Tumor-directed gene therapy in mice using a composite nonviral gene delivery system consisting of the piggyBac transposon and polyethylenimine (original) (raw)
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Cancer gene therapy, 2005
Transfer of the herpes simplex virus type I-thymidine kinase gene, followed by the administration of ganciclovir (HSV1-tk/GCV) into ovarian cancer-derived cell line either in vitro or transplanted into nude mice has been shown to provide a potential strategy for the gene therapy of ovarian cancer. We investigated the antitumor effects of HSV1-tk/GCV strategy with a chemically induced rat ovarian cancer model and a tumor-selective gene delivery by a novel nonviral gene delivery system (GE7) through the ovarian artery and tail vein. We demonstrated the expression of a reporter gene, beta-gal gene, as well as HSV1-tk gene in tumors and other organs, evaluated the overall antitumor effects after the GCV treatment and analyzed the tumor cell cycle phase distribution. Via the ovarian artery route, the expressions of beta-gal and HSV1-tk in tumors were significantly stronger than those expressed in such organs as the hearts, livers, spleens, lungs and kidneys. However, no beta-gal and HSV1...
Ovarian carcinoma cells are effectively transfected by polyethylenimine (PEI) derivatives
Cancer Gene Therapy, 2000
As a prerequisite to nonviral gene therapy approaches of ovarian carcinoma, we evaluated the possibility of transfecting established tumor cell lines (SKOV3, IGROV1) as well as primary mesothelial and tumor cells by various polyethylenimine (PEI) derivatives. Several PEI-based vectors were able to effectively transfect these cells, as shown by high luciferase expression levels (10 8 to 10 9 relative light units per milligram of cell protein) that corresponded with 25-50% of green fluorescent protein-positive cells after 24 hours. However, unpredictable differences were observed among the vectors and cell types that a posteriori justified the screening procedure. We also showed that cells that were not transfected after the first experiment remained transfectable in a subsequent transfection experiment to a level similar to that of the initial population. This experiment does not support the emergence of a transfection-resistant cell population and opens the door to multiple therapeutic gene deliveries. Although efficacy and cell targeting still remain to be improved, PEI derivatives appear to be promising molecules for the development of nonviral gene therapy of ovarian carcinoma. Cancer Gene Therapy (2000) 7, 644 -652
In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate
The FASEB Journal, 2000
To target disseminated tumors in vivo, transgenes [-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf--galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immunedeficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tu-In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate. FASEB
The Pharma Innovation Journal, 2021
Various gene expression studies based on transfection of DNA in eukaryotic cells requires suitable transfection method. After transfection procedure selection, there is need to optimize transfection efficiency which varies in each cell type depending on transfection conditions. There are few significant transfection conditions like number of cells seeded 24 hrs before transfection, cell confluence at the time of transfection, optimal transfection reagent/DNA concentration ratio, amount of media added etc., which are required to be optimized individually in each cell type to attain highest transfection efficiency. Transfection with chemical method by using cationic lipids like lipofectamine is commonly used transfection method. Reporter gene like GFP cloned in any vector can be used to optimize transfection for attaining maximum transfection efficiency. In our present study we optimized Lipofectamine 3000®/pVIVO1.GFP plasmid concentration ratio to attain highest transfection efficiency in 4T1 mice mammary tumor cells. Non structural gene of Canine Parvovirus is an oncolytic viral gene reported to be cytotoxic selectively to cancer cells without harming healthy cells. NS1 gene of CPV2 cloned in pVIVO1 vector is already available in our laboratory. To identify different oncolytic mechanisms of CPV2.NS1, there was need to transfect this gene in 4T1 cells and therefore we used GFP as reporter gene cloned in same vector of CPV2.NS1 (pVIVO1.GFP) so as to optimize for Lipofectamine/DNA ratio depicting highest transfection efficiency. The concentration of plasmid DNA (pVIVO1.GFP) was optimized to 3µg in 6 well plate with 6µl of Lipofectamine 3000® by our study. In this concentration of plasmid DNA, the 4T1 cells were showing highest transfection efficiency.
Polymer-based non-viral gene delivery as a concept for the treatment of cancer
Pharmacological Reports, 2009
Gene therapy has become a promising technique for the treatment of cancer. Nevertheless, the success of gene therapy depends on the effectiveness of the vector. The challenge of a gene carrier is to deliver exogenous DNA from the site of administration into the nucleus of the appropriate target cell. Polymer-based vectors are biologically safe, have low production costs and are efficient tools for gene therapy. Although non-degradable polyplexes exhibit high gene expression levels, their application potential is limited due to their inability to be effectively eliminated, which results in cytotoxicity. The development of biodegradable polymers has allowed for high levels of transfection without cytotoxicity. For site-specific targeting of polyplexes, further modifications, such as incorporation of ligands, can be performed. Most expectations have been addressed to polyplexes architecture according it dynamic response with the microenvironment.
Delivery of molecular cargoes in normal and cancer cell lines using non-viral delivery systems
Biotechnology Letters, 2018
Objective In this study, transfection efficiency of human papillomavirus (HPV) E7 DNA and protein constructs into HEK-293T normal cell line, and A549 and TC-1 tumor cell lines was evaluated by four delivery systems including supercharge GFP, hPP10 cell penetrating peptide, TurboFect and Lipofectamine using fluorescence microscopy and flow cytometry. Results The results indicated that Lipofectamine 2000 and TurboFect produced more effective transfection for GFP and E7-GFP DNA constructs in HEK-293T cells compared to in A549 and TC-1 cells (p \ 0.05). In contrast, the supercharge GFP was efficient for E7 DNA and E7 protein delivery in both normal cell (* 83.94 and * 77.01% for HEK-293T), and cancer cells (* 71.69 and * 67.19% for TC-1, and * 73.86 and * 67.49% for A549), respectively. Indeed, in these cell lines, transfection efficiency by ?36 GFP reached * 60-80%. Moreover, the hPP10 produced the best transfection result for E7-GFP protein in HEK-293T cells (* 63.66%) compared to TurboFect (* 32.95%); however, the efficiency level of hPP10 was only * 17.51 and * 16.36% in TC-1 and A549 cells. Conclusions Our data suggested that the supercharge GFP is the most suitable transfection vehicle for DNA and protein delivery into TC-1 and A549 tumor cell lines compared to other carriers.
Non-viral transfection methods optimized for gene delivery to a lung cancer cell line
Avicenna journal of medical biotechnology, 2013
Mehr-80 is a newly established adherent human large cell lung cancer cell line that has not been transfected until now. This study aims to define the optimal transfection conditions and effects of some critical elements for enhancing gene delivery to this cell line by utilizing different non-viral transfection Procedures. In the current study, calcium phosphate (CaP), DEAE-dextran, superfect, electroporation and lipofection transfection methods were used to optimize delivery of a plasmid construct that expressed Green Fluorescent Protein (GFP). Transgene expression was detected by fluorescent microscopy and flowcytometry. Toxicities of the methods were estimated by trypan blue staining. In order to evaluate the density of the transfected gene, we used a plasmid construct that expressed the Stromal cell-Derived Factor-1 (SDF-1) gene and measured its expression by real-time PCR. Mean levels of GFP-expressing cells 48 hr after transfection were 8.4% (CaP), 8.2% (DEAE-dextran), 4.9% (su...
Synthesis and application of a non-viral gene delivery system for immunogene therapy of cancer
Journal of Controlled Release, 2005
The synthesis and gene delivery application of a novel lipopolymer, PEG-PEI-CHOL (PPC), is described. PPC is composed of a low molecular weight branched polyethylenimine (PEI) covalently linked with functional groups methoxypolyethyleneglycol (PEG) and cholesterol (CHOL). The potential utility of PPC as a gene delivery polymer was evaluated by showing its ability to form stable nanocomplexes with DNA, protect DNA from degradation by DNase and mediate gene transfer in vitro and in vivo in solid tumors. The ratio of PEG/PEI/CHOL and nitrogen to phosphate (Polymer/DNA) was optimized for physico-chemical properties and gene delivery efficiency of PPC/DNA complexes. The gene therapy application of the polymer was shown following administration of a murine IL-12 plasmid (pmIL-12) formulated with PPC into tumors in mice which resulted in significant inhibition of tumor growth. The inhibitory effects of pmIL-12/PPC were enhanced when combined with specific chemotherapeutic agents, demonstrating the potential usefulness of pIL-12/PPC as an adjuvant therapy for cancer treatment.
Gene therapy progress and prospects: non-viral gene therapy by systemic delivery
Gene Therapy, 2006
Non-viral vectors continue to be an attractive alternative to viral vectors due to their safety, versatility and ease of preparation and scale-up. Over the past few years, investigators have been successful in developing gene carriers that can be targeted to the disease site. Several different delivery vectors for systemic use have been developed by different groups for plasmid DNA and oligonucleotide. Most of them are designed for targeted tumor therapy. The mechanism of inflammatory toxicity, the major toxicity of cationic lipoplex, has been studied and managed. In this review, we focus on the progress made over the last 2 years. We also discuss some future prospects for gene delivery.