腺病毒载体介导的靶向性肿瘤基因治疗研究
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摘要
基因转运技术是肿瘤基因治疗的重要环节之一,寻找合适的基因转运载体一直是人们的研究热点。腺病毒载体因具有宿主范围宽广、感染效率高、可插入外源基因片断大、病毒相对容易制备和不会与受染细胞发生整合等诸多优点而受到特别的重视,成为肿瘤基因治疗中应用最为广泛的载体系统。目前有多种策略和方法用于对腺病毒进行改造,使其对肿瘤细胞具有靶向性和杀伤力,包括改造病毒衣壳蛋白或利用双功能连接物把腺病毒靶向导入肿瘤细胞;利用一些肿瘤特异性启动子、组织特异性启动子和可调控性启动子进行转录水平的基因表达调控。
根据杀伤肿瘤细胞的作用机理的不同,可把腺病毒分为复制缺陷型腺病毒和条件增殖型腺病毒两大类。复制缺陷型腺病毒能起到运输载体的作用,携带外源基因(抑癌基因、细胞凋亡诱导基因、免疫调节因子、血管生成抑制因子、自杀基因等等)靶向转导入肿瘤细胞,从而引起细胞的死亡;条件增殖型腺病毒是一类经过转录调节修饰的病毒,可以以高度的特异性在靶细胞中复制增殖并最终裂解该细胞,同时释放出子代病毒再感染邻近的细胞,直至把所有肿瘤细胞杀死为止,而对非靶细胞无杀伤力。
本课题主要是基于腺病毒载体就靶向性肿瘤基因治疗开展了相关的工作。主要研究内容可分为两大部分:
第一部分:双重调控的靶向性肿瘤增殖腺病毒的构建及抗瘤研究。本课题引入两个在绝大多数肿瘤细胞中均高表达而在正常组织中低表达甚至不表达的肿瘤特异性启动子人端粒酶催化亚单位启动子和人环氧合酶-2启动子来分别调控腺病毒早期必需基因E1A和E1B,使经过改造的重组增殖腺病毒只能在同时表达人端粒酶和人环氧合酶-2的细胞内增殖并裂解该细胞,而在正常的细胞内不能有效增殖。在本实验中,通过将腺病毒穿梭质粒载体pD306/H181-C409和骨架质粒载体pBHGE3共转染工具细胞Hek293,经过在细胞内随机同源重组后,成功得到了一种新型的由人端粒酶催化亚单位启动子和人环氧合酶-2启动子共同调控的条件增殖型腺病毒Ad5-HC。通过Western blot法检测表明E1A和E1B可在多种被Ad5-HC作用后的肿瘤细胞中表达,而在正常细胞中不表达;病毒增殖试验表明Ad5-HC可在多种肿瘤细胞内大量扩增,其扩增倍数是正常细胞的上万倍;细胞杀伤试验表明Ad5-HC对多种不同类型的肿瘤细胞有强大的杀伤效果;当Ad5-HC联合化疗药物作用于肿瘤细胞时,它能与包括5-FU、表柔比星、紫杉醇在内的多种化疗药物产生协同疗效;裸鼠移植瘤抗瘤活性试验表明Ad5-HC在动物体内能有效抑制瘤体的生长。本实验采用一系列体外试验和体内试验,全面验证了由hTERT启动子和Cox-2启动子双重调控的肿瘤增殖腺病毒Ad5-HC良好的肿瘤细胞特异性和细胞杀伤作用,从而为肿瘤的病毒基因治疗提供了一种新的思路和方法。
第二部分:由AFP启动子调控且携带人工合成微小RNA的重组腺病毒的构建及抗瘤研究。本课题用人工合成的方式合成多段能直接静默细胞正常新陈代谢所必需的基因的微小RNA序列,包括磷酸甘油醛脱氢酶(glyceraldehyde phosphatedehydrogenase,GAPDH)、真核起始因子4E(eukaryotic translation initiation factor4E,eIF4E)和DNA聚合酶α(DNA polymeraseα)。通过利用复制缺陷型腺病毒载体携带这些人工合成的微小RNA进入细胞,同时利用AFP启动子在肝癌细胞内特异性高表达的特性来调控外源基因的表达,使得这些微小RNA能被靶向导入到AFP阳性的肝癌细胞内,从而下调靶基因的表达进而影响其生理功能。在本实验中,一系列携带微小RNA的复制缺陷型腺病毒被成功构建,包括Ad5-miRNA5G、Ad5-miRNA6E、Ad5-miRNA6P和Ad5-GFP。通过采用一系列体外试验(包括RT-PCR检测靶基因的mRNA水平、Western blot检测靶基因蛋白的表达、病毒细胞毒性试验、细胞活性检测)和体内试验(裸鼠移植瘤抗瘤活性试验),全面验证了重组腺病毒良好的肝癌细胞特异性和杀伤力;通过对病毒感染后的细胞进行细胞周期分析、ATP酶含量检测、总蛋白含量检测等方法进一步从作用机理上进行了分析,发现携带微小RNA的重组腺病毒不仅可导致肿瘤细胞在G2/M期阻滞,还可使细胞内ATP酶和总蛋白含量下降。总之,利用腺病毒的载体作用携带人工合成的微小RNA,通过组织特异性启动子靶向转导基因表达,可以使重组腺病毒在靶细胞内发挥明显的细胞杀伤作用。
The technology of transferring genetic materials to the targeted tissues successfully is the main objective in cancer gene therapy.During the past two decades, enormous research in the area of gene delivery has been conducted worldwide.Due to the ability of infecting both dividing and non-dividing cells,the efficient nuclear entry mechanism and its low pathogenicity for humans,adenovirus-based vectors have become widely used vehicles for cancer gene therapy.By now,quite a range of different transductional targeting strategies have been successfully developed in order to improve the efficiency of adenovirus-mediated gene delivery to tumor cells, including genetically encoded or chemically engineered structural modification of the capsid and the use of bispecific adaptor molecules directing vector attachment to alternative cell surface receptors;using some tissue-specific promoters to regulate the gene expression in target cells.
According to the mechanism of action,the adenovirus can be divided into two types,replication-defective adenovirus and conditionally replication-competent adenovirus.The replication-defective adenovirus can not replicate in cells.It always acts as a vehicle carrying some exogenous genes,e.g.,tumor suppressor genes, apoptosis inducer genes,immune regulate factors,angiogenesis inhibitors,suicide genes, to target cells and inducing cell death eventually.The conditionally replication-competent adenovirus has the ability of replicating within target cells only.It can infect,usurp host replication machinery,and release newly made progeny to infect other target cells after lysing and killing the host cell.
In our study,two types of adenovirus vectors are utilized for cancer gene therapy.
Those including:
Part one:The construction of a dually-regulated oncolytic adenovirus and its antitumor efficacy on most human solid cancers.Human telomerase reverse transcriptase(hTERT) and cyclooxygenase-2(Cox-2) are always over expressed in tumor cells but not expressed in normal tissue.In our study,we developed a novel therapy strategy using oncolytic adenovirus containing the E1A controlled by hTERT promoter and E1B controlled by Cox-2 promoter.The adenovirus vector pBHGE3 was co-transfected with adenovirus shuttle plasmid pD306/H181-C409 into Hek293 cells using the transfection reagent Lipofectamine~(TM)2000.After homologously recombining in Hek293 cells,the oncolytic adenovirus Ad5-HC was obtained.A series of in vitro and in vivo assay were performed to evaluate the specificity and cytotoxicity of Ad5-HC,including western blot analysis,adenovirus yield assay,cell viability assay and animal study.The results demonstrated that the oncolytic adenovirus Ad5-HC regulated by both hTERT promoter and Cox-2 promoter has a tumor-specific replication ability and cytolytic ability to a wide range of cancer cells which were positive for telomerase activity and Cox-2 expression.The novel dually-regulated oncolytic adenovirus Ad5-HC has a great potential of destroying tumors and improving the survival of cancer patients.
Part two:The construction of replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter and its antitumor efficacy on HCC cells.In our study,three types of artificial multi-microRNAs were constructed.Some essential genes for cells metabolism are silenced by these miRNAs, including glyceraldehyde phosphate dehydrogenase(GAPDH),which is an enzyme that catalyzes the process of glycolysis and thus serves to break down glucose for energy and carbon molecules;eukaryotic translation initiation factor 4E(eIF4E), which plays a crucial role in gene expression by controlling protein synthesis at the level of translation initiation;DNA polymerase a,which is a key replicative enzyme, plays an essential role in initiation of both leading- and lagging-strand synthesis.We systematically analyzed the tissue specificity,effect of gene silencing and cytotoxicity of these new-type replication defective adenovirus in order to explore a novel effective strategy for HCC gene therapy.The results demonstrated that the replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter can knockdown the target genes which are essential in cells metabolism effectively,and they had a potent ability of inhibiting the HCC cells growth in a tumor-specific manner.The combination of tumor-specific promoter and artificial multi-miRNAs will be a novel strategy in targeted cancer gene therapy and may become a promising approach in future.
根据杀伤肿瘤细胞的作用机理的不同,可把腺病毒分为复制缺陷型腺病毒和条件增殖型腺病毒两大类。复制缺陷型腺病毒能起到运输载体的作用,携带外源基因(抑癌基因、细胞凋亡诱导基因、免疫调节因子、血管生成抑制因子、自杀基因等等)靶向转导入肿瘤细胞,从而引起细胞的死亡;条件增殖型腺病毒是一类经过转录调节修饰的病毒,可以以高度的特异性在靶细胞中复制增殖并最终裂解该细胞,同时释放出子代病毒再感染邻近的细胞,直至把所有肿瘤细胞杀死为止,而对非靶细胞无杀伤力。
本课题主要是基于腺病毒载体就靶向性肿瘤基因治疗开展了相关的工作。主要研究内容可分为两大部分:
第一部分:双重调控的靶向性肿瘤增殖腺病毒的构建及抗瘤研究。本课题引入两个在绝大多数肿瘤细胞中均高表达而在正常组织中低表达甚至不表达的肿瘤特异性启动子人端粒酶催化亚单位启动子和人环氧合酶-2启动子来分别调控腺病毒早期必需基因E1A和E1B,使经过改造的重组增殖腺病毒只能在同时表达人端粒酶和人环氧合酶-2的细胞内增殖并裂解该细胞,而在正常的细胞内不能有效增殖。在本实验中,通过将腺病毒穿梭质粒载体pD306/H181-C409和骨架质粒载体pBHGE3共转染工具细胞Hek293,经过在细胞内随机同源重组后,成功得到了一种新型的由人端粒酶催化亚单位启动子和人环氧合酶-2启动子共同调控的条件增殖型腺病毒Ad5-HC。通过Western blot法检测表明E1A和E1B可在多种被Ad5-HC作用后的肿瘤细胞中表达,而在正常细胞中不表达;病毒增殖试验表明Ad5-HC可在多种肿瘤细胞内大量扩增,其扩增倍数是正常细胞的上万倍;细胞杀伤试验表明Ad5-HC对多种不同类型的肿瘤细胞有强大的杀伤效果;当Ad5-HC联合化疗药物作用于肿瘤细胞时,它能与包括5-FU、表柔比星、紫杉醇在内的多种化疗药物产生协同疗效;裸鼠移植瘤抗瘤活性试验表明Ad5-HC在动物体内能有效抑制瘤体的生长。本实验采用一系列体外试验和体内试验,全面验证了由hTERT启动子和Cox-2启动子双重调控的肿瘤增殖腺病毒Ad5-HC良好的肿瘤细胞特异性和细胞杀伤作用,从而为肿瘤的病毒基因治疗提供了一种新的思路和方法。
第二部分:由AFP启动子调控且携带人工合成微小RNA的重组腺病毒的构建及抗瘤研究。本课题用人工合成的方式合成多段能直接静默细胞正常新陈代谢所必需的基因的微小RNA序列,包括磷酸甘油醛脱氢酶(glyceraldehyde phosphatedehydrogenase,GAPDH)、真核起始因子4E(eukaryotic translation initiation factor4E,eIF4E)和DNA聚合酶α(DNA polymeraseα)。通过利用复制缺陷型腺病毒载体携带这些人工合成的微小RNA进入细胞,同时利用AFP启动子在肝癌细胞内特异性高表达的特性来调控外源基因的表达,使得这些微小RNA能被靶向导入到AFP阳性的肝癌细胞内,从而下调靶基因的表达进而影响其生理功能。在本实验中,一系列携带微小RNA的复制缺陷型腺病毒被成功构建,包括Ad5-miRNA5G、Ad5-miRNA6E、Ad5-miRNA6P和Ad5-GFP。通过采用一系列体外试验(包括RT-PCR检测靶基因的mRNA水平、Western blot检测靶基因蛋白的表达、病毒细胞毒性试验、细胞活性检测)和体内试验(裸鼠移植瘤抗瘤活性试验),全面验证了重组腺病毒良好的肝癌细胞特异性和杀伤力;通过对病毒感染后的细胞进行细胞周期分析、ATP酶含量检测、总蛋白含量检测等方法进一步从作用机理上进行了分析,发现携带微小RNA的重组腺病毒不仅可导致肿瘤细胞在G2/M期阻滞,还可使细胞内ATP酶和总蛋白含量下降。总之,利用腺病毒的载体作用携带人工合成的微小RNA,通过组织特异性启动子靶向转导基因表达,可以使重组腺病毒在靶细胞内发挥明显的细胞杀伤作用。
The technology of transferring genetic materials to the targeted tissues successfully is the main objective in cancer gene therapy.During the past two decades, enormous research in the area of gene delivery has been conducted worldwide.Due to the ability of infecting both dividing and non-dividing cells,the efficient nuclear entry mechanism and its low pathogenicity for humans,adenovirus-based vectors have become widely used vehicles for cancer gene therapy.By now,quite a range of different transductional targeting strategies have been successfully developed in order to improve the efficiency of adenovirus-mediated gene delivery to tumor cells, including genetically encoded or chemically engineered structural modification of the capsid and the use of bispecific adaptor molecules directing vector attachment to alternative cell surface receptors;using some tissue-specific promoters to regulate the gene expression in target cells.
According to the mechanism of action,the adenovirus can be divided into two types,replication-defective adenovirus and conditionally replication-competent adenovirus.The replication-defective adenovirus can not replicate in cells.It always acts as a vehicle carrying some exogenous genes,e.g.,tumor suppressor genes, apoptosis inducer genes,immune regulate factors,angiogenesis inhibitors,suicide genes, to target cells and inducing cell death eventually.The conditionally replication-competent adenovirus has the ability of replicating within target cells only.It can infect,usurp host replication machinery,and release newly made progeny to infect other target cells after lysing and killing the host cell.
In our study,two types of adenovirus vectors are utilized for cancer gene therapy.
Those including:
Part one:The construction of a dually-regulated oncolytic adenovirus and its antitumor efficacy on most human solid cancers.Human telomerase reverse transcriptase(hTERT) and cyclooxygenase-2(Cox-2) are always over expressed in tumor cells but not expressed in normal tissue.In our study,we developed a novel therapy strategy using oncolytic adenovirus containing the E1A controlled by hTERT promoter and E1B controlled by Cox-2 promoter.The adenovirus vector pBHGE3 was co-transfected with adenovirus shuttle plasmid pD306/H181-C409 into Hek293 cells using the transfection reagent Lipofectamine~(TM)2000.After homologously recombining in Hek293 cells,the oncolytic adenovirus Ad5-HC was obtained.A series of in vitro and in vivo assay were performed to evaluate the specificity and cytotoxicity of Ad5-HC,including western blot analysis,adenovirus yield assay,cell viability assay and animal study.The results demonstrated that the oncolytic adenovirus Ad5-HC regulated by both hTERT promoter and Cox-2 promoter has a tumor-specific replication ability and cytolytic ability to a wide range of cancer cells which were positive for telomerase activity and Cox-2 expression.The novel dually-regulated oncolytic adenovirus Ad5-HC has a great potential of destroying tumors and improving the survival of cancer patients.
Part two:The construction of replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter and its antitumor efficacy on HCC cells.In our study,three types of artificial multi-microRNAs were constructed.Some essential genes for cells metabolism are silenced by these miRNAs, including glyceraldehyde phosphate dehydrogenase(GAPDH),which is an enzyme that catalyzes the process of glycolysis and thus serves to break down glucose for energy and carbon molecules;eukaryotic translation initiation factor 4E(eIF4E), which plays a crucial role in gene expression by controlling protein synthesis at the level of translation initiation;DNA polymerase a,which is a key replicative enzyme, plays an essential role in initiation of both leading- and lagging-strand synthesis.We systematically analyzed the tissue specificity,effect of gene silencing and cytotoxicity of these new-type replication defective adenovirus in order to explore a novel effective strategy for HCC gene therapy.The results demonstrated that the replication-defective adenovirus containing artificial multi-microRNAs regulated by AFP enhancer/promoter can knockdown the target genes which are essential in cells metabolism effectively,and they had a potent ability of inhibiting the HCC cells growth in a tumor-specific manner.The combination of tumor-specific promoter and artificial multi-miRNAs will be a novel strategy in targeted cancer gene therapy and may become a promising approach in future.
引文
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