基因矫正治疗中的启动子研究

利用组织或器官特异性启动子限制目的基因只在靶肿瘤或靶器官中表达是基因治疗的一种有效途径,如AFP启动子、CEA启动子、Tyr启动子等.而利用应激刺激的可诱导启动子则可能解决组织或器官特异性启动子的弱表达问题,如Hsp启动子、hTERT启动子等.现综述基因矫正治疗在肿瘤基因治疗领域有应用价值的启动子研究进展.     

基因矫正治疗中的启动子研究

利用组织或器官特异性启动子限制目的基因只在靶肿瘤或靶器官中表达是基因治疗的一种有效途径,如AFP启动子、CEA启动子、Tyr启动子等.而利用应激刺激的可诱导启动子则可能解决组织或器官特异性启动子的弱表达问题,如Hsp启动子、hTERT启动子等.现综述基因矫正治疗在肿瘤基因治疗领域有应用价值的启动子研究进展.     

肿瘤特异性启动子驱动的RNA干扰与肿瘤基因靶向治疗

应用肿瘤特异性启动子如甲胎蛋白启动子、人端粒酶逆转录酶启动子、存活素启动子等,驱动RNA干扰(RNAi)可在肿瘤组织中发挥靶向治疗效应.该方法把肿瘤启动子的特异性和RNAi的高效性有机结合起来,可以实现对肿瘤组织的特异、高效杀伤作用.肿瘤特异性启动子驱动的RNAi在肿瘤基因靶向治疗中发挥重要作用.     

肿瘤-睾丸基因研究进展

肿瘤-睾丸(CT)基因可产生特异性抗原,诱导机体生成针对肿瘤细胞的特异性体液和(或)T淋巴细胞,在肿瘤免疫治疗中很有前景.现综述CT基因的研究进展、命名规范、数据库、分布情况、抗原表达及识别,并对研究中许多未知问题进行初步展望.     

肿瘤端粒酶靶向腺病毒载体的表达活性

目的　构建针对端粒酶阳性肿瘤的特异性腺病毒载体 ,研究其介导目的基因在肿瘤细胞内的定向表达能力。方法　将端粒酶逆转录酶 (hTERT )启动子克隆入质粒 pDC3 12的多克隆位点构成外源基因表达盒 ,在基因表达盒上下游分别插入 1个绝缘子序列 ,构建针对端粒酶阳性肿瘤的特异性腺病毒载体pSG TPE ;采用Luciferase报告基因进行hTERT启动子的活性分析 ;以pSG TPE携带绿色荧光蛋白 (EGFP)报告基因 ,重组腺病毒AdTPE EGFP ,观察其介导EGFP在肿瘤细胞内的定向表达能力 ,并与CMV启动子控制的腺病毒AdCMV EGFP作对照。结果　hTERT启动子在正常细胞内几乎没有活性 ,而在肿瘤细胞内的活性与SV 40启动子相近。腺病毒AdTPE EGFP能介导EGFP在肿瘤细胞内定向而稳定表达 ,在正常细胞内不表达 ,而对照腺病毒AdCMV EGFP在肿瘤和正常细胞内均表达EGFP。结论　靶向端粒酶阳性肿瘤的腺病毒载体 pSG TPE ,不但提高了对基因表达调控的特异性 ,而且降低了对正常细胞毒性作用。绝缘子的应用隔断了外源基因表达盒和病毒基因表达调控序列之间的互相干扰 ,进一步提高目的基因的表达效率。该载体对肿瘤的靶向基因治疗具有重要的应用价值。     

构建以hTERT、Cox-2启动子调控的肿瘤特异增殖腺病毒

目的 应用同源重组的方法构建以hTERT和Cox-2启动子调控增殖的肿瘤特异性增殖腺病毒.方法 将hTERT和Cox-2启动子从人类白细胞基因组中亚克隆出来,并将启动子分别插入到腺病毒穿梭载体pd306上的E1A和E1B基因前,使hTERT和Cox-2启动子分别调控腺病毒必须基因E1A和E1B的表达,再将构建后的pd306和腺病毒的骨架质粒BHGE3在Ad293细胞内进行同源重组,并用重组后的病毒感染Hela细胞检测病毒对肿瘤细胞的杀伤力.结果 成功构建了hTERT和Cox-2启动子,并将两个启动子连接入腺病毒载体,产生了具有感染力的可增殖腺病毒.结论 经hTERT和Cox-2启动子调控增殖的肿瘤特异性增殖腺病毒对Hela细胞具有杀伤力,为进一步研究病毒在体内、外对各种肿瘤细胞的特异性杀伤力、安全性奠定了基础.     

肿瘤基因治疗的靶向性策略

肿瘤基因治疗的靶向性问题直接关系到治疗的成败。这种靶向性的策略包括目的基因对肿瘤细胞的靶向转移，目的基因在肿瘤细胞中的特异表达，以及基因修饰细胞分泌的肿瘤靶向治疗分子。对现有病毒或非病毒载体进行改造，由与载体融合的抗体或配体与肿瘤表面抗原或受体的相互作用可以实现目的基因的靶向转移；肿瘤或组织特异性调控元件的应用，以及一些物化、生理因素的诱导则能使治疗基因特异地在肿瘤细胞中表达并发挥作用。     

靶向肿瘤的溶瘤腺病毒制备策略的研究进展

溶瘤腺病毒是指经过基因工程改造的腺病毒,其能够选择性地在肿瘤细胞中复制和表达,从而溶解肿瘤细胞;其可经过基因和衣壳蛋白层面的改造,特异性地结合和杀伤肿瘤细胞。自1996 年世界上第一例溶瘤腺病毒ONXY-015 开展临床研究以来,腺病毒在国内外广泛地应用于科学研究及转化应用,已有多个国家批准其在临床肿瘤治疗中使用,使用范围涉及到多种实体瘤。溶瘤腺病毒的改造方式多种多样,本文对溶瘤腺病毒治疗肿瘤的改造方法,如腺病毒的包膜蛋白进行修饰、腺病毒的结构基因进行改造、插入肿瘤特异性启动子、携带治疗基因与携带短发夹RNA和包括溶瘤病毒的多措施联合等研究进展作一综述。     

肿瘤-睾丸基因研究进展

肿瘤-睾丸(CT)基因可产生特异性抗原，诱导机体生成针对肿瘤细胞的特异性体液和(或)T淋巴细胞，在肿瘤免疫治疗中很有前景。现综述CT基因的研究进展、命名规范、数据库、分布情况、抗原表达及识别，并对研究中许多未知问题进行初步展望。     

CAGE基因在颅脑肿瘤中的表达及其机制

背景与目的:检测颅脑肿瘤组织中CAGE基因的表达及启动子区甲基化情况,探讨将其用于肿瘤免疫治疗的可能性.材料与方法:采用RT-PCR及甲基化特异性PCR,检测8种正常组织(17例),35例脑膜瘤和32例胶质瘤组织CAGE基因表达及启动子区甲基化情况.结果:CAGE基因在除睾丸以外的正常组织中不表达,4例脑膜瘤(11.3%)和26例胶质瘤(81.25%)CAGE基因表达阳性;CAGE基因表达阳性的组织非甲基化扩增均为阳性,表达阴性的组织甲基化扩增均为阳性.结论:CAGE基因启动子区甲基化与其表达密切相关,CAGE可能成为胶质瘤的肿瘤特异性抗原.     

Use of the PSA enhancer core element to modulate the expression of prostate- and non-prostate-specific basal promoters in a lentiviral vector context

Composite promoters combining the prostate-specific antigen (PSA) enhancer core element with promoter elements derived from gene coding for human prostate-specific transglutaminase gene, prostate-specific membrane antigen gene, prostate-specific antigen, rat probasin or phosphoglycerate kinase were characterized for their ability to specifically express the enhanced green fluorescent protein (EGFP) gene in prostate versus non-prostate cancer cell lines when transferred with a human immunodeficiency virus-1-based lentiviral vector. By themselves minimal proximal promoter elements were found to inefficiently promote relevant tissue-specific expression; in all the vectors tested, addition of the PSA enhancer core element markedly improved EGFP expression in LnCaP, a cancer prostate cell line used as a model for prostate cancer. The composite promoter was inactive in HuH7, a hepatocarcinoma cell line used as a model of neighboring non-prostate cancer cells. Among the promoters tested, the combination of the PSA enhancer and the rat probasin promoter showed both high specificity and a strong EGFP expression. Neither a high viral input nor the presence of the cPPT/CTS sequence affected composite promoter behavior. Our data suggest that composite prostate-specific promoters constructed by combining key elements from various promoters can improve and/or confer tissue specific expression in a lentiviral vector context.     

Carcinoembryonic antigen-specific suicide gene therapy of cytosine deaminase/5-fluorocytosine enhanced by the cre/loxP system in the orthotopic gastric carcinoma model

Tumor-specific gene delivery is crucial to achieving successful effects in suicide gene therapy. Carcinoembryonic antigen (CEA) promoter has been widely used for this purpose, but the expression level of tumor-specific promoters such as CEA promoter is generally low. In the previous study, we used the Cre/loxP system and showed that LacZ expression by the CEA promoter was remarkably enhanced and maintained its specificity using the Cre/loxP regulation system. In this study, the Cre/loxP system was first applied to augmentation of selective expression of the cytosine deaminase (CD) gene as a suicide gene therapy in CEA-producing cells. The double infection with AxCEANCre expressing Cre recombinase under the control of the CEA promoter and AxCALNLCD expressing the CD gene under the control of the CAG promoter by the Cre switching system rendered CEA-producing tumor cells 13-fold more sensitive to 5-fluorocytosine (5-FC) compared with the single infection with AxCEACD expressing CD gene driven by the CEA promoter. The therapeutic efficacy of the enhanced CD/5-FC suicide gene therapy was evaluated in orthotopic implantation models of human gastric carcinoma. Adenovirus vectors (1 x 10(9) plaque-forming units) were administered i.p. into mice three times, and then 5-FC was administered i.p. for the next 10 days. Tumor volume and weight in mice treated with AxCEANCre and AxCALNLCD/5-FC were significantly reduced as compared with those in mice treated not only with Mock (AxCALacZ) but also with AxCEACD/5-FC (P < 0.0001). This beneficial effect on tumor burden was also reflected in the overall survival. The survival periods of the mice treated with AxCEANCre and AxCALNLCD/5-FC were longer than those of mice treated with Mock or AxCEACD/5-FC (P < 0.01). These results suggested that application of the Cre/loxP system could provide a new approach for enhanced selective suicide gene therapy of CD/5-FC for the treatment of advanced gastric carcinoma.     

Strength and specificity of different gene promoters in oral cancer cells

Gene therapy of oral cancer will require expression of genes by promoters that are both powerful and relatively tumor specific. We compared the level of expression of a reporter gene from promoters of human cytomegalovirus (CMV), SV40 virus, mouse mammary tumor virus (MMTV), human papillomaviruses (HPV) types 16 and 18, and the human multi-drug-resistance gene (mdr1), in several lines of oral cancer cells. In the oral cancer cell line 686LN the rank order of expression levels was: CMV > SV40 > HPV > mdr1 > MMTV. Unlike in previous reports the mdr1 promoter was no more active in two cancer cell lines with mutations in the p53 gene than in two other lines with wild-type p53, and its expression level could not be increased by either doxorubicin or taxol. On the other hand, expression from the MMTV promoter was increased over 10-fold by the presence of 1 microM dexamethasone. Thus, by an appropriate choice of promoter and inducer a wide variety of expression levels, over a 3-log range, could be attained in 686LN cells. The oral cancer-specificity of each promoter was judged by comparing expression in the neuroblastoma line IMR32. The most specific promoters were those from papillomaviruses, which were up to 45 times more active in the oral cancer cells, and the least specific was the CMV promoter. In order to find if an HPV-derived promoter was sufficient to produce expression of a suicide phenotype the 686 promoter was cloned adjacent to the thymidine kinase gene of herpes simplex and the construct was expressed from an adenovirus vector. The vector reduced the growth of 686LN cells over a 5-day period by up to 32% when optimal concentrations of virus and ganciclovir were used. These data will be valuable in the design of new constructs for gene therapy of oral cancer.     

Targeted gene therapy for breast cancer with truncated Bid

We studied the efficiency of the proapoptotic factor tBid, targeted to tumor cells using the promoters of the hTERT, Survivin and Muc1 genes, in killing breast cancer cells. tBid is the active fragment of the proapoptotic protein Bid and is generated in response to death receptor activation. When placed under control of a strong CMV promoter, tBid was highly efficient in killing breast cancer cells. When expression of tBid was driven by tumor-specific promoters, the magnitude of killing was significant in cell lines with high levels of promoter activity. For successful gene therapy with targeted tBid, it is therefore crucial to be able to predict promoter activity prior to selection of the therapeutic construct. To test whether gene expression could serve as a predictor, we correlated expression of Survivin, hTERT and Muc1 genes with the activity of the corresponding promoters in a panel of breast cancer cell lines. Expression of the Muc1 gene correlated well with the activity of its promoter and the resultant tumor cell killing. For the hTERT and Survivin promoters, however, promoter activity did not correlate well with the expression of the corresponding genes. The implications and possible mechanism of these discrepancies are discussed.     

Survivin启动子与肿瘤

利用特异性启动子介导的基因治疗是肿瘤靶向性治疗的一种有效途径.Survivin启动子既有肿瘤细胞的靶向性,又有增殖性血管内皮细胞的靶向性,且能有效介导目的基因表达,展示了其在肿瘤靶向性治疗研究中的良好前景.     

The carcinoma-specific epithelial glycoprotein-2 promoter controls efficient and selective gene expression in an adenoviral context

Adenoviral vectors are widely used in cancer gene therapy. After systemic administration however, the majority of the virus homes to the liver and the expressed transgene may cause hepatotoxicity. To restrict transgene expression to tumor cells, tumor- or tissue-specific promoters are utilized. The tumor antigen epithelial glycoprotein-2 (EGP-2), also known as Ep-CAM, is expressed in many cancers from different epithelial origins. In this study, the EGP-2 promoter was shown to restrict the expression of luciferase and thymidine kinase in an adenoviral context in different cell lines. In vivo, the EGP-2 promoter mediated efficient expression of luciferase in tumors but showed a 3-log lower activity in liver tissue when compared with the cytomegalovirus (CMV) promoter. Similarly, the EGP-2 promoter mediated specific cell killing after ganciclovir treatment in EGP-2-positive cells. Moreover, in vivo, this treatment regiment did not cause any rise in the liver enzymes aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT), demonstrating absence of liver toxicity. In contrast, CMV-mediated expression of thymidine kinase in combination with ganciclovir treatment resulted in high ASAT and ALAT values. This study demonstrates the value of the EGP-2 promoter to restrict transgene expression to a broad range of tumor types, thereby preventing liver toxicity.     

Heat-inducible vectors for use in gene therapy.

The objectives of this study were to quantity and compare the activities of a minimal heat shock (HS) promoter and other promoters used in gene therapy applications, and to identify strategies to amplify the heat inducibility of therapeutic genes. Human tumour cells were transiently or stably transfected with the HS promoter driving expression of reporter genes. HS promoter activity was induced transiently, with maximum activity 16-24 h after HS, and was dependent on temperature. The activity of the minimal HS promoter was similar, after 42 degrees C HS for 1 h, to that of the cytomegalovirus (CMV) promoter. To determine if the HS promoter could be used to activate a second conditional promoter, cells were transiently transfected with vectors containing both the HS and human immunodeficiency virus type 1 (HIV1) promoters. When the IL-2 gene was placed downstream of the HIV1 promoter. IL-2 production was temperature-independent. The addition of the HIV tat gene downstream of the HS promoter caused IL-2 to be induced more than 3 fold after a single 42 degrees C HS. These data indicate that the minimal HS promoter, following activation by clinically attainable temperatures (< or = 42 degrees C), can drive expression of therapeutic genes at levels comparable to the CMV promoter and be used in conjunction with a second conditional promoter to drive temperature-dependent, gene expression.