Transfection property of a new cholesterol-based cationic lipid containing tri-2-hydroxyethylamine as gene delivery vehicle

A novel cholesterol-based cationic lipid containing a tri-2- hydroxyethylamine head group and ether linker (Chol- THEA) was synthesized and examined as a potent gene delivery vehicle. In the preparation of cationic liposome, the addition of DOPE as helper lipid significantly increased the transfection efficiency. To find the optimum transfection efficiency, we screened various weight ratios of DOPE and liposome/DNA (N/P). The best transfection efficiency was found at the Chol-THEA:DOPE weight ratio of 1:1 and N/P weight ratio of 10~15. Most of the plasmid DNA was retarded by this liposome at the optimum N/P weight ratio of 10. The transfection efficiency of Chol-THEA liposome was compared with DOTAP, Lipofectamine, and DMRIE-C using the luciferase assay and GFP expression. Chol-THEA liposome with low toxicity had better or similar potency of gene delivery compared with commercial liposomes in COS-7, Huh-7, and MCF-7 cells. Therefore, Chol-THEA could be a useful non-viral vector for gene delivery.     

Effectiveness of liposomes to transfect livestock fibroblasts

The development of an efficient transfection system in livestock cells is an important step towards investigating gene transfer and the functioning and production of transgenic animals. Important factors involved in cationic liposome mediated gene transfer were evaluated through in vitro transfection of bovine, caprine and ovine fibroblast cells. Transfection of plasmid DNA complexes of different commercially available liposomes (Lipofectamine, Lipofectin, Cellfectin and DMRIE-C; Gibco-BRL, USA) was evaluated utilizing the following parameters: DNA/liposome ratio, cell density, DNA conformation, and the effect of transfection time on the efficiency of bovine fibroblasts to express a reporter gene. The effects and concentrations of liposomes were also evaluated in caprine and ovine fibroblasts. Lipofectamine alone and Lipofectamine with Plus reagent induced high-frequency expression of beta-galactosidase and neo genes in all cells evaluated (47 and 88.3%, respectively). Regarding phenotype, chromosomal stability was similar in transfected and non-transfected cells. The parameters set in this study will establish a foundation for utilizing transfected fibroblast cells to generate transgenic animals through nuclear transfer technology and gene function studies.     

Transfer of maternally administered fusogenic liposome-DNA complexes into monkey fetuses in a pregnancy model

Background

Materno‐fetal transfer of intravenously administered liposome‐plasmid DNA complexes has been demonstrated only in mice. Studies on its materno‐fetal transfer in the pregnant monkey model is needed because of critical differences in placental structure between primates including humans and rodents.

Methods

The reporter plasmid pEGFP‐C1 was formulated in cationic lipid containing polybrene and vesicular stomatitis virus G protein. The fusogenic liposome‐plasmid DNA complexes were intradermally injected into pregnant common marmosets (N=2), a New World monkey, near term. DNA extracted from fetal tissues was subjected to PCR for detection of the egfp gene. Confocal microscopy and immunostaining were performed to determine the sites of transgene expression in the fetal organs.

Results

The egfp gene was detected in fetal blood and major organs (heart, liver, lung). The encoded protein was mainly produced in the endothelial cells of blood vessels in the fetal lungs.

Conclusions

This is the first report on materno‐fetal transfer of intradermally administered fusogenic liposome‐plasmid DNA complexes and fetal expression of a transgene in primates. Copyright © 2002 John Wiley & Sons, Ltd.

     

Gene delivery into hepatocytes with the preS/liposome/DNA system

Gene delivery into human hepatocytes remains a critical issue for the development of liver-directed gene therapy. Gene delivery based on non-viral vectors is an attractive approach relative to viral vectors. In this report, novel delivery system of preS/liposome/DNA virus-like particle (VLP) was developed for gene transfection into hepatocytes in vivo and in vitro. Plasmid pCMVbeta, expressing beta-galactosidase, was encapsulated with cationic liposome, and then the histidine-tagged preS domain of hepatitis B virus was coated on the surface of liposome/DNA to form preS/liposome/ DNA VLP. Transfection efficiencies of preS/liposome/DNA, liposome/DNA, naked DNA and preS were analyzed using several different human cell lines. The highest transfection efficiency was found using preS/liposome/DNA VLP as the transfection reagent in human hepatocyte (HH) cell line. Results show that preS domain of hepatitis B virus coated on liposome/DNA can be used for highly efficient gene transfection into human hepatocytes. Moreover, the target characteristic of preS/liposome/DNA was analyzed in vivo. After preS/liposome/DNA VLP was injected into immunocompromised (Nude) mice via the tail vein, most of beta-galactosidase was expressed in the liver; however, no significant target expression was found with the injection of liposome/ DNA or naked DNA. Our results show that preS/liposome/DNA VLP can be used as a novel liver-specific gene delivery system.     

Generation of transgenic mice by transfection of pronuclear embryos using lipid-DNA complexes

We had previously developed a methodology to introduce foreign DNA into mouse eggs and embryos using cationic lipids as vectors. In this report we use this technique to produce transgenic animals. Mouse embryos at the pronuclear stage were transfected using a mixture of a plasmid DNA, encoding for a nuclear form of beta-galactosidase, and a commercial lipid transfection reagent. Embryos were washed and incubated overnight. Those that cleaved and develop to the 2-cell stage of normal appearance were transferred to the Fallopian tubes of pseudopregnant foster mothers. We analysed a total of 158 offspring and found two, a female and a male, to be transgenic (1.27% of the total). Integration of the foreign DNA in the female was showed by Southern blot. Both animals expressed the lacz in several organs, but none of them either displayed expression in the germ cells or transmitted the transgene to their offspring. Taken together our results show that lipid transfection can generate transgenic mice, but the efficiency needs to be improved for this method to be widely applied.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendai virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nucelar protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100 - 800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenicviral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

Development of a novel fusogenic viral liposome system (HVJ-liposomes) and its applications to the treatment of acquired diseases.

Toward human gene therapy and gene analysis in vivo, a novel hybrid vector based on liposome has been developed for more efficient gene delivery and gene expression. The liposome was decorated with HVJ (Sendal virus) envelope fusion proteins to introduce DNA directly into the cytoplasm, and contained DNA and DNA-binding nuclear protein to enhance expression of the gene. Recently, several types of HVJ-liposomes were developed by altering the lipid components of the liposomes. HVJ-cationic liposomes increased gene delivery 100-800 times more efficiently in vitro than the conventional HVJ-anionic liposomes. HVJ-cationic liposomes were also more useful for gene expression in restricted portions of organs and for gene therapy of disseminated cancers. It was further discovered that the use of anionic liposomes with a virus-mimicking lipid composition (HVJ-AVE liposomes) increased transfection efficiency by several fold in vivo, especially in liver and muscle. By coupling the Epstein-Barr (EB) virus replicon apparatus to HVJ-liposomes, transgene expression was sustained in vitro and in vivo. Most animal organs were found to be suitable targets for the fusigenic-viral liposome system, and numerous gene therapy strategies using this system were successful in animals.     

The transfection of Jurkat T-leukemic cells by use of pH sensitive immunoliposomes

A gene transfer vector has been developed utilising anionic liposomes as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3+ T lymphocytes (Jurkat cells). The plasmid DNA that contained the Escherichia coli beta-galactosidase reporter gene was condensed using poly-l-lysine of molecular mass 20,700 (PLK99) to form a polyplex which was interacted with several anionic liposome formulations to form lipopolyplexes. The liposome formulations where based on dioleoylphosphatidylethanolamine (DOPE) in combination with cholesterol and dioleoylphosphatidylcholine (DOPC) and oleic acid, or dimyristoylphosphatidylethanolamine (DMPE). For targeting to the Jurkat cells distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2,000 and coupled to anti-CD3 antibody was incorporated. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, agarose gel electrophoresis and electron microscopy and the permeability of the lipopolyplexes to liposome-encapsulated glucose was determined. The polyplexes consisted of a mixed population of rod-like structures (53-160 nm long and 23-31 nm diameter) and spheres (18-30 nm diameter). The lipopolyplexes retained a permeability barrier although were more permeable to glucose than their component liposomes. The poly-l-lysine condensing agent was still susceptible to pronase digestion suggesting that the polyplex was associated with the outer surface of the liposome. The lipopolyplexes with lipid composition DOPE/cholesterol/OA/DSPE-PEG2000 anti-CD3+ PLK99-plasmid DNA had significant gene transfer activity, as monitored by beta-galactosidase expression, that depended on the charge ratio of the component polyplex and the lipid/DNA weight ratio. The anti-CD3 antibody, the liposomal lipid and pH sensitivity were essential for transfection activity.     

A new cationic liposome for efficient gene delivery with serum into cultured human cells: a quantitative analysis using two independent fluorescent probes

Cationic liposomes are useful to transfer genes into eukaryotic cells in vitro and in vivo. However, liposomes with good transfection efficiency are often cytotoxic, and also require serum-free conditions for optimal activity. In this report, we describe a new formulation of cationic liposome containing DC-6-14, O,O'-ditetradecanoyl-N-(alpha-trimethylammonioacetyl)diethan olamine chloride, dioleoylphosphatidylethanolamine and cholesterol for gene delivery into cultured human cells. This liposome, dispersed in 5% serum-containing growth medium, efficiently delivered a plasmid DNA for GFP (green fluorescent protein) into more than 80% of the cultured human cell hybrids derived from HeLa cells and normal fibroblasts. Flow cytometric analysis revealed that the efficiency of the GFP gene expression was 40-50% in a tumor-suppressed cell hybrid, while it was greatly reduced in the tumorigenic counterpart. The enhanced GFP expression in tumor-suppressed cell hybrids was quantitatively well correlated with a prolonged presence of the plasmid DNA, which had been labeled with another fluorescent probe, ethidium monoazide, within the cells. These results suggest that a newly developed cationic liposome is useful for gene delivery in serum-containing medium into human cells and the stability of the plasmid DNA inside the cell is a crucial step in this liposome-mediated gene expression. The mechanisms by which cationic liposome mediates gene transfer into eukaryotic cells are also discussed.     

Detection of transgene in progeny at different developmental stages following testis-mediated gene transfer

We recently reported that exogenous DNA injected into testis as a liposome complex can be transferred into the egg via sperm by natural mating and integrated in the genome (testis-mediated gene transfer: TMGT). Here, we studied the efficiency of each of the several liposomes in associating foreign DNA with sperm, the expression of an introduced gene in early embryos, and the presence of the DNA in fetuses and pups at different ages. The CMV/beta-actin/EGFP fusion gene, encapsulated with different liposomes, was injected into rat testis, and spermatozoa in the cauda epididymis were obtained 1, 4, and 14 days after injection. We tested each of the 8 liposomes, and found that only 2, DMRIE-C and SuperFect, led to the detection of foreign DNA on all of the days examined, with relatively higher ratios of rats having positive sperm. By means of TMGT using either of those two liposomes, more than 80% of morula-stage embryos expressed EGFP, as observed by fluorescence microscopy. Then we detected introduced DNA in the progeny by PCR and Southern dot blot, and found that the ratio of animals carrying the foreign DNA decreased as they developed, and that only a part of postpartum progeny were foreign-DNA-positive with high incidence of mosaicism. These results suggest that, although, the success rate is still limited, foreign DNA could be integrated into the genome of the progeny by TMGT at least under specific experimental conditions, the efficiency of which depends largely on the characteristics of the liposome. The results also suggest that TMGT could be applicable to fetal gene therapy as well as to the generation of transgenic animals.     

Delivery and pathway in MCF7 cells of DNA vectorized by cationic liposomes derived from cholesterol

We have investigated the delivery and the pathway in tumoral MCF7 cells of DNA carried by liposomes prepared from (trimethyl aminoethane carbamoyl cholesterol iodide (TMAE-Chol), a cholesterol-based cationic lipid with a quaternary ammonium on the polar head. The structure of DNA-liposome complexes depends on the length of DNA and on the lipid-DNA charge ratio X. Spherical beads constitute fine structures of the observed complexes even when they appear as aggregates. For oligonucleotide transfer, dissociation from liposomes after transfection, penetration of the oligonucleotides into nuclei, and a long resident time were observed. For plasmid transfer, a correlation between the variation in the transfection level and the ultrastructure of complexes was demonstrated. The results showed a cellular route of lipid/plasmid complexes from the beginning by endocytosis, entrapped into endosomes, released by the latter until entry in the perinuclear area, and then penetration of plasmids inside the nuclei resulting in the observed expression of the beta-galactosidase gene.     

阳离子脂质体转染人类骨骼肌原代细胞的初步研究

探讨不同脂质体介导基因转染人类骨骼肌原代细胞的转染效率和基因的表达.将含有β-半乳糖苷酶LacZ结构基因的质粒,用三种不同的阳离子脂质体导入人类骨骼肌原代细胞中,通过X-Gal染色观察不同的转染效率.结果发现,Fugene 6转染效率最高,蓝染细胞达10%,其脂质体与DNA的最佳比例为3∶ 2.Fugene 6可有效地将外源基因导入骨骼肌原代细胞,而且外源基因可以长效高效地表达,有望用来作为基因治疗的载体.     

Inhibitory effects of an antisense oligonucleotide in an experimentally infected mouse model of influenza A virus

The antiviral effects of a 20-mer antisense phosphorothioate oligonucleotide, PB2-as, on influenza A virus infection in mice were examined and compared to those of PB2-as encapsulated with several cationic liposomes. Intravenous injection of PB2-as, as a complex with DMRIE-C, a cationic liposome, was most effective for prolonging the mean survival time in days (MSDs) and increasing the survival rates of mice infected with the influenza A virus. In addition, the liposomal PB2-as significantly inhibited viral growth in lung tissues. These results suggest that PB2-as encapsulated with DMRIE-C may be active against the influenza A virus infection through the inhibition of virus replication in the mouse lung.     

The transfection of Jurkat T-leukemic cells by use of pH-sensitive immunoliposomes

An effective pH-sensitive gene transfer vector has been developed utilising anionic liposomes with various formulations as a carrier of plasmid DNA (pEGlacZ, 7.6 kb) to transfect CD3 T+ lymphocytes (Jurkat cells). The plasmid DNA was condensed using poly-l-lysines with a range of molecular masses to form polyplexes that were interacted with several anionic liposome formulations to form lipopolyplexes. For targeting to the Jurkat cells, distearoylphosphatidylethanolamine (DSPE) linked to poly (ethylene glycol) molecular mass 2000 and coupled to anti-CD3 antibody was incorporated in the liposomes. The polyplexes and lipopolyplexes were characterised in terms of size, zeta potential, gel electrophoresis and electron microscopy. The gene transfer activity of the lipopolyplexes, assessed from beta-galactosidase expression, depended on the charge ratio (NH(3)+/PO(4)-) of the component polyplex and the lipid/DNA weight ratio of the lipopolyplex.     

新型非病毒三元复合DNA载体的研究

基因治疗是未来临床医学最具潜力的治疗方式,目前阻碍临床基因治疗发展的主要因素是缺乏安全和高效的基因载体,因此研究理想的非病毒转基因载体具有重要的意义.构建了由质粒DNA(D)-抗DNA抗体(A)-阳离子脂质体(C)组成的三元复合纳米基因载体(DAC),研究表明,三组分在磷酸缓冲液中可通过分子组装形成复合纳米胶束,DAC在细胞培养中表现出显著高效的基因表达,DAC在血管平滑肌细胞中的基因转染效率比不含抗DNA抗体的二元组合(DC)高4倍,比不含阳离子脂质体的二元组合(DA)约高11倍.激光共聚焦荧光显微观察证明,DAC细胞摄取量和DNA进入细胞核的量均明显高于对照组,而DC二元组合(不含抗DNA抗体)的DNA很少进入细胞核,细胞在DAC存在下生长正常.未发现细胞毒性.研究结果提示,DAC的作用机理主要是三元复合胶束中DNA的装载量比二元载体大得多,抗DNA抗体与阳离子脂质体的协同作用明显有利于DNA被细胞摄取和胞吞,从而提高了基因的转染和表达.     

Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer

A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.     

Selective gene therapy for prostate cancer cells using liposomes conjugated with IgM type monoclonal antibody against prostate-specific membrane antigen

Prostate cancer cells express prostate-specific membrane antigen (PSMA). We developed an IgM type monoclonal antibody against PSMA. The antibody was coupled to poly-L-lysine and thereafter this conjugate was mixed with cationic liposomes containing plasmid DNA. The antibody-liposome complex was tested whether it could deliver the gene of interest selectively to the PSMA positive cells. As assessed by beta-galactosidase reporter gene, the transfection efficiency was 13.2% with anti-PSMA-liposome complex as compared to 4% with control IgM liposome complex. In contrast, no such differences were observed in PSMA negative PC-3, DU145 and T24 cells. Furthermore, in the suicide gene therapy in vitro with thymidine kinase gene plus ganciclovir system, anti-PSMA liposome complex demonstrated a selective growth inhibitory effect on PSMA positive LNCaP cells but not on PSMA negative cell lines.     

Protamine Sulfate Provides Enhanced and Reproducible Intravenous Gene Transfer by Cationic Liposome/DNA Complex

Abstract

A novel lipid/polycation/DNA (LPD) formulation has been developed for in vivo gene transfer. It involves the condensation of plasmid DNA with protamine sulfate, a cationic polypeptide, followed by the addition of DOTAP cationic liposomes. Compared with DOTAP/DNA complex, LPD offers greater protection of plasmid DNA against enzymatic digestion and gives consistently higher gene expression in mice via tail vein injection. The in vivo efficiency of LPD was dependent upon charge ratio and was also affected by the lipid used. Increasing the amount of DNA delivered induced an increase in gene expression. The optimal dose was approximately 50 μg per mouse, at which concentration approximately 10 ng luciferase protein per mg extracted tissue protein could be detected in the lung. Gene expression in the lung was detected as early as 1 h after injection, peaked at 6 h, and declined thereafter. Using LacZ as a reporter gene, it was shown that endothelial cells were the primary locus of transgene expression in both lung and spleen. No sign of inflammation in these organs was noticed. Since protamine sulfate has been proven to be non-toxic and only weakly immunogenic in humans, this novel vector may be useful for the clinical use of gene therapy.