• Tuberculosis and Respiratory Diseases
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• v.46 no.2
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• pp.229-240
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• 1999

Background: The impact of the immune response on cancer gene therapy using viral vectors to deliver a "suicide gene" is currently unclear. A vigrous immune response targeted at viral proteins or transgene may enhance the efficacy of tumor destruction and even augment responses to tumor antigens. These responses may involve the release of cytokines and stimulation of tumor specific cytotoxic T-lymphocytes that enhance therapeutic efficacy. On the other hand, a vigorous rapid cellular immune response may destroy cells expressing the therapeutic gene and attenuate the response to therapy. Furthermore, development of neutralizing antibody responses may prevent readministration of virus, a potentially significant limitation. Evaluating the significance of these limitations in animal models and developing solutions are therefore of obvious importance. Methods: After retroviral transduction of mouse mesothelioma cell line(AB12) with Herpes Simplex Virus thymidine kinase (HSVtk) gene in vitro, subcutaneous flank tumors were established. To study the effect of intact immune system on efficacy of tumor erradication, the ability of the HSVtk/ganciclovir system to inhibit tumor growth was compared among normal Balb/c mice, immunodeficient Balb/c-nude and SCID mice, and Balb/c mice immunosuppressed with cyclosporin. Results: Ganciclovir treatment resulted in greater inhibition of tumor growth in Balb/c mice compared with immunodeficient Balb/c-nude mice and SCID mice(in immunodeficient mice, there were no growth inhibition by ganciclovir treatment). Ganciclovir treatment resulted in greater inhibition of tumor growth in noncyclosporin (CSA) treated Balb/c mice compared with CSA treated Balb/c mice. On day 8, mean ganciclovir-treated tumor volume were 65% of control tumor volume in Balb/c mice versus 77% control tumor volume in CSA-treated Balb/c mice. This effect was still evident during therapy (day 11 and 13). On day 13, non-CSA treated tumor volume was 35% of control tumor volume versus 60% of control tumor volume in CSA treated Balb/c mice. Duration of expression of HSVtk was not affected by the immunosuppression with CSA. Conclusion: These results indicate that the immune responses against retrovirally transduced cells enhance the efficacy of the HSVtk/ganciclovir system. These findings have important implications for clinical trials using currently available retrovirus vectors as well as for future vector design.

• Reproductive and Developmental Biology
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• v.29 no.1
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• pp.57-62
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• 2005

One of the critical problems to be solved in transgenic animal production is uncontrollable constitutive expression of foreign genes, which usually results in serious physiological disturbances in the transgenic animal. To circumvent this problem, we constructed and tested two retrovirus vectors designed to express the GFP(green fluorescent protein) gene under the control of the tetracycline-inducible promoters. To maximize the GFP gene expression at turn-on state, WPRE(woodchuck hepatitis virus posttranscriptional regulatory element) sequence was introduced into the retrovirus vectors at downstream region of either the GFP gene or the sequence encoding rtTA(reverse tetracycline-controlled transactivator). Transformed cells were cultured in the medium supplemented with or without doxycycline(tetracycline derivative) for 48 hours, and induction efficiency was measured by comparing the GFP gene expression level using fluorometry and western blotting. Higher GFP expression was observed from the vector carrying the WPRE sequence at 3' side of the GFP gene, while tighter expression control(up to 20 fold) was obtained from the vector in which the WPRE sequence was placed at 3' side of rtTA sequence. The resulting tetracycline inducible vector system may be used in transgenic animal production and gene therapy.

• Korean Journal of Animal Reproduction
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• v.27 no.3
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• pp.259-268
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• 2003

Lactadherin (formerly known as BA46), a major glycoprotein of the human milk fat globule membrane, is abundant in human breast milk and breast carcinoma cells and is known to prevent symptomatic rotavirus infections. In this study, we tried to transfer the human lactadherin gene to the Chinese Hamster Ovary (CHO) cells using retrovirus vector system and tested inducible expression of the gene under the tetracycline-controllable promoter. At first, tetracycline-mediated inducibility was tested using E.coli LacZ marker gene. NIH3T3 cells co-infected with RevTet-On and RevTRE-LacZ retrovirus vectors showed that the cells responded to doxycycline (a derivative of tetracycline) in a dose-dependent manner, and prominent induction of the lacZ gene expression was observed from 1 $\mu\textrm{g}$/ml of doxycycline concentration. Based on the results of the pilot experiment, inductional expression of the human lactadherin gene was conducted using RevTet-On and RevTRE-Ltd retrovirus vectors. Analysis with the RT-PCR demonstrated successful inductional expression of the lactadherin gene in the Chinese Hamster Ovary (CHO) cells. Considering that constitutive overexpression of the exogenous genes in the target cells causes serious physiological imbalance, the results obtained in this study will be very useful especially in the studies of gene therapy and transgenic animal production.

• Tuberculosis and Respiratory Diseases
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• v.44 no.1
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• pp.162-174
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• 1997

Background : Metabolic cooperation via gap junctional intercellular communication (GJIC) is an important mechanism of the bystander effect in gene therapy using the Herpes Simplex Virus thymidine kinase/ganciclovir (HSVtk) "prodrug" system. Since retinoids have been reported to increase GJIC by induction of connexin 43 expression, we hyporthesized that treatment of tumor cells with retinoic acid could augment the bystander effect of the HSVtk/GCV system and result in improved tumor cell killing by enhancing GJIC. Methods : We transferred HSVtk gene to SKHep-J cell line that does not express connexin43, and also transferred the gene to human and murine mesothelioma cell lines that express connexin43. We verified that retinoic acid enhanced GJIC utilizing a functional double-dye transfer study and evaluated the effects of retinoic acid on the growth rate of tumor cells. We then tested the effects of retinoic acid on bystander-mediated cell killing. Results : Addition of all-trans retinoic acid (RA) increased GJIC in cell lines expressing connexin 43 and was asspciated with more efficient in vitro bystander killing in cells transduced with HSVtk via adenoviral and retroviral vectors. In contrast, there was no increase in the efficiency of the bystander effect after exposure to RA in a cell line which had no delectable connexin 43. Conclusion : These results provide evidence that retinoids can augment the efficiency of cell killing with the HSVtk/GCV system by enhancing bystander effect and may thus be a promising new approach to improve responses in gene therapy utilizing the HSVtk system to treat tumors.

• Korean Journal of Microbiology
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• v.46 no.1
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• pp.15-20
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• 2010

Xenotransplantation of pig organs is complicated by the existence of polytropic replication-competent porcine endogenous retroviruses (PERV) capable of infecting human cells. Two classes of infectious human-tropic replication-competent PERVs (PERV-A and PERV-B) and one class of ecotropic PERV-C are known. The potential for recombination between ecotropic PERV-C and human-tropic PERVs adds another level of infectious risk. A recombinant PERV-A/C (PERV-A14/220) virus is 500-fold more infectious than PERV-A. Two determinants of this high infectivity was identified; one was isoleucine-to-valine substitution at position 140 in RBD (receptor binding domain), and the other lies within the PRR (proline rich region) of the envelope protein. To examine whether the effects of the cytoplasmic tail of the PERV-C Env on fusogenesity also influences infectivity, we constructed a pseudotype retroviral vectors containing MoMLV core protein and PERV envelopes. Pseudotyping experiments with the PERV envelope glycoproteins indicated that recombinant PERV-A/C virus is 10-fold more infectious than PERV-A by lacZ staining. This result supports the suggestion that viral transduction of PERV-A/C is enhanced by a membrane-proximal cytoplasmic amphiphilic ${\alpha}$-helix in PERV-C Env tail.