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Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

ABSTRACT: Transgenic genome integration using non-viral vehicles is a promising approach for gene therapy. Previous studies reported that asparagine is a key regulator of cancer cell amino acid homeostasis, anabolic metabolism and cell proliferation. The depletion of asparagine would inhibit the growth of many cancer cells. In this study, we develop a nanoparticle delivery system to permanently integrate the asparaginase gene into the genome of human lung adenocarcinoma cells. The asparaginase plasmid and the Sleeping Beauty plasmid were co-transfected using amine-functionalized mesoporous nanoparticles into the human lung adenocarcinoma cells. The intracellular asparaginase expression led to the cell cytotoxicity for PC9 and A549 cells. In addition, the combination of the chemotherapy and the asparaginase gene therapy additively enhanced the cell cytotoxicity of PC9 and A549 cells to 69% and 63%, respectively. Finally, we showed that the stable cell clones were successfully made by puromycin selection. The doxycycline-induced expression of asparaginase caused almost complete cell death of PC9 and A549 asparaginase-integrated stable cells. This work demonstrates that silica-based nanoparticles have great potential in gene delivery for therapeutic purposes.

SUBMITTER: Chang JH

PROVIDER: S-EPMC6686048 | biostudies-literature | 2019 Aug

REPOSITORIES: biostudies-literature

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Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

Chang Jen-Hsuan JH Mou Kurt Yun KY Mou Chung-Yuan CY

Scientific reports 20190807 1

Transgenic genome integration using non-viral vehicles is a promising approach for gene therapy. Previous studies reported that asparagine is a key regulator of cancer cell amino acid homeostasis, anabolic metabolism and cell proliferation. The depletion of asparagine would inhibit the growth of many cancer cells. In this study, we develop a nanoparticle delivery system to permanently integrate the asparaginase gene into the genome of human lung adenocarcinoma cells. The asparaginase plasmid and ...[more]

PMID: 31391525

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Project description:A Sleeping Beauty (SB) transposon system is made of a transposon plasmid (containing gene encoding a desired functional or therapeutic protein) and a transposase plasmid (encoding an enzyme capable of cutting and pasting the gene into the host cell genome). It is a kind of natural, nonviral gene delivery vehicle, which can achieve efficient genomic insertion, providing long-term transgenic expression. However, before the SB transposon system could play a role in promoting gene expression, it has to be delivered efficiently first across cell membrane and then into cell nuclei. Towards this end, we used a nanoparticle-like lipid-based protocell, a closed bilayer of the neutral lipids with the DNA encapsulated inside, to deliver the SB transposon system to cancer cells. The SB transposon system was amplified in situ inside the protocells by a polymerase chain reaction (PCR) process, realizing more efficient loading and delivery of the target gene. To reach a high transfection efficiency, we introduced two targeting moieties, folic acid (FA) as a cancer cell-targeting motif and Dexamethasone (DEX) as a nuclear localization signaling molecule, into the protocells. As a result, the FA enabled the modified targeting protocells to deliver the DNA into the cancer cells with an increased efficiency and the DEX promoted the DNA to translocate to cell nuclei, eventually leading to the increased chromosome insertion efficiency of the SB transposon. In vivo study strongly suggested that the transfection efficiency of FA-modified protocells in the tumor tissue was much higher than that in other tissues, which was consistent with the in vitro results. Our studies implied that with the targeting ligand modification, the protocells could be utilized as an efficient targeting gene carrier. Since the protocells were made of neutral lipids without cationic charges, the cytotoxicity of protocells was significantly lower than that of traditional cationic gene carriers such as cationic liposomes and polyethylenimine, enabling the protocells to be employed in a wider dosage range in gene therapy. Our work shows that the protocells are a promising gene carrier for future clinical applications.

Project description:Insertional mutagenesis techniques with transposable elements have been popular among geneticists studying model organisms from E. coli to Drosophila and, more recently, the mouse. One such element is the Sleeping Beauty (SB) transposon that has been shown in several studies to be an effective insertional mutagen in the mouse germline. SB transposon vector studies have employed different functional elements and reporter molecules to disrupt and report the expression of endogenous mouse genes. We sought to generate a transposon system that would be capable of reporting the expression pattern of a mouse gene while allowing for conditional expression of a gene of interest in a tissue- or temporal-specific pattern.Here we report the systematic development and testing of a transposon-based gene-trap system incorporating the doxycycline-repressible Tet-Off (tTA) system that is capable of activating the expression of genes under control of a Tet response element (TRE) promoter. We demonstrate that the gene trap system is fully functional in vitro by introducing the "gene-trap tTA" vector into human cells by transposition and identifying clones that activate expression of a TRE-luciferase transgene in a doxycycline-dependent manner. In transgenic mice, we mobilize gene-trap tTA vectors, discover parameters that can affect germline mobilization rates, and identify candidate gene insertions to demonstrate the in vivo functionality of the vector system. We further demonstrate that the gene-trap can act as a reporter of endogenous gene expression and it can be coupled with bioluminescent imaging to identify genes with tissue-specific expression patterns.Akin to the GAL4/UAS system used in the fly, we have made progress developing a tool for mutating and revealing the expression of mouse genes by generating the tTA transactivator in the presence of a secondary TRE-regulated reporter molecule. A vector like the gene-trap tTA could provide a means for both annotating mouse genes and creating a resource of mice that express a regulable transcription factor in temporally- and tissue-specific patterns for conditional gene expression studies. These mice would be a valuable resource to the mouse genetics community for purpose of dissecting mammalian gene function.

Dataset Information

Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

Publications

Sleeping Beauty Transposon-Mediated Asparaginase Gene Delivery by a Nanoparticle Platform.

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