溶瘤腺病毒肿瘤靶向治疗：从实验室到临床

过去几十年从实验室到临床对溶瘤腺病毒进行了广泛的研究.有多种策略增强溶瘤腺病毒的肿瘤靶向性,包括将腺病毒基因组中参与细胞周期节点调控的功能基因(如E1A或E1B)进行突变或(和)利用肿瘤特异性启动子调控E1A基因的靶向转录调控,利用不同血清型腺病毒或RGD基序改变溶瘤腺病毒进入肿瘤细胞方式的靶向转导调控,以及利用细胞载体将溶瘤腺病毒传送至远处的肿瘤部位.溶瘤腺病毒作为一种载体,输送免疫调节基因或治疗性基因,通过增强抗肿瘤免疫,或引起肿瘤细胞凋亡、自杀等产生协同抗肿瘤效应.溶瘤腺病毒临床试验涉及到多种实体瘤,显示其临床应用是安全的,毒性作用轻至中度,患者能很好耐受,但有明确客观抗肿瘤反应的临床病例较少见,联合诸如化、放疗等治疗方法有助于提高临床效果. 未来的方向应强化溶瘤腺病毒免疫学相关机制的研究、突破妨碍溶瘤腺病毒研究的一些技术性瓶颈、优化细胞载体、提高溶瘤腺病毒远处传递的靶向性,以及寻找更具潜能的肿瘤干细胞作为靶点.此外,需要扩大临床试验的研究范围和加强与其他治疗方式特别是免疫治疗联合应用的研究.     

间质干细胞荷载溶瘤腺病毒治疗肿瘤进展

溶瘤腺病毒是具有高度的肿瘤靶向性和良好的转染率,但全身应用后机体的免疫防御机制明显限制了溶瘤腺病毒的治疗效果。细胞荷载溶瘤腺病毒可能是克服这一瓶颈的有效策略,间质干细胞对肿瘤细胞有天然的靶向性同时可荷载溶瘤腺病毒提高抗肿瘤效果,本文主要对间质干细胞荷载溶瘤腺病毒治疗肿瘤的研究领域及研究进展进行综述。      

转录靶向性溶瘤腺病毒治疗肺癌研究的现状与思考

目的:总结分析国内外应用转录靶向性溶瘤腺病毒治疗肺癌的进展和面临的困难。方法:以"腺病毒、肺癌、启动子"为关键词,检索CNKI及PubMed数据库,年限为2000-2010,相关文献72篇,其中中文文献16篇,英文56篇。纳入标准:1)以腺病毒为载体治疗肺癌;2)用肺癌基因启动子取代腺病毒复制必须基因启动子。根据纳入标准分析23篇文献。结果:转录靶向性溶瘤腺病毒是应用肿瘤特异性启动子调控腺病毒复制必须基因的表达,使腺病毒只在特定的肿瘤细胞中复制、扩增,从而能最大程度地靶向杀伤肿瘤细胞,而不杀伤正常细胞。可应用于肺癌的肿瘤特异性启动子有10余种,用这些特异性启动子取代腺病毒复制起始基因启动子后,腺病毒复制必须基因序列因受其特异性调控,只在肺癌细胞中复制、扩增,从而最大程度地靶向杀伤肺癌细胞,不杀伤正常细胞;同时,腺病毒载体的优越性在于其能保证启动子和治疗基因安全、稳定、高效地在肺癌组织中表达。这使得靶向性溶瘤腺病毒在肺癌治疗研究中取得了显著进展,但这些研究多集中于体外实验及动物体内实验,临床应用尚面临诸多困难。结论:转录靶向性溶瘤腺病毒为肺癌的治疗提供了新思路,如能突破目前研究中所面临的种种困难,进一步全面、深入研究肺癌的发病机制,探寻特异性更强的肺癌启动子,增强溶瘤腺病毒的靶向性,降低毒副反应,可望广泛应用于临床。     

溶瘤腺病毒联合化疗治疗肿瘤的研究

近年来用溶瘤腺病毒联合化疗治疗肿瘤的研究备受人们关注.肿瘤细胞对化疗药物的耐药性是目前临床上有效应用化疗方案的主要限制.溶瘤腺病毒能明显提高传统化疗对肿瘤细胞的杀伤效果,克服了传统肿瘤基因治疗中复制缺陷型腺病毒的转染效率低、靶向性差、抗癌基因表达量低的缺点.在肿瘤治疗中溶瘤腺病毒联合化疗将可能成为潜在有效的临床治疗方案.     

双启动子调控的条件复制腺病毒制备及其体外抑瘤作用

目的:构建并制备survivin及hTERT双启动子调控的条件复制腺病毒,探讨其特异性溶瘤作用.方法:PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,分别克隆入腺病毒载体pXC1的两个复制必需基因E1A和E1B序列上游启动子区,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXC1-SP-TP;脂质体法与pBHGE3骨架质粒共转染293E细胞进行重组腺病毒包装,稀释法测定腺病毒滴度;应用MTT、活细胞计数等方法观察其对肝癌细胞HepG2的特异性溶瘤作用并以正常人的血管内皮细胞ECV304作为对照.结果:测序及双酶切鉴定结果证实,成功构建了双肿瘤特异性启动子调控复制腺病毒载体;在293E细胞中获得了重组腺病毒Ad-SP-TP,滴度测定显示病毒滴度达到3.9×1010TCID50/ml;MTT结果显示,Ad-SP-TP可有效抑制肝癌细胞增殖而对正常细胞无增殖抑制作用;活细胞计数及细胞形态观察结果显示,重组腺病毒在肝癌细胞中选择性复制并发挥溶细胞作用.结论:双启动子调控的腺病毒具有显著的溶瘤作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为肝癌靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略.     

携带人端粒酶反转录酶启动子的溶瘤腺病毒RCA-TERT-Ad35对乳腺癌干细胞的靶向作用

目的 探讨携带人端粒酶反转录酶启动子的溶瘤腺病毒RCA-TERT-Ad35在靶向乳腺癌干细胞中的作用。方法 乳腺癌细胞系MCF-7以球体形式在加入生长因子的无血清培养基中生长。同时构建携带TERT启动子的溶瘤腺病毒RCA-TERT-Ad35,并通过溶瘤、MTT及体内抗肿瘤实验观察RCA-TERT-Ad35对干细胞样癌细胞的杀伤效果。结果 与亲代细胞相比,MCF-7球体细胞显示出干细胞特性,且MCF-7球体细胞中hTERT基因过表达,其对细胞毒性剂紫杉醇具有耐药性。溶瘤腺病毒RCA-TERT-Ad35在TERT过表达的细胞中进行复制并抑制细胞生长。在裸鼠移植瘤模型中,与RCA-Ad35相比,RCA-TERT-Ad35显著抑制肿瘤生长并改善小鼠的存活状态。结论 溶瘤腺病毒RCA-TERT-Ad35可优先杀伤TERT过表达的乳腺癌干细胞。     

溶瘤病毒联合放疗的基础与临床研究

溶瘤病毒可通过病毒复制导致肿瘤细胞裂解.目前已有大量针对呼肠孤病毒、麻疹病毒、单纯疱疹病毒及腺病毒等溶瘤病毒的作用机制、结构改造、临床安全性的研究,同时有研究证实溶瘤病毒联合放疗具有协同作用,并已进入临床研究阶段,故该治疗方法将成为有应用前景的抗肿瘤策略.     

溶瘤腺病毒的安全性研究进展

溶瘤腺病毒又称条件复制性腺病毒（conditionally replicating adenovirus,CRAD）,是一种用于肿瘤治疗的生物制剂,其安全性备受人们的关注,如何系统化评价其毒性是应用于临床的关键步骤。近年来,对于溶瘤腺病毒安全性的报道越来越多,包括从特异性杀伤肿瘤细胞的细胞实验到动物的毒理实验等。本文对溶瘤腺病毒的细胞实验、荷瘤小鼠治疗的毒性、动物急性毒性实验及长期毒性实验、生殖毒性实验的研究及其评估方法和目前临床研究状况进行综述。     

干细胞介导溶瘤腺病毒治疗脑胶质瘤研究进展

溶瘤腺病毒治疗具有肿瘤特异性、安全性和反复表达治疗基因等特点,通过溶瘤腺病毒治疗胶质细胞瘤已获得令人鼓舞的成果,但局部应用溶瘤腺病毒无法作用于散在分布的肿瘤细胞,影响了治疗效果.干细胞对脑胶质瘤具有内在趋化作用,介导溶瘤腺病毒治疗胶质瘤可进一步提高疗效.     

双启动子调控的条件复制腺病毒制备及其体外抑瘤作用

目的：构建并制备survivin及hTERT双启动子调控的条件复制腺病毒,探讨其特异性溶瘤作用。方法：PCR方法分别扩增肿瘤特异性survivin及hTERT启动子,分别克隆入腺病毒载体pXCl的两个复制必需基因E1A和E1B序列上游启动子区,构建出双肿瘤特异性启动子调控的条件复制腺病毒载体pXCl-SP—TP;脂质体法与pBHGE3骨架质粒共转染293E细胞进行重组腺病毒包装,稀释法测定腺病毒滴度;应用MTT、活细胞计数等方法观察其对肝癌细胞HepG2的特异性溶瘤作用并以正常人的血管内皮细胞ECV304作为对照。结果：测序及双酶切鉴定结果证实,成功构建了双肿瘤特异性启动子调控复制腺病毒载体;在293E细胞中获得了重组腺病毒Ad—sP—TP,滴度测定显示病毒滴度达到3．9×10^10TCID50／ml;MTT结果显示,Ad—sP—TP可有效抑制肝癌细胞增殖而对正常细胞无增殖抑制作用;活细胞计数及细胞形态观察结果显示,重组腺病毒在肝癌细胞中选择性复制并发挥溶细胞作用。结论：双启动子调控的腺病毒具有显著的溶瘤作用但对正常人血管内皮细胞不发挥溶细胞作用,实验结果为肝癌靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略。     

表达LAG-3抗体的溶瘤腺病毒对成胶质细胞瘤的抗瘤性

目的:探讨共表达淋巴细胞活化基因3(lymphocyte activation gene 3,LAG-3)抗体(LAG-3 antibody,aLAG)的溶瘤腺病毒对成胶质细胞瘤的抗肿瘤活性.方法:在溶瘤腺病毒Ad3的骨架中插入aLAG序列,获得重组溶瘤腺病毒Ad3-aLAG.应用WB法检测感染成胶质细胞瘤GL261细...     

Recent advances in genetic modification of adenovirus vectors for cancer treatment

Adenoviruses are widely used to deliver genes to a variety of cell types and have been used in a number of clinical trials for gene therapy and oncolytic virotherapy. However, several concerns must be addressed for the clinical use of adenovirus vectors. Selective delivery of a therapeutic gene by adenovirus vectors to target cancer is precluded by the widespread distribution of the primary cellular receptors. The systemic administration of adenoviruses results in hepatic tropism independent of the primary receptors. Adenoviruses induce strong innate and acquired immunity in vivo. Furthermore, several modifications to these vectors are necessary to enhance their oncolytic activity and ensure patient safety. As such, the adenovirus genome has been engineered to overcome these problems. The first part of the present review outlines recent progress in the genetic modification of adenovirus vectors for cancer treatment. In addition, several groups have recently developed cancer‐targeting adenovirus vectors by using libraries that display random peptides on a fiber knob. Pancreatic cancer‐targeting sequences have been isolated, and these oncolytic vectors have been shown by our group to be associated with a higher gene transduction efficiency and more potent oncolytic activity in cell lines, murine models, and surgical specimens of pancreatic cancer. In the second part of this review, we explain that combining cancer‐targeting strategies can be a promising approach to increase the clinical usefulness of oncolytic adenovirus vectors.     

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.     

Potent antitumor activity of double-regulated oncolytic adenovirus-mediated ST13 for colorectal cancer

Following targeted gene virotherapy, the suppression of tumorigenicity 13 ( ST13 ) gene was inserted into the double-regulated oncolytic adenovirus SG500 to ensure more safety and potent antitumor activity against colorectal cancer in vitro and in vivo . We generated the ST13-expressing oncolytic adenovirus SG500-ST13, with which colorectal carcinoma cell lines SW620 and HCT116, and the lung fibroblast cell line WI38, were infected. Crystal violet staining was carried out to detect the cytopathic effect in cells, and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method was used to assay cell viability. The effect of apoptosis induced by SG500-ST13 was confirmed by Hoechst staining and the TdT-mediated dUTP-biotin nick-end labeling method. To further identify the antitumor effects of SG500-ST13 on HCT116 xenografts in Balb/c nude mice, the induction of cell death was assessed by hematoxylin–eosin staining. Immunohistochemical study was also carried out. ( Cancer Sci 2009; 100: 678–683)     

Combination effect of oncolytic adenovirotherapy and TRAIL gene therapy in syngeneic murine breast cancer models

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and oncolytic adenovirotherapy have been investigated extensively in xenografic human tumor models established in immunocompromised nude mice. However, the effects of these therapies on syngeneic murine tumors in immunocompetent settings were not well documented. We hypothesized that TRAIL gene therapy used with an oncolytic adenovirus would overcome the weaknesses of the two therapies used individually. In this study, we evaluated the antitumor effects of an oncolytic adenovirus, Delta24, in both human and murine breast cancer cell lines. We also analyzed the effects of TRAIL gene therapy combined with oncolytic virotherapy in these cancer cells. Our results showed that Delta24 can replicate and help the E1-deleted adenovector replicate in murine cancer cells. We also found that these two therapies combined had greater antitumor activity than either one alone in both human and murine breast cancer cells lines and in the syngeneic breast cancer models established in immunocompetent mice. Moreover, Delta24 virotherapy alone and combined with TRAIL gene therapy dramatically reduced the spontaneous liver metastasis that originated in the subcutaneous 4T1 tumor established in Balb/c mice. These findings provide important considerations in the development and preclinical assessments of oncolytic virotherapy.     

Survivin promoter-regulated oncolytic adenovirus with Hsp70 gene exerts effective antitumor efficacy in gastric cancer immunotherapy

Gene therapy is a promising adjuvant therapeutic strategy for cancer treatment. To overcome the limitations of current gene therapy, such as poor transfection efficiency of vectors, low levels of transgene expression and lack of tumor targeting, the Survivin promoter was used to regulate the selective replication of oncolytic adenovirus in tumor cells, and the heat shock protein 70 (Hsp70) gene was loaded as the anticancer transgene to generate an AdSurp-Hsp70 viral therapy system. The efficacy of this targeted immunotherapy was examined in gastric cancer. The experiments showed that the oncolytic adenovirus can selectively replicate in and lyse the Survivin-positive gastric cancer cells, without significant toxicity to normal cells. AdSurp-Hsp70 reduced viability of cancer cells and inhibited tumor growth of gastric cancer xenografts in immuno-deficient and immuno-reconstruction mouse models. AdSurp-Hsp70 produced dual antitumor effects due to viral replication and high Hsp70 expression. This therapeutic system used the Survivin promoter-regulated oncolytic adenovirus vector to mediate targeted expression of the Hsp70 gene and ensure safety and efficacy for subsequent gene therapy programs against a variety of cancers.