein high mobility group 1 (HMG1) by the hemagglutinating virus of Japan (Sendai virus; HVJ) -liposome method, human Bcl-2 protein (hBcl-2) was efficiently expressed. Electron microscopy and fluorescence microscopy revealed that hepatocytes expressing exogenous hBcl-2 were almost completely protected the hypoxic cell necrosis. The expression of the hBcl-2 also inhibited activation of caspase-3 (-like) proteases and liver dysfunction. Thus, we conclude that transfection of thehbcl-2gene through HVJ-liposome method is useful to prevent liver cell necrosis induced by hypoxia. This finding could lead to new strategies to avoid the hypoxic cell death, the major problem in liver transplantation.

Antisense Cdk2 Kinase Oligodeoxynucleotide Inhibits ICAM-1 Expression in Murine Cardiac Allograft Arteriopathy

CARDIAC transplantation as a treatment for end-stage cardiac disease has been established. However, the problem of accelerated graft coronary disease in long-term survivors remains. Adhesion molecules play critical roles in graft arteriopathy; that is, intercellular adhesion molecule 1 (ICAM-1) is known to be one of the most important molecules in atherogenesis. Inhibition of arterial neointimal formation by antisense phosphorothioate oligodeoxynucleotide (ODN) directed to cell-cycle-regulatory genes after balloon angioplasty in the rat carotid injury model has been reported. The enzyme, cycline-dependent kinase (cdk) 2 kinase, plays a critical role in transition through the G1/S phase. The hemagglutination virus of Japan (HVJ) (liposome method) has been shown to increase the efficiency of cellular uptake of ODN. To test the hypothesis that inhibition of cdk2 kinase expression can inhibit ICAM-1 expression in graft arteries, we studied the effect of antisense cdk2 kinase ODN by intraluminal delivery using HVJ-liposome–mediated transfer. In this study, we demonstrate clearly that antisense cdk2 ODN treatment results in near-complete inhibition of ICAM-1 expression in neointimal formation in murine cardiac allografts.

A novel method for DEAE-dextran mediated transfection of adherent primary cultured human macrophages

Primary cultured human macrophages are difficult to transfect. We have developed a DEAE-dextran DNA transfection method that mediates the reproducible transfection of primary cultured adherent human macrophages. Three factors essential for successful transfection were identified: DEAE-dextran concentration, the quantity of DNA per transfection and the incubation time of the macrophages with the transfection medium. Maximum levels of luciferase expression were attained within 24 h and maintained for at least 56 h after transfection. While serum in the transfection medium attenuated transfection, the treatment of the macrophages with chloroquine, DMSO, or glycerol did not enhance transfection within this system. A CMV enhancer/promoter mediated substantially greater luciferase expression in the macrophages than either HIV or RSV LTRs. DEAE-dextran facilitated superior transfection compared to either cationic liposome and calcium phosphate methods, and was more practical compared to electroporation for multiple transfections. This transfection protocol provides a simple, inexpensive, reproducible method for the evaluation of gene expression in primary cultured adherent human macrophages.

Transformation of rat glioma cells with the M-CSF gene inhibits tumorigenesis in vivo.

Macrophage colony-stimulating factor (M-CSF) is a potent stimulator of the effector cells such as monocytes and macrophages. To evaluate the effect of M-CSF on malignant gliomas, we transfected the rat gliosarcoma cell line (9L) with human M-CSF expression vector (pCEF-MCSF) by a liposome method. Transfectants were selected using G418-containing medium. As a control, 9L cells transfected with pRc/CMV and selected by G418 were used. The effects of M-CSF gene transfection on tumor cell proliferation in vitro and in vivo were examined. All growth rate did not change in vitro. While the control 9L cells formed progressively enlarging masses, 9L cells transfected with the M-CSF gene did not develop into tumors after the injection into rats. On the other hand, in rats receiving anti-asialo GM1 antibody, 9L cells transfected with M-CSF gene developed into tumors, though at a slower rate than control 9L cells. Histologic examination after transplantation of 9L cells transfected with M-CSF gene disclosed intense infiltration of macrophages in the tumor. Thus M-CSF gene transfection into glioma cells stimulates an antitumor effect.

Molecular Delivery System for Antisense Oligonucleotides: Enhanced Effectiveness of Antisense Oligonucleotides by HVJ-liposome Mediated Transfer.

BACKGROUND: The effectiveness of antisense oligodeoxynucleotides for in vitro and in vivo studies is limited by a low efficiency of cellular uptake and instability due to degradation by nucleases. To overcome some of these problems, we recently developed a transfer method that utilizes inactivated Sendai virus (hemagglutinating virus of Japan [HVJ]) complexed with liposomes to deliver antisense oligodeoxynucleotides. In this study, we compared the effectiveness of the HVJ-liposome method versus a cationic liposome method versus passive uptake to deliver antisense oligodeoxynucleotides against basic fibroblast growth factor on angiotensin (Ang) II-induced rat vascular smooth muscle cell growth. METHODS AND RESULTS: Twenty to twenty-eight hours after transfection, antisense fibroblast growth factor oligodeoxynucleotides introduced by passive uptake and HVJ-liposome method decreased basal DNA synthesis significantly as compared to the sense, control, and scrambled oligodeoxynucleotides groups; however, 60-68 hours after transfection, only antisense fibroblast growth factor oligodeoxynucleotides transduced by the HVJ-liposome method resulted in a significant inhibition of DNA synthesis under basal and Ang II-(10(-;6)M) stimulated conditions. The IC(25) of oligodeoxynucleotides assessed by the inhibition of thymidine incorporation was significantly lower using the HVJ-liposome method than those using the other transfer methods. To clarify the mechanisms of cellular uptake of oligodeoxynucleotides with the HVJ-liposome method, we studied the cellular fate of fluorescein isothiocyanate (FITC)-labeled oligodeoxynucleotides FITC-oligodeoxynucleotides was localized in nuclei at 5 minutes after transfection with the HVJ-liposome method. In contrast, FITC-oligodeoxynucleotides introduced by passive uptake was detected in nonnuclear cellular compartments, possibly endosomes, but not the nuclei. Cellular fluorescence of oligodeoxynucleotides introduced by passive uptake disappeared within 24 hours, while that introduced by the HVJ-liposome method could be observed up to 72 hours. CONCLUSION: These results demonstrate that the HVJ-liposome transfer enhanced the effectiveness of AS-fibroblast growth factor via its specific molecular mechanisms of transfer.

Preparation of Liposomes Encapsulating Water-Soluble Compounds Using Supercritical Carbon Dioxide

In this paper the development of a new preparation method of liposomes containing a water soluble marker (fluorescein isothiocyanate–dextran (FITC–dextran) or zinc phthalocyanine tetrasulfonic acid (TSZnPc) using supercritical carbon dioxide (called "the supercritical liposome method") is described. The apparatus used consisted of two main parts: the high-pressure part, in which the lipid components 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and cholesterol (Chol) (7:3 molar ratio) were dissolved under pressure in supercritical carbon dioxide, and a low-pressure part, in which the homogeneous supercritical solution is expanded and simultaneously mixed with the aqueous phase to yield liposomes encapsulating the water soluble marker. Addition of 7% absolute ethanol to carbon dioxide at 25MPa and 60°C and the use of a high-pressure recycling system during 30min form the homogeneous solution with high reproducibility of both lipid components and resulted in an equal expansion profile (recovery after expansion versus time) of POPC and Chol. Incubation of the lipid components during 60min at the above mentioned conditions generated only 3% degradation. The average size of the liposomes was about 200nm and could not be influenced by the experimental conditions used. Optimal values for encapsulated volume (1.25L/mol) and efficiency (20%) of the liposomes were obtained using statistical experimental design by using the water soluble marker TSZnPc and an encapsulation capillary with 5.0cm length and 0.5mm inner diameter. The total amount of ethanol used to obtain an encapsulation efficiency of 20% was 15-fold reduced compared to the ethanol injection method of Batzri and Korn (Biochim. Biophys. Acta1973, 298, 1015–1019).

Linkup of a Fast Protein Liquid Chromatography System with a Stirred Thermostated Cell for Sterile Preparation of Liposomes by the Proliposome–Liposome Method: Application to Encapsulation of Antibiotics, Synthetic Peptide Immunomodulators, and a Photosensitizer

The proliposome–liposome method is based on the conversion of the initial proliposome preparation into a liposome dispersion by dilution with the aqueous phase. This technique is characterized by an extremely high entrapment efficiency and is suitable for the encapsulation of a wide range of drugs with different water and alcohol solubility. A description of a home-made stirred thermostated cell and its linkup with an FPLC system for a rapid and automated preparation of multilamellar liposomes under strictly controlled conditions (temperature, dilution rate, and schedule) is presented. The highly reproducible procedure yields multilamellar liposomes with a high encapsulation efficiency for various drugs. Carboxyfluorescein, as a model hydrophilic compound, was entrapped with an efficiency of 81 ± 2%. The antibiotics neomycin and gentamycin were entrapped with efficiencies of 65 and 69%, respectively. Synthetic immunomodulators adamantylamide dipeptide, muramyl dipeptide, and β-d-GlcNAc-norMurNAc-l-Abu-d-isoGln were entrapped with efficiencies of 87, 62, and 85%, respectively. The photosensitizer mesotetra-(parasulfophenyl)-porphin was entrapped with an efficiency of 65%. The cell has been designed for laboratory-scale preparation of liposomes (300–1000 mg of phospholipid per run) in a procedure taking less than 90 min. The method can be readily scaled up and linked with secondary processing methods, such as pressure extrusion through polycarbonate filters.

Wild-type p53 gene-induced morphological changes and growth suppression in hepatoma cells.

The human hepatocellular carcinoma (HCC) cell line, HLF, expresses only mutant-type p53 (mt-p53), which has an amino acid substitution at the 244th residue from glycine to alanine. HLF cells were transfected with wild-type p53 (wt-p53) cDNA construct pC53-SN3, mt-p53 cDNA construct pC53-SCX [which differs by a single nucleotide, resulting in alanine instead of valine at the 143rd residue in p53 (p53-143)], or pCMV-Neo-Bam, as a control, by a liposome method. After G418 selection, three wt-p53 stable transformants (WT), four mt-p53 transformants (MT), and three control vector transformants (VT) were obtained. We analyzed the cell growth and morphological changes of these transformants under different culture conditions [fetal calf serum (FCS), 10%, 1%, and 0%]. Whereas no difference from control in the growth rate and morphology was observed under the 10% FCS conditions, serum starvation induced remarkable phenotypical changes in all three WTs, but not in the other transformant. Corresponding to these phenotypical changes, the transcriptional activity of wt-p53 was increased more than nine fold. These results indicated that serum starvation would induce wt-p53 biological function, which is tightly linked to morphological changes and growth suppression. To induce these changes, the introduction of the wt-p53 gene itself was not sufficient, and additional triggering, i.e., serum starvation, was indispensable.

Inwardly rectifying potassium channels expressed by gene transfection into the green Monkey kidney cell line COS-1.

1. cDNA encoding a functional inwardly rectifying K+ (IRK1) channel was transfected into COS-1 cells (a Green Monkey kidney cell line) using the liposome method, and voltage clamp experiments were done after 48-72 h. 2. Transfected cells showed inward rectification under whole-cell recording. The unitary current-voltage relationships in the inside-out configuration were almost linear in the absence of internal Mg2+ and polyamines, and the channel conductance averaged 34.1 +/- 2.0 pS (n = 15) at 23-26 degrees C. 3. Internal Mg2+ (2-10 microM) induced sublevels in the outward current with one-third and two-thirds of the unitary amplitude as in native channels. 4. To determine the subunit stoichiometry, we constructed tandem multimeric cDNAs consisting of the coding sequences of the IRK1 gene linked in a head-to-tail fashion. Cells transfected with tandem homomultimers up to octamers showed similar inwardly rectifying K+ channels. 5. A mutation (E138Q) eliminated the ionic conductance of the channel. Channels expressed by dimeric constructs containing a single mutant have a conductance ranging between 5 and 35 pS. 6. The E138Q mutant cotransfected with a wild-type dimeric, trimeric or tetrameric construct did not alter the channel conductance. The results do not support the notion that IRK1 channel proteins consist of four subunits.

In VivoTransfer Efficiency of Antisense Oligonucleotides into the Myocardium Using HVJ–Liposome Method

Although antisense strategy has been at the center of interest in gene therapy, little is known about application of this strategy to cardiac diseases because of the lack of a suitable delivery method into the heart. Therefore, we compared the transfection efficiency of antisense oligodeoxynucleotides (ODN) using HVJ–liposome method and direct transfer inin vivotransfer into heart. To investigate the cellular fate and localization of ODN, transfer of “naked” FITC-labeled antisense ODN or ODN enwrapped in HVJ–liposome complex were examined. Followingin vivotransfer using direct injection as well asin vitrotransfer, fluorescence rapidly disappeared within 1 day, whereas transfer by HVJ–liposome method resulted in sustained fluorescence localized in the nucleus for at least 1 week. Measurement of fluorescence also demonstrated a significantly higher level in myocardium transfected by HVJ–liposome method than direct transfer. The present study demonstrated that HVJ–liposome method is more efficacious for ODN delivery by prolongation of half-life of ODN, suggesting its usefulness for gene therapy in cardiac diseases.

In Vivo Transfer of Foreign Gene to Keratinocytes Using the Hemagglutinating Virus of Japan-Liposome Method

The hemagglutinating virus of Japan (HVJ)-liposome method involves the entrapment of DNA and nuclear protein within liposomes and the use of HVJ to enhance liposome fusion with cell membranes. This method has been used successfully for in vivo gene transfer to various types of tissue. In this study, we investigated whether this method transfers genes effectively to normal and malignantly transformed keratinocytes in vivo. We applied HVJ-liposome complex (HLC) containing the beta-galactosidase gene to the tape-stripped skin of hairless rats and detected the enzyme activity in the keratinocytes of the treated skin. Comparison of this method with the naked DNA injection method, which was shown recently to be useful for in vivo gene transfer to keratinocytes, demonstrated that the transfer efficiency of the latter was about 5 times higher than that of the former. We assessed the efficacy of the HVJ-liposome method for gene transfer to transformed keratinocytes by examining the effect of HLC containing the herpes simplex virus thymidine kinase gene on the growth of mouse squamous cell carcinomas. Local injection of HLC into the tumors followed by administration of ganciclovir to mice resulted in tumor growth inhibition. These results indicate that the HVJ-liposome method is suitable for in vivo gene transfer to keratinocytes; also that this method may prove a good tool for basic research into keratinocyte biology and future keratinocyte gene therapy.

Stearylamine Liposome as a New Efficient Reagent for DNA Transfection of Eukaryotic Cells

The efficiency of stearylamine (SA) liposome in transfecting DNA into eukarytic cells was evaluated and compared with that of the Lipofectin reagent and the calcium phosphate transfection method. Results with the 21 cell lines tested varied, depending on the cell type. The overall indication was that SA liposomes had certain advantages over the Lipofectin reagent and both of them were superior to the calcium phosphate method, showing the greatest effect in 8 different cell lines, while the calcium phosphate method was the best in only 4 cell lines. There were 2 cell lines which could only be transfected by SA liposomes, another 2 only by the calcium phosphate method, but none depended solely on the Lipofectin reagent. The number of cell lines failed to be transfected was 2 for SA liposomes, 3 for calcium phosphate method, and 4 for the Lipofectin reagent. The advantage of the SA liposome method is discussed.

5417978 Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use : Tari Ana Maria; Lopez-Berestein Gabriel; Deisseroth Albert B Houston, TX, United States Assigned to Board of Regents The University of Texas System

5417978 Liposomal antisense methyl phosphonate oligonucleotides and methods for their preparation and use : Tari Ana Maria; Lopez-Berestein Gabriel; Deisseroth Albert B Houston, TX, United States Assigned to Board of Regents The University of Texas System