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Abstract
Human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV), and herpes simplex virus (HSV) have been incurable to date because effective antiviral therapies target only replicating viruses and do not eradicate latently integrated or nonreplicating episomal viral genomes. Endonucleases that can target and cleave critical regions within latent viral genomes are currently in development. These enzymes are being engineered with high specificity such that off-target binding of cellular DNA will be absent or minimal. Imprecise nonhomologous-end-joining (NHEJ) DNA repair following repeated cleavage at the same critical site may permanently disrupt translation of essential viral proteins. We discuss the benefits and drawbacks of three types of DNA cleavage enzymes (zinc finger endonucleases, transcription activator-like [TAL] effector nucleases [TALENs], and homing endonucleases [also called meganucleases]), the development of delivery vectors for these enzymes, and potential obstacles for successful treatment of chronic viral infections. We then review issues regarding persistence of HIV-1, HBV, and HSV that are relevant to eradication with genome-altering approaches.
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Vanlandschoot P, Stortelers C, Beirnaert E, Ibañez LI, Schepens B, Depla E, Saelens X. Nanobodies®: New ammunition to battle viruses. Antiviral Res 2011; 92:389-407. [DOI: 10.1016/j.antiviral.2011.09.002] [Citation(s) in RCA: 83] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2011] [Revised: 08/30/2011] [Accepted: 09/06/2011] [Indexed: 01/23/2023]
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Qing Y, Chen M, Zhao J, Hu H, Xu H, Ling N, Peng M, Ren H. Construction of an HBV DNA vaccine by fusion of the GM-CSF gene to the HBV-S gene and examination of its immune effects in normal and HBV-transgenic mice. Vaccine 2010; 28:4301-7. [PMID: 20430121 DOI: 10.1016/j.vaccine.2010.04.023] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2009] [Revised: 03/30/2010] [Accepted: 04/13/2010] [Indexed: 01/04/2023]
Abstract
BACKGROUND The hepatitis B virus (HBV) DNA vaccine can generate both HBsAg-specific humoral and cellular immune responses. The immune response can be improved by inclusion of an adjuvant, such as the cytokine GM-CSF which is known to be a very good adjuvant. METHODS To investigate the ability of GM-CSF to enhance HBV-DNA vaccines, we constructed the plasmids by fusion of GM-CSF gene to the HBV-S gene. Normal and HBV-transgenic mice were then immunized with these plasmids. RESULTS Our results show that pCDNA3.1-GM-CSF-S induced the most powerful HBsAg-specific humoral and cellular immune response, and that it was able to overcome the non-response to HBsAg in HBV-transgenic mice. In contrast, pCDNA3.1-S-GM-CSF was able to induce only a very poor immune response. CONCLUSIONS When the HBV-S gene is fused to the GM-CSF gene, the immune effects of the HBV DNA vaccine both in normal and HBV-transgenic mice can be strengthened and HBV-DNA plasmids fused with GM-CSF may be useful for both preventative and therapeutic purposes.
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Affiliation(s)
- Yuling Qing
- Key Laboratory of Molecular Biology for Infectious Diseases, Ministry of Education, Institute for Viral Hepatitis, Second Affiliated Hospital, Chongqing Medical University, Chongqing 400010, China
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Serruys B, Van Houtte F, Farhoudi-Moghadam A, Leroux-Roels G, Vanlandschoot P. Production, characterization and in vitro testing of HBcAg-specific VHH intrabodies. J Gen Virol 2009; 91:643-52. [PMID: 19889923 DOI: 10.1099/vir.0.016063-0] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022] Open
Abstract
Hepatitis B virus (HBV) infections represent a global health problem, since these account for 350 million chronic infections worldwide that result in 500,000-700,000 deaths each year. Control of viral replication and HBV-related disease and mortality are of utmost importance. Because the currently available antiviral therapies all have major limitations, new strategies to treat chronic HBV infection are eagerly awaited. Six single-domain antibodies (VHHs) targeting the core antigen of HBV (HBcAg) have been generated and three of these bound strongly to HBcAg of both subtype ayw and adw. These three VHHs were studied as intrabodies directed towards the nucleus or the cytoplasm of a hepatoma cell line that was co-transfected with HBV. A speckled staining of HBcAg was observed in the cytoplasm of cells transfected with nucleotropic VHH intrabodies. Moreover, an increased intracellular accumulation of hepatitis B e antigen (HBeAg) and a complete disappearance of intracellular HBcAg signal were observed with nuclear targeted HBcAg-specific VHHs. These results suggest that HBcAg-specific VHHs targeted to the nucleus affect HBcAg and HBeAg expression and trafficking in HBV-transfected hepatocytes.
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Affiliation(s)
- Benedikte Serruys
- Center for Vaccinology, Ghent University and Hospital, Ghent, Belgium
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Tang H, Liu L, Liu FJ, Chen EQ, Murakami S, Lin Y, He F, Zhou TY, Huang FJ. Establishment of cell lines using a doxycycline-inducible gene expression system to regulate expression of hepatitis B virus X protein. Arch Virol 2009; 154:1021-1026. [PMID: 19495936 DOI: 10.1007/s00705-009-0402-0] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/10/2008] [Accepted: 05/14/2009] [Indexed: 12/31/2022]
Abstract
The hepatitis B virus (HBV) X gene plays an important role in HBV-associated pathogenesis, especially hepatocarcinogenesis. Establishment of a stable and regulable HBx expression system will allow study of the function of this gene. Here, we describe the development of a doxycycline-inducible recombinant plasmid (pBPSTR3-FlagX) with the full-length HBV X gene and all components of the tetracycline-on ("Tet-on") gene expression system. This vector exhibited dose-dependent doxycycline-dependent induction of the Flag-HBx protein in HepG2 and Hep3B cells. We also observed dose-dependent doxycycline transactivation of HBx in HepG2 cells. After transfecting HepG2 cells with the pBPSTR3-FlagX plasmid, we isolated five puromycin-resistant cell clones with stable HBx expression, two of which exhibited stable and tight control of HBx expression by doxycycline. This new system has great potential for functional studies of the HBV X gene.
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Affiliation(s)
- Hong Tang
- Center of Infectious Diseases, West China Hospital of Sichuan University, Chengdu, People's Republic of China
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Gonzalez-Aseguinolaza G, Crettaz J, Ochoa L, Otano I, Aldabe R, Paneda A. Gene therapy for viral hepatitis. Expert Opin Biol Ther 2006; 6:1263-78. [PMID: 17223736 DOI: 10.1517/14712598.6.12.1263] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022]
Abstract
Hepatitis B and C infections are two of the most prevalent viral diseases in the world. Existing therapies against chronic viral hepatitis are far from satisfactory due to low response rates, undesirable side effects and selection of resistant viral strains. Therefore, new therapeutic approaches are urgently needed. This review, after briefly summarising the in vitro and in vivo systems for the study of both diseases and the genetic vehicles commonly used for liver gene transfer, examines the existing status of gene therapy-based antiviral strategies that have been employed to prevent, eliminate or reduce viral infection. In particular, the authors focus on the results obtained in clinical trials and experimental clinically relevant animal models.
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MESH Headings
- Animals
- Disease Models, Animal
- Genetic Therapy/methods
- Genetic Therapy/trends
- Hepatitis B, Chronic/genetics
- Hepatitis B, Chronic/prevention & control
- Hepatitis C, Chronic/genetics
- Hepatitis C, Chronic/prevention & control
- Hepatitis, Viral, Animal/genetics
- Hepatitis, Viral, Animal/prevention & control
- Hepatitis, Viral, Human/genetics
- Hepatitis, Viral, Human/prevention & control
- Humans
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Affiliation(s)
- Gloria Gonzalez-Aseguinolaza
- University of Navarra, Division of Gene Therapy and Hepatology, Center for Applied Medical Research (CIMA), 31008 Pamplona, Spain.
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Zhang Y, Rong Qi X, Gao Y, Wei L, Maitani Y, Nagai T. Mechanisms of co-modified liver-targeting liposomes as gene delivery carriers based on cellular uptake and antigens inhibition effect. J Control Release 2006; 117:281-90. [PMID: 17196291 DOI: 10.1016/j.jconrel.2006.11.006] [Citation(s) in RCA: 36] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/26/2006] [Revised: 09/04/2006] [Accepted: 11/09/2006] [Indexed: 11/23/2022]
Abstract
In order to deliver antisense oligonucleotides (asODN) into hepatocytes orientedly in the treatment of hepatitis B virus (HBV) infection, the liver-targeting cationic liposomes was developed as a gene carrier, which was co-modified with the ligand of the asialoglycoprotein receptor (ASGPR), beta-sitosterol-beta-d-glucoside (sito-G) and the nonionic surfactant, Brij 35. Flow cytometry (FCM) analysis and enzyme-linked immunosorbent assay (ELISA) showed that the asODN-encapsulating cationic liposomes exhibited high transfection efficiency and strong antigens inhibition effect in primary rat hepatocytes and HepG2.2.15 cells, respectively. With the help of several inhibitors acting on different steps during the targeting lipofection, the cellular uptake mechanisms of the co-modified liver-targeting cationic liposomes were investigated through antigens inhibition effect assay and confocal laser scanning microscopy (CLSM) analysis. The cellular uptake with high transfection efficiency seemed to involve both endocytosis and membrane fusion. The ligand sito-G was confirmed to be able to enhance ASGPR-mediated endocytosis, the nonionic surfactant Brij 35 seemed to be able to facilitate membrane fusion, and the co-modification resulted in the most efficient transfection but no enhanced cytotoxicity. These results suggested that the co-modified liver-targeting cationic liposomes would be a specific and effective carrier to transfer asODN into hepatocytes infected with HBV orientedly.
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Affiliation(s)
- Yuan Zhang
- School of Pharmaceutical Sciences, Peking University, Beijing, China
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Khan AU. Ribozyme: A clinical tool. Clin Chim Acta 2006; 367:20-7. [PMID: 16426595 DOI: 10.1016/j.cca.2005.11.023] [Citation(s) in RCA: 32] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2005] [Revised: 11/18/2005] [Accepted: 11/22/2005] [Indexed: 01/15/2023]
Abstract
Catalytic RNAs (ribozymes) are capable of specifically cleaving RNA molecules, a property that enables them to act as potential antiviral and anti-cancer agents, as well as powerful tools for functional genomic studies. Recently, ribozymes have been used successfully to inhibit gene expression in a variety of biological systems in vitro and in vivo. Phase I clinical trials using ribozyme gene therapy to treat AIDS patients have been conducted. Despite initial success, there are many areas that require further investigation. These include stability of ribozymes in cells and designing highly active ribozymes in vivo, identification of target sequence sites and co-localization of ribozymes and substrates, and their delivery to specific tissues and maintenance of its stable long-term expression. This review gives a brief introduction to ribozyme structure, catalysis and its potential applications in biological systems as therapeutic agents.
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Affiliation(s)
- Asad U Khan
- Interdisciplinary Biotechnology unit, Aligarh Muslim University, Aligarh 202002, India.
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Zhao YG, Peng B, Deng H, Chen G, Yang F, Shao M, Lu H, Li Y, Peng J, Xu L, Xu Y. Anti-HBV immune responses in rhesus macaques elicited by electroporation mediated DNA vaccination. Vaccine 2006; 24:897-903. [PMID: 16253404 DOI: 10.1016/j.vaccine.2005.08.093] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2005] [Revised: 05/25/2005] [Accepted: 08/25/2005] [Indexed: 11/27/2022]
Abstract
Electroporation has been shown to be an effective method to improve the efficiency of gene expression and the immunogenicity of DNA vaccines. In order to optimize the procedure and test for its efficacy in more clinically relevant large animal models, we examined the detailed immune responses in rhesus macaques after vaccination intramuscularly with electroporation using the plasmid encoding for HBV preS(2)-S antigen and an adjuvant plasmid encoding for hIL-2 and hIFN-gamma. Several important factors were examined, including the dose response relationships, the effect of various prime and boost regimens, and different combinations of electro-pulse parameters. The immune responses were closely followed for more than a year. The results showed that in rhesus macaques, electroporation can significantly enhance the immunogenicity of the DNA vaccines, resulting in greatly improved antibody responses as well as peptide-stimulated IFN-gamma producing T cell responses. In addition, we also reported the different antibody response behaviors resulted from different electro-pulse parameters. The detailed data would be useful to suggest possible optimization strategies for better DNA vaccine efficacy.
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Affiliation(s)
- Yong-Gang Zhao
- School of Life Science and Biotechnology, Shanghai Jiao Tong University, Shanghai, PR China
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Lu YY, Cheng J, Yang YP, Liu Y, Wang L, Li K, Zhang LX. Cloning and characterization of a novel hepatitis B virus core binding protein C12. World J Gastroenterol 2005; 11:5666-71. [PMID: 16237762 PMCID: PMC4481485 DOI: 10.3748/wjg.v11.i36.5666] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To elucidate the biological function of HBV core antigen (HBcAg) on pathogenesis of hepatitis B, a novel gene C12 coding for protein with unknown function interacting with HBcAg in hepatocytes was identified and characterized.
METHODS: HBcAg bait plasmid pGBKT7-HBcAg was constructed and transformed into yeast AH109, then the transformed yeast was mated with yeast Y187 containing liver complementary DNA (cDNA) library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α -gal for screening twice. After extracting and sequencing of plasmid from blue colonies, we isolated a cDNA clone encoding a novel protein designated as C12 that directly interacted with HBcAg. The interaction between HBcAg and C12 was verified again by re-mating. pEGFP-N1-C12 fluorescent protein fusion gene was transfected in 293 and L02 cell, and observed by fluorescent microscope. MTT reduction assay was used to study the action of C12 protein effect on metabolism of mammal cell. Yeast two-hybrid and cDNA microarray were performed to search binding protein and differential expression genes regulated by C12 protein.
RESULTS: C12 gene was screened and identified by yeast two-hybrid system 3. The interaction between HBcAg and the novel protein coded by the new gene C12 was further confirmed by re-mating. After 48 h, fluorescence of fusion protein could be observed steadily in the 293 and L02 cell plasma. Under MTT assay, we found that the expression of C12 did not influence the growth of liver cells. Seventeen differential expression genes in HepG2 cells transfected with C12 protein expression plasmid by cDNA microarray, of which 16 genes were upregulated and 1 gene was downregulated by C12 protein. Twenty-one colonies containing 16 different genes coding for C12 protein binding proteins were isolated by yeast two-hybrid, there were 2 new genes with unknown function.
CONCLUSION: The novel protein C12 is located in cell plasma, and its overexpression has no significant effect on the metabolism of liver cell. It interacts with many proteins in hepatocytes and may be involved in many processes of gene expression.
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Affiliation(s)
- Yin-Ying Lu
- Gene Therapy Research Center, Institute of Infectious Diseases, The 302 Hospital of PLA, 100 Xisihuanzhong Road, Beijing 100039, China
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Abstract
Multicellular organisms have evolved under relentless attacks from pathogens, and as a consequence have spiked their genomes with numerous genes that serve to thwart these threats, notably through the building of the innate and adaptive arms of the immune system. The innate immune system is by far the most ancient, being found as widely as in plants and Drosophila, while adaptive immunity arose with the emergence of cartilaginous fishes. Innate immunity enters rapidly into the game during the course of an infection and generally involves the recognition by specific cellular receptors of common pathogen-associated patterns to elicit broad defensive responses, mediated in humans by interferons, macrophages, and natural killer cells, amongst others. When innate immunity fails to eradicate the infection quickly, adaptive immune responses enter into play, to generate exquisitely specific defenses to virtually any pathogen, thanks to a quasi-infinite repertoire of nonself receptors and effectors. A specific form of innate immunity, coined "intrinsic immunity," completes this protection by providing a constant, always-on, line of defense, generally through intracellular obstacles to the replication of pathogens. This component of the immune system has gained much attention as it was discovered that it is a cornerstone of the resistance of mammals against retroviruses. One of these newly discovered intracellular molecular weapons, the APOBEC family of proteins, is active against several classes of retroelements. We present here the current state of knowledge on this rapidly evolving field and discuss implications for gene therapy.
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Affiliation(s)
- Bastien Mangeat
- School of Life Sciences and Frontiers in Genetics National Center for Competence in Research, Swiss Federal Institute of Technology, Lausanne, Switzerland
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12
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Mangeat B, Trono D. Lentiviral Vectors and Antiretroviral Intrinsic Immunity. Hum Gene Ther 2005. [DOI: 10.1089/hum.2005.16.ft-101] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
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Pan WH, Xin P, Morrey JD, Clawson GA. A self-processing ribozyme cassette: utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus. Mol Ther 2004; 9:596-606. [PMID: 15093190 DOI: 10.1016/j.ymthe.2003.12.013] [Citation(s) in RCA: 25] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2003] [Accepted: 12/26/2003] [Indexed: 12/29/2022] Open
Abstract
We have been developing a self-processing triple-ribozyme cassette, which consists of two cis-acting hammerhead ribozymes flanking an internal, trans-acting hammerhead ribozyme (ITRz). Here, the single ITRz was replaced by two contiguous ITRz (dITRz), and a short poly(A) tail was designed onto the 3' end of the liberated dITRz, to produce the "SNIP(AA)" cassette. Self-processing of the cassette appeared to proceed efficiently in cells: The only region of the cassette identified in cells was the liberated dITRz, with approximately 10-20% of the dITRz found within the nucleus. We tested this reagent against two therapeutically important targets, human papillomavirus 11 E6/E7 mRNA and hepatitis B virus (HBV). Library selection protocols were utilized to define accessible target sites, and ribozymes targeted to these sites were very active in vitro. Pairs of the selected ribozymes were then inserted into the SNIP(AA) cassette. SNIP(AA) constructs targeted to the E6/E7 mRNA were tested in cell culture using a cotransfection approach. Significant reductions were produced in E6/E7 target, with 80-90% reductions observed at 5 days following cotransfection. SNIP(AA) constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIP(AA) constructs were packaged in liposomes, which were targeted to hepatocytes using asialofetuin, and administered ip. After 2 weeks, a >80% reduction in viral liver DNA was observed. Immunohistochemical staining for core antigen showed a similar decrease in the number of hepatocytes staining positively, compounded by a concomitant loss of residual staining intensity. These results demonstrate the in vivo utility of the self-processing SNIP(AA) cassette against HBV.
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Affiliation(s)
- Wei-Hua Pan
- Department of Pathology, The Gittlen Cancer Research Institute, Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033, USA
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Kan QC, Yu ZJ, Lei YC, Hao LJ, Yang DL. Lethiferous effects of a recombinant vector carrying thymidine kinase suicide gene on 2.2.15 cells via a self-modulating mechanism. World J Gastroenterol 2003; 9:2216-20. [PMID: 14562381 PMCID: PMC4656466 DOI: 10.3748/wjg.v9.i10.2216] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To determine the lethiferous effects of a recombinant vector carrying thymidine kinase (TK) suicide gene on 2.2.15 cells and the possible self-modulating mechanism.
METHODS: A self-modulated expressive plasmid pcDNA3-SCITK was constructed by inserting the fragments carrying hepatitis B virus antisense-S (HBV-anti-S) gene, hepatitis C virus core (HCV-C) gene, internal ribosome entry site (IRES) element of HCV and TK gene into the eukaryotic vector pcDNA3, in which the expression of TK suicide gene was controlled by the HBV S gene transcription. 2.2.15 cells that carry the full HBV genome and stably express series of HBV antigen were transfected with pcDNA3-SCITK or vector pcDNA3-SCI which was used as the mock plasmid. The HepG2 cells transfected with pcDNA3-SCITK were functioned as the negative control. All the transfected cells were incubated in DMEM medium supplemented with 10 μg/mL. of ganciclovir (GCV). The HBsAg levels in the supernatant of cell culture were detected by ELISA on the 1st, 3rd and 6th day post-transfection. Meanwhile, the morphology of tranfected cells was recorded by the photograph and the survival cell ratio was assessed by the trypan blue exclusion test on the 6th day post-transfection.
RESULTS: The structural accuracy of pcDNA3-SCITK was confirmed by restriction endonuclease digestion, PCR with specific primers and DNA sequencing. The HBsAg levels in the supernatant of transfected 2.2.15 cell culture were significantly decreased on the 6th day post-transfection as compared with that of the mock control (P < 0.05). The lethiferous effect of pcDNA3-SCITK expression on 2.2.15 cells was initially noted on the 3rd day after transfection and aggravated on the 6th day post transfection, in which the majority of transfected 2.2.15 cells were observed shrunken, round in shape and even dead. With assessment by the trypan blue exclusion test, the survival cell ratio on the 6th day post transfection was 95% in the negative control and only 11% in the experimental group.
CONCLUSION: The results indicate that suicide gene expression of pcDNA3-SCITK can only respond to HBV-S gene transcription, which may be potentially useful in the treatment of HBV infection and its related liver malignancies.
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Affiliation(s)
- Quan-Cheng Kan
- Division of Clinical Immunology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei Province, China
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