• Journal of The Korean Society of Inherited Metabolic disease
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• v.5 no.1
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• pp.9-17
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• 2005

Homocystinuria is a metabolic disorder caused by a deficiency of cystathionine ${\beta}$-synthase (CBS). Patients with homocystinuria show clinical symptoms such as mental retardation, lens dislocation, vascular disease with life-threatening thromboembolisms and skeletal deformities. Generally, the major treatments for CBS deficiency include pharmacologic doses of pyridoxine or dietary restriction of methionine. However, there is no effective treatment for this disease up till today and gene therapy can be an attractive novel approach to treatment of the disease. We investigated whether a recombinant adeno-associated virus could be used as a CBS gene transfer vector to reduce the excessive homocysteine level in the homocystinuria mouse model. Recombinant adeno-associated virus vector encoding the human CBS gene (rAAV-hCBS), driven by EF1-a promoter, was infused into CBS-deficient mice ($CBS^{-/-}$) via intramuscular (IM) and intraperitoneal (IP) injection. IP injection was more efficient than IM injection for prolongation of lives and reduction of plasma homocysteine levels. After 2 weeks of gene transfer by IP injection, serum homocysteine level was significantly decreased in treated mice compared with the age-matched controls and the life span was extended about 1.5 times. Also, increased expression of CBS gene was observed by immunohistochemical staining in livers of treated $CBS^{-/-}$ mice and microvesicular lipid droplets was decreased in cytoplasm of liver. These results demonstrate the possibility and efficacy of gene therapy by AAV gene transfer in homocystinuria mice.

• Journal of Veterinary Science
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• v.22 no.1
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• pp.8.1-8.11
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• 2021

Background: Porcine circovirus type 2 (PCV2) is an important infectious pathogen implicated in porcine circovirus-associated diseases (PCVAD), which has caused significant economic losses in the pig industry worldwide. Objectives: A suitable viral vector-mediated gene transfer platform for the expression of the capsid protein (Cap) is an attractive strategy. Methods: In the present study, a recombinant adeno-associated virus 8 (rAAV8) vector was constructed to encode Cap (Cap-rAAV) in vitro and in vivo after gene transfer. Results: The obtained results showed that Cap could be expressed in HEK293T cells and BABL/c mice. The results of lymphocytes proliferative, as well as immunoglobulin G (IgG) 2a and interferon-γ showed strong cellular immune responses induced by Cap-rAAV. The enzyme-linked immunosorbent assay titers obtained and the IgG1 and interleukin-4 levels showed that humoral immune responses were also induced by Cap-rAAV. Altogether, these results demonstrated that the rAAV8 vaccine Cap-rAAV can induce strong cellular and humoral immune responses, indicating a potential rAAV8 vaccine against PCV2. Conclusions: The injection of rAAV8 encoding PCV2 Cap genes into muscle tissue can ensure long-term, continuous, and systemic expression.

• KSBB Journal
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• v.16 no.6
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• pp.579-591
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• 2001

The cell growth inhibitor effect of cervical cancer cells was investigated by liposome mediated transfection (pRcCMVp53/lipofectin) and by transfection using adenovirus (AdCMVp57). The papilloma virus cancer cell lines we used in this study were HPV16 positive, having inhibiter gene, wild p53 gene, CaSki, SiHa, HPV18 positive HeLa, HeLaS3 and HPV negative C33A, HT3. LacZ gene of E.coli was used as the marker gene for the transfection efficiency. The effect on the inhibition of tumor cell growth was measured by cell count and cell viability though ELISA analysis and MTT assay. The inhibition of tumor cell growth was confirmed by measuring each assay for six days, comparing with the normal control cell growth. The cell growth of cervical cancer calls by transfection was significantly reduced and showed tittle differences among the cell lines. To eliminate the potential problem of Ad(adenovirus) contamination during rAAV production, rAAV can be produced by a triple transfection of vector plasmic, packaging plasmid, and adenovirus helper plasmid. To examine the helper functions of Ad plasmids on the production of rAAV vector, we carried out cotransfection of three plasmids, AAV vector, packaging construct, and Ad helper plasmids. The optimized transfection condition for calcium phosphate method is 25ug of total DNA per 10-cm-diameter plate of 293 cell. We found that rAAV yields peaked at 48hr after Ad infection. The titer of rAAV was measured by the dot blot analysis to measure the number of particles/ml based on the quantification of viral DNA. Recent1y, Kombucha(fungi) was identified as a very potent antileukefic agent. In the present study, effect of natural toxin(plankton) and Kombucha is PSP(GTXI-3, neoSTX), on various MTT assay cervical cancer cell line. Toxin(GTX 1-3, neoSTX) also inhibited the proliferation in primary cervical cancer calls in a dose-dependent toxin concentration. These results showed that toxin was very potent in inhibiting the proliferation of cervical cancer calls in vitro. Toxins and Kombuoha exhibited a dose dependent inhibition of cellular proliferation in cancer cell line.

• Journal of Korean Neurosurgical Society
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• v.63 no.5
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• pp.579-589
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• 2020

Objective : No optimum genetic rat Huntington model both neuropathological using an adeno-associated virus (AAV-2) vector vector has been reported to date. We investigated whether direct infection of an AAV2 encoding a fragment of mutant huntingtin (AV2-82Q) into the rat striatum was useful for optimizing the Huntington rat model. Methods : We prepared ten unilateral models by injecting AAV2-82Q into the right striatum, as well as ten bilateral models. In each group, five rats were assigned to either the 2×10¹² genome copies (GC)/mL of AAV2-82Q (×1, low dose) or 2×10¹³ GC/mL of AAV2-82Q (×10, high dose) injection model. Ten unilateral and ten bilateral models injected with AAV-empty were also prepared as control groups. We performed cylinder and stepping tests 2, 4, 6, and 8 weeks after injection, tested EM48 positive mutant huntingtin aggregates. Results : The high dose of unilateral and bilateral AAV2-82Q model showed a greater decrease in performance on the stepping and cylinder tests. We also observed more prominent EM48-positive mutant huntingtin aggregates in the medium spiny neurons of the high dose of AAV2-82Q injected group. Conclusion : Based on the results from the present study, high dose of AAV2-82Q is the optimum titer for establishing a Huntington rat model. Delivery of high dose of human AAV2-82Q resulted in the manifestation of Huntington behaviors and optimum expression of the huntingtin protein in vivo.

• Proceedings of the Microbiological Society of Korea Conference
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• 2002.10a
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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.

• BMB Reports
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• v.42 no.1
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• pp.59-64
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• 2009

Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by $87{\pm}4$, $80.3{\pm}2.6$ and $86.2{\pm}7%$ respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance.

• Journal of Embryo Transfer
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• v.25 no.1
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• pp.35-42
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• 2010

Many studies propose that dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I) is associated with neurodegenerative disorders, such as Parkinson's disease and Huntington's disease. Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. With a recombinant adeno-associated virus vector carrying the NDI1 gene (rAAV-NDI1) as the gene delivery method, we were able to attain high transduction efficiencies even in the human epithelial cervical cancer cells that are difficult to transfect by lipofection or calcium phosphate precipitation methods. Using a rAAV-NDI1, we demonstrated that the Ndi1 enzyme is successfully expressed in HeLa cells. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced HeLa cells were not affected by rotenone which is inhibitor of complex I, but was inhibited by flavone and antimycin A. The NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. In particular, in the NDI1-transduced cells, the yeast enzyme becomes integrated into the human respiratory chain. It is concluded that the NDI1 gene provides a potentially useful tool for gene therapy of mitochondrial diseases caused by complex I deficiency.

• Reproductive and Developmental Biology
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• v.35 no.4
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• pp.427-434
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• 2011

Mitochondria diseases have been reported to involve structural and functional defects of complex I-V. Especially, many of these diseases are known to be related to dysfunction of mitochondrial proton-translocating NADH-ubiquinone oxidoreductase (complex I). The dysfunction of mitochondria complex I is associated with neurodegenerative disorders, such as Parkinson's disease, Huntington's disease, and Leber's hereditary optic neuropathy (LHON). Mammalian mitochondrial proton-translocating NADH-quinone oxidoreductase (complex I) is largest and consists of at least 46 different subunits. In contrast, the NDI1 gene of Saccharomyces cerevisiae is a single subunit rotenone-insensitive NADH-quinone oxidoreductase that is located on the matrix side of the inner mitochondrial membrane. The Saccharomyces cerevisiae NDI1 gene using a recombinant adeno-associated virus vector (rAAV-NDI1) was successfully expressed in AML12 mouse liver hepatocytes and the NDI1-transduced cells were able to grow in media containing rotenone. In contrast, control cells that did not receive the NDI1 gene failed to survive. The expressed Ndi1 enzyme was recognized to be localized in mitochondria by confocal immunofluorescence microscopic analyses and immunoblotting. Using digitonin-permeabilized cells, it was shown that the NADH oxidase activity of the NDI1-transduced cells was not affected by rotenone which is inhibitor of complex I, but was inhibited by antimycin A. Furthermore, these results indicate that Ndi1 can be functionally expressed in the AML12 mouse liver hepatocytes. It is conceivable that the NDI1 gene is powerful tool for gene therapy of mitochondrial diseases caused by complex I deficiency. In the future, we will attempt to functionally express the NDI1 gene in mouse embryonic stem (mES) cell.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.8
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• pp.4031-4036
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• 2012

Background: The negative signaling provided by interactions of the co-inhibitory molecule, programmed death-1 (PD-1), and its ligands, B7-H1 (PD-L1) and B7-DC (PD-L2), is a critical mechanism contributing to tumor evasion; blockade of this pathway has been proven to enhance cytotoxic activity and mediate antitumor therapy. Here we evaluated the anti-tumor efficacy of AAV-mediated delivery of the extracellular domain of murine PD-1 (sPD-1) to a tumor site. Material and Methods: An rAAV vector was constructed in which the expression of sPD-1, a known negative regulator of TCR signals, is driven by human cytomegalovirus immediate early promoter (CMV-P), using a triple plasmid transfection system. Tumor-bearing mice were then treated with the AAV/sPD1 construct and expression of sPD-1 in tumor tissues was determined by semi quantitative RT-PCR, and tumor weights and cytotoxic activity of splenocytes were measured. Results: Analysis of tumor homogenates revealed sPD-1 mRNA to be significantly overexpressed in rAAV/sPD-1 treated mice as compared with control levels. Its use for local gene therapy at the inoculation site of H22 hepatoma cells could inhibit tumor growth, also enhancing lysis of tumor cells by lymphocytes stimulated specifically with an antigen. In addition, PD-1 was also found expressed on the surfaces of activated CD8+ T cells. Conclusion: This study confirmed that expression of the soluble extracellular domain of PD-1 molecule could reduce tumor microenvironment inhibitory effects on T cells and enhance cytotoxicity. This suggests that it might be a potential target for development of therapies to augment T-cell responses in patients with malignancies.