CDglyTK自杀基因系统治疗小鼠慢性粒细胞白血病皮下移植瘤的研究

为了探讨逆转录病毒介导的CDglyTK自杀基因系统时K562细胞的体内外杀伤作用，将逆转录病毒介导的CDglyTK自杀基因转染入K562细胞，体外实验用MTT法观察5-氟胞嘧彰丙氧鸟苷（5-fluorocytosine／ganciclovir，5-FC／GCV）时K562／CDglyTK细胞的生长抑制率。体内实验时将K562／CDglyTK细胞和K562细胞接种于裸鼠皮下，使用GCV和5-FC后，观察裸鼠肿瘤体积的变化及裸鼠的生存率。体外实验表明，GCV联合5-FC时K562／CDglyTK细胞具有明显的杀伤作用；体内实验结果显示，皮下注射K562细胞和K562／CDglyTK细胞后小鼠成瘤率无明显区别；使用5-FC／GCV可明显抑制裸鼠体内的肿瘤形成；经5-FC／GCV治疗后K562／CDglyTK组的肿瘤体积较对照组明显缩小，裸鼠生存率也较时照组明显提高。结论：双自杀基因在体内外对K562细胞均有杀伤作用。     

逆转录病毒介导Fcy::Fur融合基因联合5-FC治疗胶质瘤的体内试验研究

目的构建含融合自杀基因Fcy::Fur重组逆转录病毒,用自杀基因治疗系统Fcy::Fur/5-氟胞嘧啶(5-FC)对裸鼠胶质瘤进行体内抑瘤作用的实验研究。方法扩增Fcy::Fur基因并构建Fcy::Fur基因重组逆转录病毒载体;载体转染包装细胞获得高滴度病毒并转染鼠胶质瘤细胞C6,筛选并鉴定阳性转基因克隆;构建裸鼠荷胶质瘤动物模型,腹腔注射5-FC,观察裸鼠肿瘤重量变化及电镜、流式细胞仪(FCM)检测肿瘤的凋亡。结果PCR法扩增出全长Fcy::Fur基因,经测序证实序列正确;构建了PLXSN-Fcy::Fur逆转录病毒载体,载体转染包装细胞PT67,获得滴度为3.5×106CFU/mL的逆转录病毒;转染C6获转基因阳性克隆C6-Fcy::Fur,检测C6-Fcy::Fur有Fcy::Fur基因的mRNA表达;裸鼠前肢背部接种转基因细胞,成瘤后腹腔注射5-FC,转基因肿瘤的生长较对照组明显抑制。FCM法检测到凋亡峰,电镜观察到转基因肿瘤细胞有凋亡小体。结论AdE1CMVCD与5-FC联合在体内对胶质瘤有明显的抑制作用,为临床胶质瘤基因治疗提供了可靠的理论及应用依据。     

HSV-TK和IL-12联合基因对宫颈癌的杀伤作用

目的观察单纯疱疹病毒胸苷激酶(HSV-TK)丙氧鸟苷(GCV)自杀基因系统联合IL-12基因对人宫颈癌hela细胞系体外杀伤作用。方法采用脂质体转染法将HSV-TK及IL-12联合基因转染hela细胞,并将其用于体外实验,观察GCV对转染联合基因的Hela的杀伤作用及旁观者效应等。结果加入GCV后体外培养联合基因转染的Hela细胞,24 h后细胞明显发生形态上改变,正常对照组细胞生长旺盛;随着GCV剂量的加大,转染联合基因的Hela细胞的存活率呈逐渐下降的趋势,GCV对转染细胞的杀伤作用较对正常细胞的杀伤作用明显增强。并观察到自杀基因系统具有明显的旁观者效应。结论脂质体可介导HSV-TK及IL-12联合基因转入人宫颈癌hela细胞并获得稳定表达,GCV对HSV-TK及IL-12联合基因转染的hela细胞具有明显的杀伤作用。     

疮疹病毒Ⅰ型胸苷激酶基因和更昔洛韦系统治疗脑恶性胶质瘤的临床观察及护理

目的 探讨和观察单纯疱疹病毒Ⅰ型胸苷激酶基因(TK)逆转录病毒载体生产细胞(PLTKcSN／VPC)结合羟甲基无环鸟苷(GCV)系统治疗(基因治疗)恶性胶质瘤的方法在人体内的安全性。方法在围手术期内对接受该治疗的患进行临床观察和护理。结果 19例患接受该治疗后产生一些轻微的毒副作用，包括发热、神经系统症状、心血管及消化系统反应，对症处理后均可控制。结论 通过密切观察和护理．可有效地预防和减轻治疗后不良反应。     

聚焦超声介导阳离子脂质体携带HSV-TK增强基因转染及联合更昔洛韦对大鼠胶质瘤C6细胞的影响

目的探讨聚焦超声对载单纯疱疹病毒胸苷激酶(herpes simplex virus thymidine kinase,HSV-TK)基因阳离子脂质体体外基因转染的影响,及其联合更昔洛韦(ganciclovir,GCV)对大鼠脑胶质瘤C6细胞的影响。方法制备含HSV-TK基因质粒,利用阳离子脂质体转染至对数期大鼠胶质瘤C6细胞,并随机分为脂质体组和超声1、2、3、4min组,脂质体组加入含9μg阳离子脂质体-HSV-TK基因质粒的100μL培养液;超声1、2、3、4min组在脂质体组基础上分别给予体外超声(1 MHz,功率3W/cm2,电压150mvPP)辐照1、2、3、4min。5组C6细胞均于37℃、体积分数5%CO2培养箱,含体积分数10%胎牛血清的高糖DMEM完全培养液,继续培养48h。转染48h后,荧光显微镜下观察载HSV-TK基因阳离子脂质体转染效率。将对数生长期C6细胞接种于96孔板,依据转染效率实验结果分为GCV组、超声3min+GCV组,超声3 min+GCV组加入9μg阳离子脂质体-HSV-TK基因质粒的100μL培养液,并超声辐照3min,GCV组转染空白阳离子脂质体;转染24h后,2组均分别加入0.1、1、10、50、100、1 000mg/L GCV作用48h,采用分光光度法检测C6细胞存活率。结果转染48h后,超声1、2、3、4min组C6细胞HSV-TK基因转染效率[(0.14±0.07)%、(1.39±0.11)%、(19.61±4.80)%、(18.81±7.92)%]均高于脂质体组[(0.05±0.01)%](P0.05),且超声3、4min组转染效率高于超声1、2min组,超声2 min组转染效率高于超声1 min组(P0.05),超声3 min组与超声4min组转染效率比较差异无统计学意义(P0.05);随GCV浓度增加,GCV组和超声3min+GCV组C6细胞存活率均逐渐降低;GCV为50、100、1 000mg/L时,超声3min+GCV组C6细胞存活率[(85.88±1.80)%、(75.51±1.43)%、(68.17±5.26)%]均低于GCV组[(95.40±0.85)%、(92.62±4.50)%、(83.66±0.98)%](P0.05);GCV为0.1、1、10mg/L时,超声3min+GCV组C6细胞存活率[(99.32±3.01)%、(97.01±1.53)%、(94.50±1.15)%]与GCV组[(99.56±5.14)%、(98.60±5.18)%、(97.39±8.06)%]比较差异均无统计学意义(P0.05)。结论聚焦超声体外辐照可明显提高载HSV-TK基因阳离子脂质体转染效率,增强GCV的抗胶质瘤作用。     

提高多药耐药基因导入人CD_(34)~ 细胞转导效率的探讨

目的　探讨逆转录病毒介导的多药耐药基因 (mdr1)导入人CD34 细胞的影响因素 ,以提高基因转导效率。方法　用流式细胞术 (FCM)检测不同组合细胞因子及人骨髓基质细胞 细胞因子支持的基因转导效率 ;用造血祖细胞集落培养观察耐药性 ;用FCM检测不同浓度紫杉醇素对基因转导细胞的作用。结果　细胞因子SCF Flt配体 (FL) IL 3组合支持的基因转导效率高于其它组合 (SCF IL 6 IL 3,SCF IL 6 IL 3 Tpo ,SCF IL 3)。骨髓基质细胞 细胞因子 (SCF FL IL 3)支持的基因转导效率 (2 0 .5 % )又高于单纯用该组细胞因子的转导效率 (15 .2 % ) ,并且前者形成的抗性集落形成细胞数多于后者。在 10ng ml紫杉醇作用下基因转导CD34 细胞的阳性率可达 38.5 %。结论　骨髓基质细胞 细胞因子 (SCF FL IL 3)对基因转导有较强的促进作用 ;一定浓度的紫杉醇具有富集基因转导细胞的作用     

单纯疱疹病毒胸苷激酶基因转导在供者淋巴细胞输注中的应用

供者淋巴细胞输注(DLI)作为治疗BMT后白血病复发的重要手段已被广泛应用,但GVHD是DLI的主要并发症。将单纯疱疹病毒胸苷激酶基因转导至供者外周血淋巴细胞,输给去T细胞的alloBMT术后复发患者,这样不仅建立了移植物抗白血病作用,而且如有严重的GVHD发生方可用丙氧鸟苷选择性去除体内供者淋巴细胞。     

逆转录病毒双顺反子策略有效转导和共表达ALDH1基因与mdr1基因的研究

目的研究含内在核糖体进入位点(IRES) 的逆转录病毒双顺反子载体介导的醛脱氢酶基因(ALDH1)与多药耐药基因(mdr1)转导和共表达.方法以携带ALDH1与mdr1基因的重组逆转录病毒双顺反子G1Na-ALDH1-IRES-mdr1(G1Na-AIM)为载体,电穿孔法转导双嗜型包装细胞PA317,长春新碱筛选;所得病毒生产细胞PA317/ AIM与单嗜型包装细胞GP+E86乒乓感染提高病毒滴度;以重组病毒上清转染K562细胞,应用聚合酶链反应(PCR)和Southern blot检测转移基因的整合,流式细胞术(FCM)及MTT分析基因表达,集落培养法测定转导效率.结果双嗜型病毒上清滴度达1.0×105cfu/ml.基因修饰细胞K562/ AIM经PCR 和Southern blot证实双基因稳定整合至基因组,对4-氢过氧化环磷酰胺(4-HC)及长春新碱(VCR)耐药 (提高3～10倍),FCM及集落法检测基因转导效率为62%～70%.结论逆转录病毒IRES双顺反子载体能引导不同类型的耐药基因有效共表达,可用于扩大耐药范围,进行体内显性选择.     

腺病毒介导的KDR启动子-胸苷激酶系统对血管内皮细胞旁观者效应的实验研究

目的：研究腺病毒介导的血管内皮因子受体（KDR）启动子-胸苷激酶系统对血管内皮细胞的旁观者效应。方法：采用新型腺病毒载体AdEasy系统，构建由KDR启动子调控的、腺病毒介导的、单纯疱疹病毒胸苷酸激酶基因系统（AdK—DR—HSv-tk），在人胚胎肾细胞系293细胞中包装、扩增后，体外感染表达KDR的人脐静脉血管内皮细胞（HUVEC），建立HUVEC／tk转化细胞株。观察丙氧鸟苷（GCV）对HUVEC、HUVEC／tk细胞的存活率及旁观者效应。结果：从病毒滴度及PCR检查，证实tk基因随病毒的感染已成功地导入293细胞及HUVEC细胞。HUVEC／tk细胞对丙氧鸟苷的敏感性显著高于亲代HUVEC细胞。HUVEC／tk及HUVEC＋HUVEC／tk混合细胞还随丙氧乌苷浓度的增加受到明显的抑制。HUVEC＋HUVEC／tk混合细胞的存活率，随HUVEC／tk细胞比例的增加而降低，说明腺病毒介导的KDR启动子-单纯疱疹病毒胸苷激酶／丙氧鸟苷系统不但能杀伤tk^＋细胞，也能杀伤未导入tk的细胞，存在明显的旁观者效应。结论：腺病毒介导的KDR启动子单纯疱疹病毒胸苷激酶／丙氧乌苷系统对血管内皮细胞具有明显的杀伤及旁观者效应。     

载自杀基因的靶向微泡联合超声辐照抑制视网膜母细胞瘤的实验研究

目的探讨载自杀基因的靶向微泡联合超声辐照对视网膜母细胞瘤(RB)的抑制作用。方法制备携带单纯疱疹病毒Ⅰ型胸苷激酶(HSV1-tk)质粒和血管内皮细胞生长因子受体2抗体的靶向微泡,分为空白对照组、细胞+质粒组、细胞+质粒+Sono Vue组、细胞+靶向微泡组、细胞+质粒+超声辐照组、细胞+质粒+Sono Vue+超声辐照组及细胞+靶向微泡+超声辐照组进行实验。荧光显微镜观察各组基因转染情况,流式细胞仪检测转染率,加入丙氧鸟苷(GCV)后检测细胞的抑制率。结果细胞+靶向微泡+超声辐照组的转染率为(24.78±1.04)%,高于细胞+质粒+Sono Vue+超声辐照组(14.31±0.69)%,差异有统计学意义(P0.05)。随着GCV浓度的增加和培养时间的延长,各组抑制率逐渐升高。当GCV浓度为100 mg/L,培养96 h后,细胞+靶向微泡+超声辐照组对RB细胞的抑制率达(92.91±1.71)%。结论加入GCV后,携带HSV1-tk基因的靶向微泡联合超声能有效地抑制RB细胞。     

慢病毒介导的突变型胸苷激酶对T淋巴细胞杀伤作用的实验研究

目的观察慢病毒介导的突变型单纯疱疹病毒胸苷激酶(HSV-sr39tk)及野生型HSV-tk对T细胞的体外杀伤作用,比较HSV-sr39tk/更昔洛韦(GCV)、HSV-sr39tk/阿昔洛韦(ACV)、HSV-tk/GCV、HSV-tk/ACV对T细胞存活率的影响。方法采用改良的磷酸钙沉淀法将包装质粒、包膜蛋白质粒和含目的基因的转移质粒共转染293T包装细胞,收集的病毒上清感染T细胞后,分别与不同浓度梯度的前体药物GCV和ACV作用4d后,MTT法测定细胞存活率。结果共转染293T细胞后获得较高滴度的慢病毒(2×106IU/ml)。前体药物GCV和ACV浓度在0～10.0μmol/L时转染HSV-sr39tk细胞(39tk+T细胞)存活率下降比较明显,GCV组细胞存活率由(96.04±3.23)%下降为(36.76±4.38)%,ACV组细胞存活率由(97.31±4.61)%下降为(43.75±8.99)%,而GCV和ACV浓度大于10.0μmol/L时,39tk+T细胞存活率下降趋势则减缓。统计显示39tk+T细胞对GCV、ACV均有敏感性(P值均<0.05);转染HSV-tk T细胞(tk+T细胞)对GCV有敏感性(P<0.05),而对ACV不具有敏感性(P>0.05);同一浓度时39tk+T细胞+GCV与tk+T细胞+GCV两组间T细胞存活率差异具有统计学意义(P<0.05)。结论慢病毒载体可高效、稳定地感染T细胞,同时不影响细胞的增殖,与野生型HSV-tk基因比较,表达突变型HSV-sr39tk的T细胞对GCV具有更高的敏感性,而且对ACV也具有敏感性。     

Regression of hepatocellular carcinoma in vitro and in vivo by radiosensitizing suicide gene therapy under the inducible and spatial control of radiation.

To improve the efficacy and selectivity of gene therapy for hepatocellular carcinoma (HCC), we designed a strategy for suicide gene therapy in conjunction with radiation therapy using an HVJ-liposome vector system. The radio-inducible suicide gene was constructed by insertion of the early growth response gene 1 (Egr-1) promoter upstream of the HSV-tk gene (EGF-tk). First, to test the tumor specificity of Egr-1, RT-PCR and immunohistochemistry were performed. The Egr-1 gene was highly expressed in HCC compared with normal liver, where expression was barely detectable. Next, radiation-inducible activity of the Egr-1 promoter was examined in primary cultured normal hepatocytes and human hepatoma cell lines Huh7, HepG2, and PLC/PRF/5 by luciferase assay as a reporter gene system. Egr-1 promoter activity was markedly increased in hepatoma cell lines in a radiation dose-dependent manner, with maximum activation (15- to 28-fold) 12 hr after irradiation. In contrast, only a twofold increase in activation was noted in normal hepatocytes. An in vitro gene therapy experiment showed that EGR-tk-transduced hepatoma cells became highly sensitive to ganciclovir (GCV) after irradiation, but not without irradiation. GCV with or without irradiation did not show any cytotoxic effects against control gene-transfected cells. In addition, a "radiosensitization effect" was also demonstrated by combination therapy with the HSV-tk/GCV system and irradiation. To examine the efficacy of this EGR-tk/GCV gene therapy in vivo, xenografted liver tumors in nude mice were targeted using the HVJ-liposome vector system. EGR-tk-transfected tumors regressed significantly after a combination therapy of irradiation and GCV in all mice (n = 8), and almost disappeared in 3 weeks without any side effects. In comparison, tumors continued to grow in all mice (n = 8 in each group) treated by transfer of EGR-tk followed by either irradiation without GCV or GCV without irradiation. Our data indicate that HSV-tk gene therapy under the control of a radioinducible promoter is effective, and might be selective for hepatoma cells because of its inducible and radiosensitive capacity after radiation exposure as well as its tumor-specific activation.     

以KDR为启动子的双自杀基因系统诱导结肠癌细胞凋亡的实验研究

目的研究以KDR为启动子的双自杀基因系统CDglyTK诱导结肠癌细胞凋亡的实验研究。方法用联合基因AdKDR-CDglyTK腺病毒转染表达KDR不同的人结肠癌细胞HCT116和LS174T,检测转染效率;RT-PCR检测双自杀基因CDglyTK的表达;用梯度浓度的前药5-FC及GCV处理,MTT法检测双自杀基因系统对HCT116和LS174T的细胞毒性作用;流式细胞术观察细胞凋亡变化;免疫组织化学法检测caspase-3蛋白的表达。结果携带双自杀基因的重组腺病毒载体成功转染结肠癌细胞。RT-PCR结果显示：表达KDR的HCT116细胞能表达目的基因,而不表达KDR的LS174T细胞不表达目的基因。5-FC和GCV对转染腺病毒的HCT116有明显的细胞毒作用而对LS174T细胞不敏感。流式细胞仪结果显示：给予5-FC和GCV后HCT116细胞出现凋亡峰,G0/G1升高、S期下降。Caspase-3表达增强（P〈0.01）。结论 KDR启动子驱动的双自杀基因系统CDglyTK能特异性杀伤结肠癌HCT116细胞,KDR可作为结肠癌基因治疗的靶点,并通过增强caspase-3途径诱导细胞凋亡。     

HSV vector cytotoxicity is inversely correlated with effective TK/GCV suicide gene therapy of rat gliosarcoma

Herpes simplex virus (HSV)-mediated delivery of the HSV thymidine kinase (tk) gene to tumor cells in combination with ganciclovir (GCV) administration may provide an effective suicide gene therapy for destruction of malignant glioblastomas. However, because HSV is a highly cytotoxic agent, gene expression from the virus is short-lived which may limit the effectiveness of HSVtk/GCV therapy. Using different replication-defective HSVtk gene vectors, we compared HSV vector backgrounds for their cytotoxic activity on infection of 9L gliosarcoma cells in culture and brain tumors in rats and evaluated the impact of vector toxicity on the effectiveness of tk/GCV-mediated suicide gene therapy. As reported previously for other cell lines, a vector deleted for both copies of the immediate-early (IE) gene ICP4 (SOZ.1) was highly toxic for 9L cells in culture while a vector deleted in addition for the ICP22 and ICP27 IE genes (T.1) reduced or arrested 9L cell proliferation with more limited cell killing. Nevertheless, both vectors supported widespread killing of uninfected cells in the presence of GCV following low multiplicity infections, indicating that vector cytotoxicity did not preempt the production of vector-encoded TK enzyme necessary for the killing of uninfected cells by the HSV-tk/GCV bystander effect. Although an SOZ.1-related vector (SHZ.2) caused tumor cell necrosis in vivo, injection of SHZ.2 at multiple coordinates thoughout the tumor followed by GCV administration failed to prolong markedly the survival of tumor-bearing rats. In contrast, a single injection of T.1 produced a life-extending response to GCV. These results indicate that vector cytotoxicity can limit the efficacy of HSV-tk/GCV treatment in vivo, which may be due to premature termination of tk gene expression with attendant abortion of the bystander effect.     

双自杀基因慢病毒转移载体系统的构建及其对K562细胞的杀伤作用

为了探讨双自杀基因慢病毒转移载体系统对K562细胞的杀伤作用,采用分子生物学方法构建了含大肠杆菌胞嘧啶脱氨酶(E.coli CD)和单纯疱疹病毒胸苷激酶(HSV-TK)的双自杀基因慢病毒转移载体,将慢病毒基因转移系统中的3种成分质粒(目的基因转移载体、包装结构及包膜结构质粒)用脂质体分为2组(实验组、对照组)共转染入病毒包装细胞293T,在荧光显微镜下观察转染效果,在透射电镜下观察上清中的病毒颗粒.大量收集病毒上清,浓缩后感染K562细胞,荧光显微镜下观察感染效果,RT-PCR鉴定目的基因在K562细胞中的整合转录.给予前体药物5-氟胞嘧啶(5-FC)和(或)无环鸟苷(GCV)后,用MTT法测定体外杀伤效应,扫描电镜下观察使用前体药物后K562细胞的变化.结果表明成功构建了双自杀基因慢病毒转移载体,脂质体法可有效将上述慢病毒表达质粒转入293T细胞.荧光显微镜下观察到对照组大量绿色荧光蛋白(green fluorescenceprotein,GFP)表达.透射电镜下观察到大量浓缩后病毒颗粒.荧光显微镜下观察到此基因转移载体系统对293T细胞及K562细胞的感染率均很高,单独使用GCV或5-FC对转染自杀基因的K562细胞的生长抑制率(growth inhibition ration,GIR)分别为48.73%、50.16%,与未转染K562细胞比较明显升高,有显著性差异(P＜0.01),联合使用5-FC和GCV时K562细胞的GIR为87.69%,比单独使用5-FC或GCV时明显升高,有显著性差异(P＜0.01).结论双自杀基因慢病毒基因转移载体系统可将双自杀基因高效转移至K562细胞,是一种有效的基因转移载体系统.     

Enhancement of thymidine kinase-mediated killing of malignant glioma by BimS,a BH3-only cell death activator

Herpes simplex virus thymidine kinase (HSV-tk)/gancyclovir (GCV) therapy has the ability to inhibit tumor formation in animal models but the results of clinical trials have been disappointing. To improve the performance of tk/GCV therapy, we tried combination therapy designed to enhance its cytotoxic effects by introducing genes that induce apoptosis of the tumor cells through different pathways. We concentrated our efforts on the use of Bim, a BH3-only member of death activators in the Bcl-2 superfamily, because Bim is not involved in the pathways through which HSV-tk/GCV therapy induces apoptosis in malignant glioma cells. Among three alternative splicing variants, BimEL, BimL, and BimS, BimS lacks the binding domain for the dynein light chain LC8, which negatively regulates the proapoptotic function of BimEL and BimL. All four malignant glioma cell lines, U251, A172, T-430, and U373 underwent cell death after transfer of BimS using an adenovirus vector (AVC2). Intriguingly, combination of AVC2-BimS with AVC2-tk markedly increased the sensitivity of U251 cells to GCV both in vitro and in vivo. In contrast, AVC2-BimL did not induce significant cell death. These results indicated that BimS had the ability to improve the efficiency of HSV-tk/GCV therapy in the treatment of malignant glioma and suggested that the targeting of different proapoptotic pathways may be a useful strategy for the development of an effective gene therapy approach to treatment.     

GCV modulates the antitumoural efficacy of a replicative adenovirus expressing the Tat8-TK as a late gene in a pancreatic tumour model

Replication-competent adenoviruses carrying the herpes simplex thymidine kinase (TK) gene have shown contradictory evidence with regard to their antitumoural efficacy in combination with ganciclovir (GCV) treatment. We generated a replication-competent adenovirus carrying Tat8-TK, a modified form of the TK gene, under the control of the adenoviral major late promoter (AdRGDTat8-TK-L). Pancreatic cancer cell lines with different sensitivity to the TK/GCV system were infected with AdRGDTat8-TK-L, both in the presence and absence of GCV, and tested for treatment efficacy. We observed that, although the presence of GCV reduced viral replication in all infected cell lines, in three out of four GCV significantly enhanced the efficacy of the virotherapy. Interestingly, the cytotoxicity of the AdRGD-Tat8-TK-L/GCV was found more potent than that of a first generation AdTK/GCV system. In tumour xenografts from BxPC-3 and NP-18 pancreatic cells, both AdRGDTat8-TK-L and AdRGDTat8-TK-L/GCV treatment showed antitumoural activity. In BxPC-3 tumours scheduling of virus and prodrug was a key factor to determine the outcome of the therapy. Importantly, the addition of GCV enhanced the antitumoural effect of AdRGDTat8-TK-L only when applied in two rounds of virus+GCV. Interestingly, in spite of interfering with viral replication in vitro, GCV treatment of NP-18 tumours did not compromise the antitumoural efficacy of the AdRGDTat8-TK-L adenovirus. Thus, our results show that the combination therapy of a replicative adenovirus and the Tat8-TK/GCV suicide system can prove beneficial, when the appropriate regimen of virus and GCV is applied.     

Adeno-Associated Viral-Mediated Gene Transfer to Hepatoma: Thymidine Kinase/Interleukin 2 Is More Effective in Tumor Killing in Non-Ganciclovir (GCV)-Treated Than in GCV-Treated Animals

Interleukin 2 (IL-2) enhancement of herpes simplex virus-thymidine kinase (HSV-TK)/ganciclovir (GCV)-induced tumor killing was studied by cloning the human interleukin 2 gene into an HSV-TK-bearing adeno-associated viral (AAV) vector (TK/IL-2). The mouse hepatocellular carcinoma cell line Hepa 1-6 was used as a model in this study. We found that TK/IL-2-transduced Hepa 1-6 cells were more susceptible to ganciclovir treatment than tumor cells transduced with only TK in both nude mice and immunocompetent C57L/J mice. TK/IL-2-transduced tumors also showed shrinkage without GCV treatment. The tumor-killing effect of AAV-mediated TK/IL-2 gene transfer was further studied by inoculating animals with TK/IL-2- or TK-transduced tumor cells mixed with unmodified cells with or without GCV treatment. Although tumor growth in each group was inhibited, the best result was obtained from the TK/IL-2-transduced group without GCV treatment. In this group, 10% of the transduced tumor cells could eradicate the whole tumor in 50% of the animals tested as well as provide long-term protection against tumor cell rechallenge. When this group was treated with GCV, the antitumor effect of TK/IL-2 was reduced. We attribute this to the early ablation of transgene-bearing tumor cells by GCV treatment, which thus reduces the duration of IL-2 expression. We conclude that (i) TK/IL-2 plus GCV treatment generates a stronger tumor-killing effect than HSV-TK plus GCV and (ii) tumor killing of TK/IL-2 is more effective in non-GCV-treated animals than in GCV-treated animals.     

Effect of combined cytostatic cyclosporin A and cytolytic suicide gene therapy on the prevention of experimental graft-versus-host disease

The immunosuppressive drug cyclosporin A (CsA) represents the standard preventive treatment of graft-versus-host disease (GVHD), the main complication of allogeneic hematopoietic stem cell transplantation (HSCT). However, its efficacy is only partial and many patients develop lethal GVHD despite CsA. A strategy of genetic immunosuppression based on conditional elimination of donor T cells expressing the Herpes simplex type 1 thymidine kinase (TK) suicide gene was recently developed. In this system, ganciclovir (GCV) selectively kills dividing but not quiescent TK T cells. Since CsA is known to have a cytostatic effect on T cells, it could negatively interfere with the division-dependent TK gene therapy. We thus tested whether administration of CsA would antagonize elimination of alloreactive donor TK T cells mediated by GCV in a murine model of GVHD. In vivo experiments revealed that, contrary to GCV, CsA only transiently controlled alloactivation-induced T cell proliferation, and likewise could not prevent lethal GVHD. When T cells resumed proliferation under CsA, they were however still sensitive to GCV. Survival, as well as immune reconstitution, was excellent in mice treated with GCV alone or in combination with CsA. These observations should help to design improved suicide gene therapy trials in the field of allogeneic HSCT.     

Eradication of breast cancer xenografts by hyperthermic suicide gene therapy under the control of the heat shock protein promoter

To investigate the usefulness of heat shock protein (HSP) promoter for breast cancer gene therapy, hyperthermia and HSV thymidine kinase (tk) suicide gene combination therapy was examined with mouse mammary cancer cell line FM3A. HSP promoter activity was markedly increased after heat shock (41-45 degrees C), with maximum activation (about 400-fold) at 3 hr. An in vitro cytotoxic assay showed that HSP-tk-transduced FM3A cells became more sensitive (more than 50,000 times) to ganciclovir (GCV) with heat shock, but untreated cells showed no increased cytotoxic sensitivity to GCV compared with control FM3A cells. In addition to promoter-oriented selective cell killing, a "chemosensitization effect" as a bystander effect was demonstrated by hyperthermia and suicide gene combination therapy, using a non-heat-inducible promoter. Immunohistochemical analysis revealed that this synergistic killing effect was dependent on apoptotic cell death with upregulation of both Fas and FasL (Fas ligand) expression. We also examined the efficacy of HSP-tk gene therapy in vivo by implanting breast cancer in subcutaneous and intraperitoneal models of BALB/c nude mice targeted by the HVJ-anionic liposome method. Significant tumor regression was observed in HSP-tk-transduced tumors followed by hyperthermia therapy, but no such inhibition was noted in either the mock vector transfection or hyperthermia group compared with control tumor-bearing mice. Our results demonstrate that this combination system is synergistically effective in mediating Fas-dependent apoptosis for a specific gene therapy targeting HSP-expressing mammary carcinomas, even in advanced and heat-resistant breast cancer.