Cooperative Therapeutic Effects of Herpes Simplex Virus Thymidine Kinase Gene/Ganciclovir System and Chemotherapeutic Agents on Prostate Cancer in vitro

The killing effects of herpes simplex virus thymidine kinase gene/ganciclovir （HSV-tk/GCV） approach by the addition of several commonly clinical chemotherapeutic agents on hormone refractory prostate cancer （HRPC） cells PC-3m were investigated. After transferring of the HSV-tk gene into PC-3m cells, mRNA and protein expression of HSV-tk was detected by reverse-transcript polymerase chain reaction （RT-PCR） and strept avidin-biotin complex （SABC） im- munohistochemical method. The killing effect of GCV, cisplatin （CDDP）, etoposide （VP-16）, vincristine （VCR）, methotrexate （MTX）, 5-fluorouracil （5-Fu）, and suramin on PC-3m cells was evaluated by morphological assessment analysis, trypan blue exclusion assay and MTT assay respectively. Additionally, the cooperative effect of HSV-tk/GCV system combined with the above agents on the target cancer cells was determined by MTT. Furthermore, apoptosis and necrosis induced by GCV plus 5-Fu or suramin was analyzed by flow cytometry （FCM）. The results showed that that there was HSV-tk mRNA and protein expression in pDR2-tk plasmid transduced PC-3m cell. Combination of GCV with VP-16, VCR, 5-Fu or suramin led to an enhanced cellular killing effect, but with CDDP resulted in a reduced one and with MTX in an approximate one. FCM revealed that synergistic use of GCV and 5-fu or suramin resulted in a rather large proportion of apoptosis and necrosis with the apoptosis index being 36.38 % and 35.51%, and the proportion of necrosis being 33.05 % and 28.87 %, respectively. In conclusion, HSV-tk/CGV approach by addition of certain clinical available chemotherapeutic drugs brings on statistically significant enhanced cell killing over single-agent treatment. Our results highlight the potential for such new combination therapies for future treatments of HRPC.

Vascular damage and anti-angiogenic effects of tumor vessel-targeted adenovirus-mediated herpes simplex virus thymidine kinase gene

AIM： To explore the therapeutic efficacy and mechanism of herpes simplex virus-thymidine kinase （HSV-tk） targeting angiogenesis against hepatocellular carcinoma in vivio and in vitro. METHODS： Recombinant adenovirus containing kinase domain insert with receptor （KDR） or cytomegalovirus （CMV） promoter-controlled HSV-tk gene （AdKDR-tk and AdCMV-tk） was constructed using pAdeasy system. The expression of KDR antigen in human umbilical venous endothelial cells （HUVEC） and HepG2 was detected with histological analysis of cells. The virus was used to infect HUVEC and HepG2. Following administration of ganciclovir （GCV）, the survival rate of gene-transfected HUVEC and HepG2 was evaluated by MTT method. To develop hepatocarcinomas in 32 Balb/C mice with HepG2 cells, the mice were divided into four groups： ganciclovir group （Ⅰ）, Ad group （Ⅱ）, AdCMV-tk group （Ⅲ） and AdKDR-tk group （Ⅳ）. Then selective administration of recombinant adenovirus or Ad via the intratumorial was given to all rats. Ganciclovir （GCV） was given at a dose of 100 mg·kg^-1·d^-1 （ip） started on the following day and lasted 10 d. Microvessel density （MVD） of tumor in all the treated animals were examined by the immunohistochemical methods and tumor burden was evaluated 10 d before and alter the last GCV dose.RESULTS： Immunocytochemical staining indicated the expression of KDR antigen in HUVEC. Under adenovirus infection index of 100, with increasing GCV concentration from 0 up to 50 mg/L, the survival rate of AdKDR-tk- transfected HUVEC and HepG2 decreased from 100% to （28.94 ± 5.67）% and （75.45 ± 2.91）% at proper order, respectively （P 〈 0.01）, while the survival rate of AdCMV- tk-transfected HUVEC and HepG2 declined from 100% to （17.56 ± 2.48）% and （23.15± 5.72）%, respectively （P 〉 0.05）. Compared with group I, there was a decrease of tumor weight by 14.7% in group Ⅲ and by 23.6% in group Ⅳ. And there was a distinct difference between group M and Ⅳ （P 〈 0.05）. The median MVD for all groups was 37.4 ± 8.6, 30.6 ± 7.8, 27.6 ± 7.1, and 10.7 ± 4.1 （microvessels/mm^2） in group Ⅰ, Ⅱ, M and IV, respectively. And there was a marked difference between group M and Ⅱ （P 〈 0.05）, Ⅳ and Ⅱ （P 〈 0.01）, and Ⅳ and M （P 〈 0.01）. CONCLUSION： KDR promoter-HSV-tk gene may effectually restrain the growth of tumor via targeting angiogenesis for hepatocellular carcinoma with treatment of GCV.

Construction of the recombinant vector carrying herpes simplex virus thymidine kinase and cytokine genes expressed in cell line Tca8113

Objective: To construct expression vector containing fusion genes of herpes simplex virus thymidine kinase(Hsv-tk), Interleukin-2(IL-2) with internal ribosome entry sites(IRES), and to assess their expression in cell line Tca8113. Methods: IL-2 cDNA was obtained by reverse transcription. Hsv-tk, IL-2 and IRES genes were amplified by PCR. The purified amplification products were inserted into pGEM-T-Easy, and transformed into E.coli JM109. The purified recombinant plasmids were identified by restriction endonucleases. The recombinant plasmids were digested and pEGFP-N 3 were linearized, DNA fragments of Hsv-tk, IRES and IL-2 were ligated into linearized pEGFP-N 3, and then transferred into E.coli JM109. The recombinant tk-IL-2 genes were cloned separately and introduced into the expression vector pEGFP-N 3 containing GFP. The recombinant vectors were identified by their restriction sites through PCR. The plasmids pEGFP-TI was also transfected into Tca8113 cells by calcium phosphate method for the expression of fusion proteins. Fusion genes expressing vector PL(TI)SN was generated by the fusion of HSV-tk, IRES and IL-2 with the use of DNA recombination technology. The recombinant retroviruses were transferred into Tca8113 cells by lipofectamine. The positive clones were obtained after G418 selection and named Tca/TI respectively. Results: The pEGFP-TI pasmid was identified respectively by restriction endonucleases, and their fragment sizes were 1 120 bp and 450 bp. The pEGFP-TI pasmid as templates were amplified respectively by PCR, and their PCR products were 1 120 bp and 450 bp. The pEGFP-TI vectors were used to transfect Tca8113 cell, and the cells with fluorescence accounted for 60% of the total amount. Conclusion: pFGFP-tk-IRES-IL-2 expressing vector is easy to assess the expression of tk-IRES-IL-2-GFP fusion protein localization in transfected cells. The successful construction of expressing vector containing fusion genes of Hsv-tk, IRES and IL-2 may be beneficial for gene therapy in cell line Tca8113.

Effects of herpes simplex virus thymidine kinase/acyclovir system on growth of human pulmonary adenocarcinoma A549 cell line in vitro and in vivo

Objective: To observe the effect of anciclovir (ACV) treatment on tumors induced by inoculation of TK gene-transfected human pulmonary adenocarcinoma A549 cells in nude mice. Methods: A recombinant plasmid containing TK gene was constructed and transfected into A549 cells by electroporation. The sensitivity of the transgenic cells (A549-TK) to ACV was examined by MTT assay in vitro and for in vivo observation, inoculation of A549-TK and A-549 cells into nude mice was separately performed to induce tumor growth, the response of which to ACV treatment was observed, and the tumor tissues were pathologically examined. Results: A recombinant plasmid containing TK gene was successfully constructed and transfected into A549 cells. The sensitivity of A549-TK cells to ACV was 43 times higher than that of A549 cells. The tumors induced by A549-TK cells showed no significant increase in size after ACV treatment (P>0. 05) , and light microscopy revealed local tissue necrosis, karyoklasis, and nuclei disappearance. Conclusion: A549-TK cells acquires sensitivity to ACV both in vitro and in vivo, and ACV can inhibit the growth of tumors induced by A549-TK cell inoculation in nude mice.

An Oncolytic Adenovirus Expressing Herpes Simplex Virus-Thymidine Kinase for Targeting Cancer Therapy:An in vitro Evaluation

Objective： Oncolytic adenovirus, also called conditionally replicating adenovirus （CRAD）, has been developed for the treatment of cancer. However, there is a tremendous need to enhance their antitumor efficacy. Here we wish to evaluate whether a strategy that combines the herpes simplex virus-thymidine kinase with oncolytic effects offers a therapeutic advantage. Methods： A novel adenovirus Ad-ETK containing a sequentially positioned promoter of human telomerase reverse transcriptase （hTERT）, the coding sequence of E1A gene, an internal ribosome entry site sequence （IRES） and the coding sequence of herpes simplex virus-thymidine kinase （HSV-TK） was constructed. Infection of various cells with Ad-ETK followed by RT-PCR confirmed the expression of E1A and HSV-TK. The oncolytic ability and synergism between oncolytic effects and HSV-TK system was measured. The infection efficiency was determined by flow cytometry. Results： Ad-ETK deliverys E1A and HSV-TK gene, which selectively replicates in hTERT-positive tumor cells, and the progeny virus can reach up to 150 IU/cell. Our in vitro study showed that Ad-ETK plus ganciclovir （GCV） induced an obvious cell death. Conclusion： An oncolytic adenovirus plus the HSV-TK/GCV suicide gene system resulted in a significant improvement in treatment efficacy and it may offer important considerations in the development and preclinical assessments of oncolytic virotherapy.

Cloning of Thymidine Kinase Gene of Duck Plague Virus Using Degenerate PCR

The DNA of duck plague virus （DPV） thymidine kinase （TK） gene was cloned and sequenced from a vaccine virus in the study. Degenerate oligonucleotide primers for the consensus site of herpesvirus UL24, TK, and glycoprotein H（gH） gene were used in the polymerase chain reaction （PCR） to amplify DNA product with 3 741-base-pairs （bp） in size. DNA sequence analysis revealed a 1 077-base-pairs （bp） open reading frame （ORF） encoding a 358 amino acid polypeptide homologous to herpesvirus TK proteins. The predicted TK protein shared 31.2, 41.3, 35.7, 37.4, and 28.4% identity with herpes simplex virus typel, equine herpesvirus type 4, Marek＇s disease virus 2, herpesvirus turkey, and infectious laryngotracheitis virus, respectively. Comparison of the amino acid sequences of other herpesvirus TK proteins showed that these proteins were not conserved on the whole, otherwise the portion of the TK proteins corresponding to the nucleotide binding domain and the nucleoside binding site were highly conserved among herpesvirus. Comparison with the amino acid sequences of the conserved nucleotide and nucleoside binding domains of other eleven herpesvirus TK proteins to the predicted DPV peptide confirmed its identity as the DPV TK protein.

Herpes simplex virus-1 infection or Simian virus 40-mediated immortalization of corneal cells causes permanent translocation of NLRP3 to the nuclei

AIM: To investigate into the potential involvement of pyrin containing 3 gene(NLRP3), a member of the nucleotide-binding oligomerization domain-like receptors with cytosolic pattern recognition, in the host defense of corneas against viruses.METHODS: The herpes viral keratitis model was utilized in BALB/c mice with inoculation of herpes simplex virus-1(HSV-1). Corneal tissues removed during therapy of patients with viral keratitis as well as a Simian vacuolating virus 40(SV40)-immortalized human corneal epithelial cell line were also examined.Immunohistochemistry was used to detect NLRP3 in these subjects, focusing on their distribution in tissue or cells. Western blot was used to measure the level of NLRP3 and another two related molecules in NLPR3 inflammasome, namely caspase-1 and IL-1β.RESULTS: The NLRP3 activation induced by HSV-1infection in corneas was accompanied with redistribution of NLRP3 from the cytoplasm to the nucleus in both murine and human corneal epithelial cells. Furthermore,in the SV40-immortalized human corneal epithelial cells,NLRP3 was exclusively located in the nucleus, and treatment of the cells with high concentration of extracellular potassium(known as an inhibitor of NLRP3activation) effectively drove NLRP3 back to the cytoplasm as reflected by both immunohistochemistry and Western blot.· CONCLUSION: It is proposed that herpes virus infection activates and causes redistribution of NLRP3 to nuclei. Whether this NLRP3 translocation occurs with other viral infections and in other cell types merit further study.

Preclinical evaluation of herpes simplex virus armed with granulocyte-macrophage colony-stimulating factor in pancreatic carcinoma

AIM: To investigate the therapeutic efficacy and mechanisms of action of oncolytic-herpes-simplex-virus encoding granulocyte-macrophage colony-stimulating factor(HSVGM-CSF) in pancreatic carcinoma.METHODS: Tumor blocks were homogenized in a sterile grinder in saline.The homogenate was injected into the right armpit of each mouse.After vaccination,the mice were randomly assigned into four groups: a control group,a high dose HSVGM-CSFgroup [1 × 107plaque forming units(pfu)/tumor],a medium dose HSVGM-CSF group(5 × 106pfu/tumor) and a low dose HSVGM-CSF group(5 × 105pfu/tumor).After initiation of drug administration,body weights and tumor diameters were measured every 3 d.Fifteen days later,after decapitation of the animal by cervical dislocation,each tumor was isolated,weighed and stored in 10% formaldehyde solution.The drug effectiveness was evaluated according to the weight,volume and relative volume change of each tumor.Furthermore,GM-CSF protein levels in serum were assayed by enzyme-linked immunosorbent assays at 1,2,3 and 4 d after injection of HSVGM-CSF.RESULTS: Injection of the recombinant mouse HSV encoding GM-CSF resulted in a significant reduction in tumor growth compared to the control group,and dosedependent effects were observed: the relative tumor proliferation rates of the low dose,medium dose and high dose groups on 15 d after injection were 45.5%,55.2% and 65.5%,respectively.The inhibition rates of the tumor weights of the low,middle,and high dose groups were 41.4%,46.7% and 50.5%,respectively.Furthermore,the production of GM-CSF was significantly increased in the mice infected with HSVGM-CSF.The increase in the GM-CSF level was more pronounced in the high dose group compared to the other two dose groups.CONCLUSION: Our study provides the first evidence that HSVGM-CSFcould inhibit the growth of pancreatic cancer.The enhanced GM-CSF expression might be responsible for the phenomenon.

The Role, Mechanism and Transcriptional Regulation of LAT in Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus (HSV) infection in the human body can be latent in neurons for long time and be reactivated leading to recurrence at high rate. Currently there is no effective clinical strategy for the prevention and treatment of the disease relapse. HSV LAT gene is expressed in large quantities and lytic genes are turned off leading to HSV latency. Disruption of the gene expression is thought to cause HSV reactivation and disease relapse. To reveal the essence of HSV latency and reactivation, we summarized and innovatively classified the role, mechanism and transcriptional regulation of LAT in HSV latency and reactivation. This review may have important implications for future studies on HSV latency and reactivation, HSV disease prevention and treatment, and safer and more effective oncolytic HSVs (oHSVs).

Regulation of Wnt/β-catenin signaling by herpesviruses

The Wnt/β-catenin signaling pathway is instrumental in successful differentiation and proliferation of mammalian cells. It is therefore not surprising that the herpesvirus family has developed mechanisms to interact with and manipulate this pathway. Successful coexistence with the host requires that herpesviruses establish a lifelong infection that includes periods of latency and reactivation or persistence. Many herpesviruses establish latency in progenitor cells and viral reactivation is linked to host-cell proliferation and differentiation status. Importantly, Wnt/β-catenin is tightly connected to stem/progenitor cell maintenance and differentiation. Numerous studies have linked Wnt/β-catenin signaling to a variety of cancers, emphasizing the importance of Wnt/β-catenin pathways in development, tissue homeostasis and disease. This review details how the alpha-, beta-, and gammaherpesviruses interact and manipulate the Wnt/β-catenin pathway to promote a virus-centric agenda.

基于293T-Cas9细胞株构建ICP34.5基因敲除的oHSV-1/Ecfp病毒

目的构建稳定表达Cas9蛋白的工具细胞,并利用短回文重复序列/相关蛋白9(CRISPR/Cas9)编辑技术改造1型单纯疱疹病毒(HSV-1),获得ICP34.5基因敲除的oHSV-1/Ecfp重组病毒。方法利用慢病毒包装体系,将包装Cas9表达质粒的病毒感染HEK293T细胞,然后经嘌呤霉素筛选获得稳定表达Cas9蛋白的293T-Cas9细胞株,通过聚合酶链式反应(PCR)及蛋白质免疫印迹技术检测Cas9基因及蛋白表达情况;利用分子克隆技术构建HSV-1病毒ICP34.5基因敲除的基因编辑质粒(pU6-Ecfp);pU6-Ecfp转染293T-Cas9细胞后感染野生型HSV-1病毒,在胞内通过CRISPR/Cas9技术敲除HSV-1病毒中ICP34.5基因,再利用增强型青色荧光(Ecfp)示踪及极限稀释法进行病毒富集纯化,通过PCR法进行oHSV-1/Ecfp病毒鉴定。结果PCR扩增、核酸电泳及蛋白质免疫印迹技术结果显示,细胞株293T-Cas9高转录Cas9基因及高表达Cas9蛋白;通过PCR鉴定及荧光示踪结果显示,基因编辑质粒pU6-Ecfp构建成功;Ecfp荧光示踪、PCR扩增及核酸电泳结果显示,Ecfp成功插入ICP34.5基因敲除位点,获得oHSV-1/Ecfp重组病毒。结论稳定表达Cas9蛋白的293T-Cas9细胞可作为CRISPR/Cas9基因编辑的工具细胞,构建的HSV-1/Ecfp病毒实现了ICP34.5基因敲除及Ecfp示踪蛋白的敲入。

Transcriptional regulation of the Herpes Simplex Virus 1α-gene by the viral immediate-early protein ICP22 in association with VP16

Herpes Simplex Virus 1 (HSV1) is capable of inducing two forms of infection in individuals, and the establishment of which type of infection occurs is linked to the transcriptional activation of viral α genes. One of the HSV1 α genes, ICP22, is known to have multiple functions during virus replication, but its distinct roles are still unclear. This study showed that ICP22 functions as a general repressor for certain viral and cellular promoters, and this transcriptional repression by ICP22 is independent of the specific upstream promoter element, as shown using the CAT enzyme assay system. Further work also found that VP16 interfered with ICP22 mediated transcriptional repression of the viral α4 gene, through interactions with specific elements upstream of the α4 gene promoter. These findings support the possibility that ICP22 and VP16 control transcription of HSV1α genes in a common pathway for the establishment of either viral lytic or latent infections.

HSV gene transfer in the treatment of chronic pain

It has proven difficult to use systemic administration of small molecules to selectively modulate nociception.Over the past decade,we and others have developed non-replicating herpes simplex virus(HSV)-based vectors to treat chronic pain.Subcutaneous inoculation of an HSV vector effectively transduces sensory neurons in the dorsal root ganglion;release of transgene-coded inhibitory neurotransmitters or anti-inflammatory peptides reduces pain-related behaviors in rodent models of chronic inflammatory and neuropathic pain.A phase 1 trial of this therapy in patients is set to begin soon.

Gene-Eden-VIR Is Antiviral: Results of a Post Marketing Clinical Study

Introduction: This paper reports the results of a post marketing clinical study that tested the antiviral properties of Gene-Eden-VIRTM. Specifically, the clinical study tested the effect of Gene-Eden-VIR on the severity, duration, and frequency of symptoms reported by individuals infected with various viruses. The viruses included the Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Epstein Barr Virus (EBV), Human Cytomegalovirus (HCMV) and Hepatitis C Virus (HCV). The symptoms included abnormal Pap smear, low and high grade cervical dysplasia, warts, blisters, cold sores, hives, skin tabs, panic attacks, depression, kidney problems, sleeping problems, liver problems, fever, fatigue, sore throat, swollen lymph nodes, diarrhea, and weight loss. Treatment: A capsule of Gene-Eden-VIR includes five natural ingredients: 100 mg of quercetin, 150 mg of green tea extract, 50 mg of a cinnamon extract, 25 mg of a licorice extract, and 100 mcg of selenium. The dosage was 1, 2, 3, or 4 capsules per day. The duration of treatment was 2 to 54 weeks. Population: The study population consisted of 60 infected individuals, ages 20 to 66. Results: The participants reported no side effects after taking Gene-Eden-VIR. Seventy three percent of the individuals treated with Gene-Eden-VIR reported a decrease in their symptoms. Specifically, they reported a decrease in the severity (p = 0.006, n = 45), duration (p = 0.009, n = 34), and frequency of their symptoms (p Conclusions: This post marketing clinical study showed that Gene-Eden-VIR is a safe and effective antiviral treatment. Specifically, the clinical study showed that Gene-Eden-VIR is a safe and effective treatment against the Human Papillomavirus (HPV), Herpes Simplex Virus (HSV), Epstein Barr Virus (EBV), Human Cytomegalovirus (HCMV), and Hepatitis C Virus (HCV). Therefore, health care practitioners should recommend Gene-Eden-VIR as a safe and effective antiviral treatment against these viruses.

Gene-Eden-VIR Decreased Physical and Mental Fatigue in a Post Marketing Clinical Study That Followed FDA Guidelines;Results Support Microcompetition Theory

Objective: The Microcompetition with Foreign DNA theory, proposed by Hanan Polansky in 2003, describes how latent viruses can cause chronic conditions, including fatigue. The Gene-Eden-VIR formula was designed to target latent viruses. Therefore, the theory predicts that treatment with Gene-Eden-VIR will decrease fatigue in individuals infected with a latent virus. The objective of this study was to test this prediction. Framework: A post marketing clinical study that followed FDA guidelines. Treatment: Gene-Eden-VIR, a dietary supplement. A capsule of Gene-Eden-VIR includes 100 mg of quercetin, 150 mg of green tea extract, 50 mg of cinnamon extract, 25 mg of licorice extract, and 100 mcg of selenium. The treatment included 1, 2, 3, or 4 capsules per day, and lasted 2 to 54 weeks. Population: 100 individuals infected with a latent virus, including the Human Papillomavirus (HPV), Epstein Barr Virus (EBV), Herpes Simplex Virus (HSV), Human Cytomegalovirus (HCMV), and Hepatitis C Virus (HCV). Ages ranged from 20 to 66. All participants reported a feeling of fatigue at the start of the study. Specifically, 98, 90, and 79 participants reported a feeling of general, physical, and mental fatigue, respectively. Results: Following treatment with Gene-Eden-VIR, 73.47%, 62.22%, and 47.36% reported a decrease in their feeling of general, physical, and mental fatigue, respectively. The participants also reported a statistically significant decrease in every aspect of fatigue tested in the study. The results also showed a duration effect, that is, those treated for 2 months or more reported a larger decrease in their feeling of fatigue (general, p = 0.03, n = 65;physical and mental, p = 0.05, n = 70). The results showed no interviewer bias, and no selection bias. In addition, the results showed therapeutic consistency under varying manufacturing conditions. The participants reported no side effects after taking Gene-Eden-VIR. Conclusions: This post marketing clinical study showed that treatment with Gene-Eden-VIR safely decreased the feeling of general, physical, and mental fatigue in individuals infected with a latent virus. Since most individuals are infected with a latent virus, health care practitioners should recommend Gene-Eden-VIR as a first line treatment for fatigue. The results of this post marketing clinical study support the Microcompetition with Foreign DNA theory.

HSVTK Gene Therapy for CarcinoembryonicAntigen-Producing Human Lung Cancer Cells

The long-term success of gene therapy for cancer relies heavily on the development of effective targeting systems. We investigate the possibility of targeted gene therapy using promoter of carcinoembryonic antigen (CEA) gene. By using luciferase reporter gene, we found that CEA promoter exhibit 16 times high activity in CEA-producing lung cancer cells, A549 than in nonproducing cells, Hela. We also constructed a recombinant expression plasmid pCEATK, in which CEA promoter drives the effector gene, thymidine kinase gene of Herpes Simplex Virus (HSVTK). A549 cells transfected with pCEATK became 865 times more sensitive to ganciclovir (GCV) than the control cells. However, Hela cells transfected with this plasmid remained resistant to GCV. These data indicate the potential for targeted gene therapy using the CEA promoter against CEA-producing tumor cells, such as lung cancer cells.

A short perspective on gene therapy:Clinical experience on gene therapy of gliomablastoma multiforme

More than two decades have passed since the first gene therapy clinical trial was conducted.During this time,we have gained much knowledge regarding gene therapy in general,but also learned to understand the fear that persists in society.We have experienced drawbacks and successes.More than 1700 clinical trials have been conducted where gene therapy is used as a means for therapy.In the very first trial,patients with advanced melanoma were treated with tumor infiltrating lymphocytes genetically modified ex-vivo to express tumor necrosis factor.Around the same time the first gene therapy trial was conducted,the ethical aspects of performing gene therapy on humans was intensively discussed.What are the risks involved with gene therapy?Can we control the technology?What is ethically acceptable and what are the indications gene therapy can be used for?Initially,gene therapy was thought to be implemented mainly for the treatment of monogenetic diseases,such as adenosine deaminase deficiency.However,other therapeutic areas have become of interest and currently cancer is the most studied therapeutic area for gene therapy based medicines.In this review I will be giving a short introduction into gene therapy and will direct the discussion to where we should go from here.Furthermore,I will focus on the use of the Herpes simplex virus-thymidine kinase for gene therapy of malignant gliomas and highlight the efficacy of gene therapy for the treatment of malignant gliomas,but other strategies will also be mentioned.

SPECT imaging of cardiac reporter gene expression in living rabbits

This work is to demonstrate feasibility of imaging the expression of herpes simplex virus 1-thymidine kinase (HSV1-tk) reporter gene in rabbits myocardium by using the reporter probe 131I-2'-fluoro-2'-deoxy-1-β-D- arabinofuranosyl-5-iodouracil (131I-FIAU) and SPECT. Rabbits of the study group received intramyocardial injection of Ad5-tk and control group received aseptic saline injection. Two sets of experiments were performed on the study group. Rabbits of the 1st set were injected with 131I-FIAU 600 μCi at Day 2 after intramyocardial transfection of Ad5-tk in 1×109, 5×108, 1×108, 5×107 and 1×107 pfu, and heart SPECT imaging was done at different hours. Rabbits of the 2nd were transferred various titers of Ad5-tk (1×109, 5×108, 1×108, 5×107, 1×107 pfu) to determine the threshold and optimal viral titer needed for detection of gene expression. Two days later, 131I-FIAU was injected and heart SPECT imaging was performed at 6, 24 and 48 h, before killing them for gamma counting of the hearts. Reverse tran- scription-polymerase chain reaction (RT-PCR) was used to verify the transferred HSV1-tk gene expression. Semi-quantitative analysis derived of region of interest (ROI) of SPECT images and RT-PCR images was performed and the relationship of SPECT images with ex vivo gamma counting and mRNA level were evaluated. SPECT images conformed 131I-FIAU accumulation in rabbits injected with Ad5-tk in the anterolateral wall. The optimal images qual- ity was obtained at 24~48 h for different viral titers. The highest radioactivity in the focal myocardium was seen at 6 h, and then declined with time. The threshold was 5×107 pfu of virus titer. The result could be set better in 1~5×108 pfu by SPECT analysis and gamma counting. ROI-derived semi-quantitative study on SPECT images correlated well with ex vivo gamma counting and mRNA levels from RT-PCR analysis. The HSV1-tk/131I-FIAU reporter gene/reporter probe system is feasible for cardiac SPECT reporter gene imaging. The optimal Ad5-tk titer is 1~5×108 pfu and optimal imaging time is 24~48 h after transferred Ad5-tk in rabbit. The imaging of transgene expression in heart might be used for noninvasive imaging of gene therapy in cardiac diseases in human.

Oncolytic herpes simplex virus vectors for the treatment of human breast cancer

Background Oncolytic herpes simplex virus (HSV) vectors can be used for cancer therapy as direct cytotoxic agents, inducers of anti-tumor immune responses, and as expressers of anti-cancer genes. In this study, the efficacy of HSV vectors, G47Delta and NV1023 were examined for the treatment of the human breast cancer. Methods Human breast cancer MDA-MB-435 cells were cultured or implanted subcutaneously in BALB/c nude mice. The cells or tumors were inoculated with G47Delta or NV1023, and cell killing or inhibition of tumor growth determined. Both viruses contained the LacZ gene and expression in infected cells was detected with X-gal histochemistry. Results G47Delta and NV1023 were highly cytotoxic to MDA-MB-435 cells in vitro at very low multiplicities of infection. X-gal staining of infected tumor cells in vitro and in vivo illustrated the replication and spread of both viruses. G47Delta and NV1023 inoculation inhibited tumor growth and prolonged mouse survival. Both vectors behaved similarly. Conclusions Oncolytic HSV vectors, G47Delta and NV1023, were extremely effective at killing human breast cancer cells in vitro and in tumor xenografts in vivo. This novel form of cancer therapy warrants further investigation and consideration of clinical application.

pLTKcSN/VPC及GCV系统的旁观者效应观察

目的：探讨单纯疱疹病毒Ⅰ型胸苷激酶基因（ＴＫ）逆转录病毒载体生产细胞（ｐＬＴＫｃＳＮ／ＶＰＣ）和更昔洛韦（ＧＣＶ）系统杀伤恶性胶质瘤细胞过程中的旁观者效应。方法：采用大鼠胶质瘤细胞Ｃ６、人恶性胶质瘤Ｕ８７ＭＧ和它们的转染ＴＫ阳性细胞Ｃ６ＴＫ，Ｕ８７ＴＫ，将Ｃ６与Ｃ６ＴＫ，Ｕ８７ＭＧ和Ｕ８７ＴＫ按比例（ＴＫ阳性细胞占细胞总体的０％～１００％，１０％梯度）分别混合培养于９６孔板中，每种混合比例设６孔。培养２４ｈ后，６孔中３孔加入浓度为０．５μｇ／ｍｌＧＣＶ，另外３孔作为空白对照。继续培养７２ｈ，直接计数各孔活细胞数并以对照组计数结果为本底计算ＧＣＶ对各混合比例的抑制率。结果：当ＴＫ阳性细胞占１０％时，Ｃ６和Ｕ８７ＭＧ组的抑制率达到或超过３０％，当ＴＫ阳性细胞占总体的５０％以上时，ＧＣＶ对各混合比例的抑制率接近１００％。结论：ｐＬＴＫｃＳＮ／ＶＰＣ和ＧＣＶ系统杀伤恶性胶质瘤细胞过程中存在明显的旁观者效应。