• Journal of Photoscience
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• 제9권2호
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• pp.451-453
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• 2002

A new concept of "photo" -antisense method has been evaluated, where the inhibition of gene expression by the conventional antisense method is enhanced by photochemical binding between antisense oligonucleotides conjugated with photo-reactive compound and target mRNA or DNA. Fluorescein labeled oligodeoxyribonucleotides (F-DNA) was delivered to cell nuclei in the encapsulated form in multilamellar lecithin liposomes with neutral charge. F-DNA was previously shown to photo-bind to the complementary stranded DNA, and the delivery system using neutral liposome to be effective in normal human keratinocytes. In the present study, we used human kidney cancer G401.2/6TG.1 cell line to be advantageous in reproducible experiments. p53 was adopted as a target gene since antisense sequence information has been accumulated. The nuclear localization ofF-DNA was identified by comparing the fluorescence ofF-DNA with that of Hoechst 33258 under fluorescence microscope. After 7hr incubation to accumulate p53 protein induced by UV -B, p53 protein was quantified by Western blot. After 2hrs from F-DNA application, about 30% of cell population incorporated F-DNA in their nuclei with some morphological change possibly due to liposomal toxicity. Irradiation of visible light longer than 400nm from solar simulator at this time enhanced the inhibitory action of antisense F-DNA. The present results suggest that photo-antisense method is promising to control gene expression in time and space dependent manner. Further improvement of F-DNA delivery to cancer cells in the stability and toxicity is in progress. progress.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.109-115
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• 2002

Gene therapy is to treat and cure diseases by an introduction of therapeutic genes in defective cells or tissues of human body. Gene delivery system, gene expression system, and therapeutic gene are three core elements for gene therapy. The efficient delivery of therapeutic genes and appropriate gene expression are the crucial issues for therapeutic outcome of gene delivery. Because it can be used in common for the treatment and cure of various diseases, gene delivery system is the most important core element for a successful gene therapy. Viruses are naturally evolved to transfer their genomes into host cells efficiently. This ability has made vectorologists exploit viruses as attractive vehicles for the delivery of therapeutic genes. Viral vectors based on adenovirus (Ad) and adeno-associated virus (AAV) have been often used for gene delivery in laboratory. Ad and AAV vectors derived from human DNA viruses differ greatly in their life cycle, expression level and duration of transgenes, immunogenicity, and vector preparation. Both vectors can be used as effective tools for gene therapy and more recently in functional genomics. Here, the characteristics of Ad and AAV vectors are discussed.

• Journal of Microbiology and Biotechnology
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• 제22권6호
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• pp.866-871
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• 2012

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.

• BMB Reports
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• 제57권1호
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• pp.19-29
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• 2024

Mitochondrial DNA (mtDNA), a multicopy genome found in mitochondria, is crucial for oxidative phosphorylation. Mutations in mtDNA can lead to severe mitochondrial dysfunction in tissues and organs with high energy demand. MtDNA mutations are closely associated with mitochondrial and age-related disease. To better understand the functional role of mtDNA and work toward developing therapeutics, it is essential to advance technology that is capable of manipulating the mitochondrial genome. This review discusses ongoing efforts in mitochondrial genome editing with mtDNA nucleases and base editors, including the tools, delivery strategies, and applications. Future advances in mitochondrial genome editing to address challenges regarding their efficiency and specificity can achieve the promise of therapeutic genome editing.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XII)
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• pp.582-586
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• 2003

There has been interest in developing gene therapy based on naked plasmid DNA for treating serum protein deficiencies and human erythropoietin (hEPO) is one of the candidate for gene therapy being Investigated most enthusiastically. We constructed novel plasmid DNA vectors pVAC-hEPOI/II/III which contain one, two, three hEPO gene(s) respectively for producing high level expression and secretion of hEPO in vitro and in vivo. NIH3T3 and COS7 cells were transfected transiently with these vectors and increase in hEPO expression in medium reached 2-5 fold in comparison with pSecTagB-hEPO. Intra muscular administrations of pVAC-hEPOI/II/III vectors into mice resulted in high level secretion of hEPO in the serum and corresponding increases in hematocrit level. In conclusion the transduction efficiency of naked plasmid vectors is one of the critical factors of a gene delivery system and these novel plasmid vectors will contribute to various gene therapy based on naked plasmid DNA.

• Molecules and Cells
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• 제22권2호
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• pp.175-181
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• 2006

Delivery of DNA vaccines to airway mucosa would be an ideal method for mucosal immunization. However, there have been few reports of a suitable gene delivery system. In this study we used a cationic emulsion to immunize mice via the intranasal route with pCMV-S coding for Hepatitis B virus surface antigen (HBsAg). Complexing pCMV-S with a cationic emulsion dramatically enhanced HBsAg expression in both nasal tissue and lung, and was associated with increases in the levels of HBs-specific Abs in serum and mucosal fluids, of cytotoxic T lymphocytes (CTL) in the spleen and cervical and iliac lymph nodes, and of delayed-type hypersensitivity (DTH) against HBsAg. In contrast, very weak humoral and cellular immunities were observed following immunization with naked DNA. In support of these observations, a higher proliferative response of spleenocytes was detected in the group immunized with the emulsion/pCMV-S complex than in the group immunized with naked pCMV-S. These findings may facilitate development of an emulsion-mediated gene vaccination technique for use against intracellular pathogens that invade mucosal surfaces.

• 한국미생물학회:학술대회논문집
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• 한국미생물학회 2002년도 추계학술대회
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• pp.129-131
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• 2002

• Archives of Pharmacal Research
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• 제28권6호
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• pp.722-729
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• 2005

With the aim to improve the specificity and to reduce the cytotoxicity of polyethylenimine (PEI), we have synthesized the conjugates of the branched PEI (25 kDa) with transferrin. The trans-ferrin-PEI (TP) conjugates with five compositions were synthesized using periodate oxidation method and confirmed by FT-IR spectroscopy and gel permeation chromatography. The free amine contents of TP conjugates, which were able to condense and deliver DNA, increased as the amount of PEI increased. TP/DNA polyplexes were characterized by measuring gel elec-trophoresis, ethidium bromide fluorescence quenching, particle size and zeta potential of complexes. Complete complexation of the polyplexes was observed above the N/P ratio of 5 in TP/DNA, and above 3 in PEI/DNA, respectively. The zeta potential of the complexes decreased as the amount of transferrin in TP conjugates increased. Transfection efficiency of TP conjugates was evaluated in HeLa cell and Jurkat cell systems. Among the five compositions of TP conjugates, TP-2 system mediated a higher $\beta$-galactosidase gene expression than PEI system in Jurkat cell which was known to express elevated numbers of transferrin receptors. From the results of the cell viability based on MTT assay, TP conjugates showed lower cytotoxicity com-pared with the PEI system. We expect that the TP conjugate can be used efficiently as a non-viral gene delivery vector.

• Journal of Pharmaceutical Investigation
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• 제34권4호
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• pp.263-267
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• 2004

Branched and linear polyethylenimines (PEIs) have been studied as efficient and versatile agents for gene delivery in vitro and in vivo. PEIs exist in a linear or branched topology and are available in a wide range of molecular weight (Mw). Most studies have been done using PEIs with Mw higher than 10Kd. This study was aimed to test the transfection efficiency and the cell viability following gene delivery using PEI of Mw 2Kd, a relatively lower Mw cationic polymer. We used murine interleukin-2(mIL-2) plasmid DNA complexed with branched PEI 2Kd or 25Kd, and transfected them into a myoblast muscle cell line, C2C12. The cellular uptake of mIL-2 plasmid DNA was determined using quantitative polymerase chain reaction. RNA transcript levels were studied in the myoblast cells. Our results show that PEI 2Kd was as effective as PEI 25Kd in celluar gene delivery and transfection efficiency in C2C12 cells. Moreover, MTT assay indicated that PEI 2Kd/DNA complexes did not significantly reduce the cell viability regardless of N/P ratios. These results suggest that PEI of Mw 2Kd might play a role as effective and low toxic nonviral vector systems for muscular cell lines.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.591-599
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• 2008

A murine model immunized by systemic and mucosal delivery of plasmid DNA vaccine expressing glycoprotein B (pCIgB) of pseudorabies virus (PrV) was used to evaluate both the nature of the induced immunity and protection against a virulent virus. With regard to systemic delivery, the intramuscular (i.m.) immunization with pCIgB induced strong PrV-specific IgG responses in serum but was inefficient in generating a mucosal IgA response. Mucosal delivery through intranasal (i.n.) immunization of pCIgB induced both systemic and mucosal immunity at the distal mucosal site. However, the levels of systemic immunity induced by i.n. immunization were less than those induced by i.m. immunization. Moreover, i.n. genetic transfer of pCIgB appeared to induce Th2-biased immunity compared with systemic delivery, as judged by the ratio of PrV-specific IgG isotypes and Th1- and Th2-type cytokines produced by stimulated T cells. Moreover, the immunity induced by i.n. immunization did not provide effective protection against i.n. challenge of a virulent PrV strain, whereas i.m. immunization produced resistance to viral infection. Therefore, although i.n. immunization was a useful route for inducing mucosal immunity at the virus entry site, i.n. immunization did not provide effective protection against the lethal infection of PrV.