Liposome Vector Containing Biosurfactant-Complexed DNA as Herpes Simplex Virus Thymidine Kinase Gene Delivery System

For injectable-sized liposome complexed with DNA (lipoplexes) with high transfection efficiency of genes, we initially prepared small-sized liposomes by addition of biosurfactant. For selectivity of gene expression, the thymidine kinase (MK-tk) gene controlled by midkine was used for herpes simplex virus thymidine kinase (HSV-tk) gene therapy. Liposomes composed of 3([N-(N′,N′–dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol), L-dioleoylphosphatidylethanolamine (DOPE), and a biosurfactant, such as β-sitosterol β-D-glucoside (Sit-G) for Sit-G-liposomes and mannosylerythrytol lipid A (MEL) for MEL-liposomes, produced about 300-nm-sized lipoplexes. Sit-G- and MEL-liposomes showed higher transfection efficiency of the luciferase marker gene and thymidine kinase activity in the presence of serum in the cells. The treatment with transfection of MK-tk gene by Sit-G-liposome and injection of ganciclovir significantly reduced tumor growth in a solid tumor model, compared with that by Sit-G-liposome alone. This finding suggested that Sit-G-liposome is a potential vector for HSV-tk gene therapy.     

带HSV—tk基因的重组腺病毒基因治疗小鼠B16黑色素瘤的实验研究

我父成功开展了携带单纯疹疹Ⅰ型病毒胸腺嘧啶激酶基因（Ｈｅｒｐｅｓｓｉｍｐｌｅｘｖｉｒｕｓｔｈｙ－ｍｎｉｄｉｎｅｋｉｎａｓｅ,ＨＳＶ－ｔｋ）的复制缺陷型重组腺病毒Ａｄ（ＨＳＶ－ｔｋ）结合使用ＧＣＶ治疗Ｃ５７ＢＬ／６小鼠Ｂ１６黑色素瘤的离体及动物试验。     

Serum-resistant gene transfer to oral cancer cells by Metafectene and GeneJammer: application to HSV-tk/ganciclovir-mediated cytotoxicity

Cationic lipids and polyamines have been used as non-viral gene transfer reagents, both in vitro and in vivo. One of the limitations to their use in vivo is the inhibition of gene delivery by serum. We showed previously that, in the absence of serum, relatively high cytotoxicity in oral cancer cell lines could be achieved via transfection of the Herpes Simplex Virus thymidine kinase (HSV-tk) gene followed by treatment with ganciclovir (GCV), despite the low efficiency of transfection (Konopka et al., Gene Ther. Mol. Biol. 8 (2004) 307-318). In this study we evaluated the effect of high concentrations (20-60%) of fetal bovine serum (FBS) on the transfection efficiency of two novel reagents, the polycationic liposome, Metafectene, and the polyamine reagent, GeneJammer, in HSC-3 and H357 human oral squamous cell carcinoma (OSCC) cells. We also examined whether the HSV-tk gene delivered in the presence of FBS (up to 60%, could induce cell death following treatment with GCV. Transfection was optimized using a luciferase-expressing plasmid. Both Metafectene- and GeneJammer-mediated luciferase gene expression in HSC-3 cells was reduced by 40-50% when transfection was performed in the presence of 20-60% FBS. The delivery of the HSV-tk gene by Metafectene in the absence and the presence of 60% FBS, followed by GCV treatment for 9 days, resulted in 95% and 70% cytotoxicity, respectively. With GeneJammer, transfection in 0% and 60% FBS resulted in 90% and 40% cytotoxicity, respectively, after 9 days. In contrast, very low transfection activity and a much higher inhibitory effect of serum were observed in H357 cells. Nevertheless, about 35% GCV-mediated cytotoxicity was observed with H357 cells at both 0% and 60% FBS, using GeneJamer. Thus, Metafectene and GeneJammer can be used in the delivery of genes in biological milieu and in the gene therapy of OSCC in animal models.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

Transfection efficiency boost by designer multicomponent lipoplexes

Cationic liposome-DNA complexes (lipoplexes) have emerged as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. We investigated the correlation between structural evolution of multicomponent lipoplexes when interacting with cellular lipids, the extent of DNA release and the efficiency in transfecting mouse fibroblast (NIH 3T3), ovarian (CHO) and tumoral myofibroblast-like (A17) cell lines. We show, for the first time, that the transfection pattern increases monotonically with the number of lipid components and further demonstrate by means of synchrotron small angle X- ray scattering (SAXS) that structural changes of lipoplexes induced by cellular lipids correlate with the transfection efficiency. Specifically, inefficient lipoplexes either fused too rapidly upon interaction with anionic lipids or, alternatively, are found to be extremely resistant to solubilization. The most efficient lipoplex formulations exhibited an intermediate behaviour. The extent of DNA unbinding (measured by electrophoresis on agarose gel) correlates with structural evolution of the lipoplexes but DNA-release does not scale with the extent of transfection. The general meaning of our results is of broad interest in the field of non-viral gene delivery: rational adjusting of lipoplex composition to generate the proper interaction between lipoplexes and cellular lipids may be the most appropriate strategy in optimizing synthetic lipid transfection agents.     

Verapamil enhances the efficiency of DNA-mediated gene transfer in mammalian cells

Treatment of Ltk^? cells with the calcium antagonists, verapamil and diltiazem, but not nifedipin, causes a 3-fold enhancement of the frequency of transfer of the cloned gene for herpes simplex virus thymidine kinase (HSV-tk). The frequency of phenotypic expression of the HSV-tk DNA was 20 to 34 times higher than that of genotypic transformation. Phenotypic expression was also 2.3 to 2.6 times increased when 20 μg/ml of verapamil was present during calcium phosphate-mediated DNA transfection.     

Lipid composition of cationic nanoliposomes implicate on transfection efficiency

Abstract

Cationic liposome (CL)-DNA complexes (lipoplexes) have appeared as leading nonviral gene carriers in worldwide gene therapy clinical trials. Arriving at therapeutic dosages requires the full understanding of the mechanism of transfection. However, using CLs to deliver therapeutic nucleic acids and drugs to target organs have some problems, including low transfection efficiency. The aim of this study was developing novel CLs containing four neutral lipids; cholesterol, 1,2-dioleoyl phosphatidylethanolamine, distearoylphosphatidylcholine and dipalmitoylphosphatidylcholine as a helper lipid and dimethyl dioctadecyl ammonium bromide as a cationic lipid to increase transfection efficiency. We have investigated the correlation between number of lipid composition and transfection efficiency. The morphology, size and zeta potential of liposomes and lipoplexes were measured and lipoplexes formation was monitored by gel retardation assay. Transfection efficiency was assessed using firefly luciferase reporter assay. It was found that transfection efficiency markedly depended on liposome to plasmid DNA (pDNA) weight ratio, lipid composition and efficiency of pDNA entrapment. High transfection efficiency of plasmid by four component lipoplexes was achieved. Moreover, lipoplexes showed lower transfection efficiency and less cytotoxicity compared to Lipofectamine?. These results suggest that lipid composition of nanoliposomes is an important factor in control of their physical properties and also yield of transfection.     

Retrovirus—mediated herpes simplex virus thymidine kinase gene therapy approach for hepatocellular carcinoma

INTRODUCTI0NHepatocellularcarcinoma(HCC)is0ne0fthem0stc0mm0nhumanmalignancies,causinganestimatedl,250,OOOdeatht0llperyearworldwide[1].Thep0orprognosisencounteredintreatment0fsuchcarcinomaismainlycausedbylatediagn0sisandinsufficiency0feffectivestrategies,especiallyforadvanced-stagedpatients.However,recentknowledge0fpathogenesisofHCCatm0lecularlevelprovidesanalternativeappr0achwhenc0nsideringgenetherapyastreatmelltf0rHCC.Am0ngthevari0usgenetherapystrategiesincancer)itwaJsrep0rtedthatth…     

Ovarian cancer gene therapy using HPV-16 pseudovirion carrying the HSV-tk gene

Ovarian cancer is the leading cause of death from all gynecological cancers and conventional therapies such as surgery, chemotherapy, and radiotherapy usually fail to control advanced stages of the disease. Thus, there is an urgent need for alternative and innovative therapeutic options. We reason that cancer gene therapy using a vector capable of specifically delivering an enzyme-encoding gene to ovarian cancer cells will allow the cancer cell to metabolize a harmless prodrug into a potent cytotoxin, which will lead to therapeutic effects. In the current study, we explore the use of a human papillomavirus (HPV) pseudovirion to deliver a herpes simplex virus thymidine kinase (HSV-tk) gene to ovarian tumor cells. We found that the HPV-16 pseudovirion was able to preferentially infect murine and human ovarian tumor cells when administered intraperitoneally. Furthermore, intraperitoneal injection of HPV-16 pseudovirions carrying the HSV-tk gene followed by treatment with ganciclovir led to significant therapeutic anti-tumor effects in murine ovarian cancer-bearing mice. Our data suggest that HPV pseudovirion may serve as a potential delivery vehicle for ovarian cancer gene therapy.     

The piggyBac transposon is an integrating non-viral gene transfer vector that enhances the efficiency of GDEPT

Gene-directed enzyme prodrug therapy (GDEPT) is a strategy developed to selectively target cancer cells. However, the clinical benefit is limited due to its poor gene transfer efficiency. To overcome this obstacle, we took advantage of piggyBac (PB) transposon, a natural non-viral gene vector that can induce stable chromosomal integration and persistent gene expression in vertebrate cells, including human cells. To determine whether the vector can also mediate stable gene expression in ovarian cancer cells, we constructed a PB transposon system that simultaneously expresses the Herpes simplex virus thymidine kinase (HSV-tk) gene and the monomeric red fluorescent protein (mRFP1) reporter gene. The recombinant plasmid, pPB/TK, was transfected into ovarian adenocarcinoma cells SKOV3 with FuGENE HD reagent, and the efficiency was given by the percentage of mRFP1-positive cells detected by flow cytometry and confocal microscopy. The specific expression of HSV-tk in transfected cells was confirmed by RT-PCR and western blotting. The sensitivity of transfected cells to pro-drug ganciclovir (GCV) was determined by methylthiazoletetrazolium (MTT) assay. A total of 56.4 ± 8.4% cells transfected with pPB/TK were mRFP1 positive, compared to no measurable mRFP1 expression in pORF-HSVtk-transfected cells. The expression level of HSV-tk in pPB/TK-transfected cells was ∼10 times higher than in pORF-HSVtk-transfected cells. The results show that pPB/TK transfection increases the sensitivity of cells to GCV in a dose-dependent manner. Our data indicate that the PB transposon system could enhance the anti-tumor efficiency of GDEPT in ovarian cancer.     

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.     

Transfection of oral cancer cells mediated by transferrin-associated lipoplexes: Mechanisms of cell death induced by herpes simplex virus thymidine kinase/ganciclovir therapy

The Herpes Simplex Virus thymidine kinase (HSV-tk) suicide gene/ganciclovir (GCV) approach has been used for the treatment of a variety of cancers. The purpose of the present study was to evaluate the cytotoxic effect of ganciclovir in oral squamous cancer cells, previously transfected with HSV-tk gene delivered by transferrin-associated complexes (Tf-lipoplexes), as well as to investigate the mechanisms involved in the bystander effect and in the process of cell death. The delivery of HSV-tk gene to the oral cancer cells, HSC-3 and SCC-7, mediated by Tf-lipoplexes followed by ganciclovir treatment resulted in essentially 100% cytotoxicity, the observed toxic effect being dependent both on GCV dose and incubation time. Cell death was shown to occur mainly by an apoptotic process. Different experimental approaches demonstrated that the observed cytotoxicity was mainly due to diffusion of the toxic agent into neighbouring, non-transfected cells, via gap junctions. Preliminary in vivo studies in a murine model for oral squamous cell carcinoma have shown a significant inhibition of tumor growth upon injection of Tf-lipoplexes carrying HSV-tk followed by intraperitonal injection of GCV, as compared to controls.     

Transfection efficiency of lipoplexes for site-directed delivery

Targeted gene delivery is a promising strategy to cure disease on its basic level at the site of interest. The ultrastructure, internalization, and transfection efficiency of lipoplexes was investigated. We found that at a charge ratio (ρ) of 4.0 lipoplexes had optimum characteristics for gene delivery in vitro. To decrease the size of lipoplexes, we used a method of continuous-flow microfluidics. PEGylation of lipoplexes did not hinder internalization, but was found to hamper transfection. To discriminate between uptake and transfection efficiency of lipoplexes, we used fluorescence-based approaches: microscopy and FACS. To this end, GFP plasmid was labeled with Alexa 594, and, in parallel experiments, GFP plasmid was combined with rhodamine-labeled lipid. Our studies confirm that cellular uptake does not imply transfection efficiency, and that hurdles in cellular processing have to be taken before targeted gene delivery becomes an established therapeutic option.     

Small interfering RNA delivery into the liver by cationic cholesterol derivative-based liposomes

Purpose: Previously, we reported that the cationic liposomes composed of a cationic cholesterol derivative, cholesteryl (2-((2-hydroxyethyl)amino)ethyl)carbamate (OH-C-Chol) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) (termed LP-C), could deliver small interfering RNAs (siRNAs) with high transfection efficiency into tumor cells. In this study, to develop a liposomal vector for siRNA delivery in vivo, we prepared the poly(ethyleneglycol) (PEG)-modified cationic liposomes (LP-C-PEG) and evaluated their transfection efficiency in vitro and in vivo.

Materials and methods: We prepared LP-C-PEG/siRNA complexes (LP-C-PEG lipoplexes) formed in water or 50?mM NaCl solution, and evaluated their siRNA biodistribution and gene silencing effect in mice after intravenous injection.

Results: LP-C-PEG lipoplexes strongly exhibited in vitro gene silencing effects in human breast tumor MCF-7 cells as well as LP-C lipoplexes. In particular, formation of LP-C and LP-C-PEG lipoplexes in the NaCl solution increased the cellular association. When LP-C-PEG lipoplexes with Cy5.5-labeled siRNA formed in water or NaCl solution were injected into mice, accumulation of the siRNA was observed in the liver. Furthermore, injection of LP-C-PEG lipoplexes with ApoB siRNA could suppress ApoB mRNA levels in the liver and reduce very-low-density lipoprotein/low-density lipoprotein levels in serum compared with that after Cont siRNA transfection, although the presence of NaCl solution in forming the lipoplexes did not affect gene silencing effects in vivo.

Conclusions: LP-C-PEG may have potential as a gene vector for siRNA delivery to the liver.     

Tanshinone IIA Increases the Bystander Effect of Herpes Simplex Virus Thymidine Kinase/Ganciclovir Gene Therapy via Enhanced Gap Junctional Intercellular Communication

The bystander effect is an intriguing phenomenon by which adjacent cells become sensitized to drug treatment during gene therapy with herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV). This effect is reported to be mediated by gap junctional intercellular communication (GJIC), and therefore, we postulated that upregulation of genes that facilitate GJIC may enhance the HSV-tk/GCV bystander effect. Previous findings have shown Tanshinone IIA (Tan IIA), a chemical substance derived from a Chinese medicine herb, promotes the upregulation of the connexins Cx26 and Cx43 in B16 cells. Because gap junctions are formed by connexins, we hypothesized that Tan IIA might increase GJIC. Our results show that Tan IIA increased GJIC in B16 melanoma cells, leading to more efficient GCV-induced bystander killing in cells stably expressing HSV-tk. Additionally, in vivo experiments demonstrated that tumors in mice with 10% HSV-tk positive B16 cells and 90% wild-type B16 cells became smaller following treatment with the combination of GCV and Tan IIA as compared to GCV or Tan IIA alone. These data demonstrate that Tan IIA can augment the bystander effect of HSV-tk/GCV system through increased gap junction coupling, which adds strength to the promising strategy that develops connexins inducer to potentiate the effects of suicide gene therapy.     

Retrovirus-mediated gene therapy for hepatocellular carcinoma with reversely oriented therapeutic gene expression regulated by alpha-fetoprotein enhancer/promoter

In the present study, to achieve more selective and efficient therapeutic gene expression in hepatoma cells, we compared the therapeutic efficacies of the retroviral vectors expressing the herpes simplex virus thymidine kinase (HSV-tk) gene by the alpha-fetoprotein (AFP) enhancer/promoter in the forward (LNAFE0.3TK) and reverse (LN[AFE0.3TK]R) orientation to the vector long terminal repeats. By Northern blotting, the level of the HSV-tk mRNA in LN[AFE0.3TK]R-infected HepG2 human hepatoma cells was much higher than that in LNAFE0.3TK-infected cells. Consistent with this, LN[AFE0.3TK]R infection into HepG2 cells caused a greater cytotoxicity by ganciclovir exposure together with a stronger bystander effect than LNAFE0.3TK infection. In an animal model, intratumorous injection of LN[AFE0.3TK]R with ganciclovir treatment resulted in pronounced growth inhibition of HepG2 tumor. Thus, the reversely oriented therapeutic gene expression under the control of AFP enhancer/promoter is a possible candidate for the retrovirus-mediated gene therapy for hepatocellular carcinoma.     

In vivo antitumor effect of herpes simplex virus thymidine kinase gene therapy in rat hepatocellular carcinoma: feasibility of adenovirus-mediated intra-arterial gene delivery

Transfer of the herpes simplex virus-thymidine kinase gene, followed by the administration of ganciclovir (HSV-tk/GCV), has been a major approach for cancer gene therapy. We investigated the antitumor effect of the HSV-tk/GCV strategy with the rat orthotopic hepatocellular carcinoma (HCC) model and the tumor-selective gene delivery by an adenovirus-mediated gene transfer through the hepatic artery. The complete antitumor effect was demonstrated, after the treatment with GCV in rat HCC established by the implantation of HSV-tk transferred rat HCC cells. The in vivo bystander effect was also observed. The marked infiltration of CD4+ and CD8+ T lymphocytes, macrophages and NK cells were found in the tumor area. After the injection of adenovirus carrying the LacZ gene into the hepatic artery, the selective expression of transgene in the tumor cell was achieved. These findings indicate that the HSV-tk/GCV strategy, using an adenoviral vector, could be a promising avenue for the treatment of hepatocellular carcinoma.     

Transfection with fluorinated lipoplexes based on fluorinated analogues of DOTMA,DMRIE and DPPES

Fluorinated double-chain (poly)cationic lipids (one or both of these chains being ended by a highly fluorinated tail) which are close analogues of DOTMA, DMRIE or DPPES were designed as synthetic vectors for gene delivery. For N/P ratios (N=number of amine functions of the lipid; P=number of DNA phosphates) from 0.8 to 5, these fluorinated cationic lipids condensed DNA, with or without the use of DOPE, to form fluorinated lipoplexes. No specific cell toxicity was evidenced for these new fluorinated lipoplexes. The efficiency of some of the fluorinated lipoplexes to transfect lung epithelial A549 cells was comparable to that of the first generation of fluorinated lipoplexes made from fluorinated analogues of DOGS (Transfectam) [Bioconjug. Chem. 12 (2001) 114]. These results, combined with the higher in vivo transfection potential found for fluorinated lipoplexes than for conventional lipoplexes or PEI polyplexes [J. Gene Med. 3 (2001) 109], confirm that fluorinated lipoplexes are very promising gene transfer systems.     

Interference of serum with lipoplex-cell interaction: modulation of intracellular processing

We have investigated the mechanism of lipoplex-mediated transfection, employing a dialkyl pyridinium surfactant (SAINT-2), and using serum as a modulator of complex stability and processing. Particle size and stability determine lipoplex internalization, the kinetics of intracellular processing, and transfection efficiency. Clustered SAINT-2 lipoplexes are obtained in the absence of serum (-FBS lipoplexes), but not in its presence (+FBS lipoplexes), or when serum was present during lipoplex formation [FBS], conditions that mimic potential penetration of serum proteins. The topology of DNA in [FBS] lipoplexes shifts from a supercoiled, as in -FBS lipoplexes, to a predominantly open-circular conformation, and is more prone to digestion by DNase. Consistently, atomic force microscopy revealed complexes with tubular extensions, reflecting DNA that protrudes from the lipoplex surface. Interestingly, the internalization of [FBS] lipoplexes is approximately three-fold higher than that of -FBS and +FBS lipoplexes, yet their transfection efficiency is approximately five-fold lower. Moreover, in contrast to -FBS and +FBS complexes, [FBS] complexes were rapidly processed into the late endosomal/lysosomal degradation pathway. Intriguingly, transfection by [FBS] complexes is greatly improved by osmotic rupture of endocytic compartments. Our data imply that constraints in size and morphology govern the complex' ability to interact with and perturb cellular membranes, required for gene release. By extrapolation, we propose that serum may regulate these parameters in an amphiphile-dependent manner, by complex 'penetration' and modulation of DNA conformation.