The therapy and mechanisms of replication-deficient recombinant adenovirus Ad-p14ARF in hepatocellular carcinoma

Objective: To study the therapeutic effect and mechanisms of recombinant adenovirus Ad-p14ARF in hepatocel- lular carcinoma cell lines. Methods: Morphology and trypan blue assay were adopted to evaluate the proliferation of different liver cancer cells after Ad-p14ARF infection. Cell apoptosis was confirmed by detecting phosphatidylserine (PS) externaliza- tion with Annexin V/PI double staining. Western blotting assay analyzed the expression of related proteins. Subcutaneous tumor model of BEL7402 was established to evaluate the therapeutic ability of Ad-p14ARF. Results: Ad-p14ARF suppressed cell growth, proliferation and promoted cell apoptosis of cancer cell lines with different genetic background. Ad-p14ARF in- hibited growth of liver cancer cells (HepG2, BEL7402) in a dose-dependent manner. Ad-p14ARF leaded to overexpression of Bax and p21, which were the downstream regulating genes of p53. Ad-p14ARF suppressed tumor growth significantly in the experimental therapy in nude mice bearing subcutaneous tumor of BEL7402. Conclusion: P14ARF gene is a powerful tumor suppressor gene to be used in cancer gene therapy. It may play an important role in gene therapy against the malignancies in the future.

Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

Objective: To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-l8 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1) IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus. and the concentration of IL-18 in lung lavage was higher than that in peripheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on expermental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL- 18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.

Adenovirus-mediated human β-nerve growth factor gene transfer has a protective effect on cochlear spiral ganglion after blast exposure

Objective:To study whether adenovirus-mediated human β-nerve growth factor (Ad-hNGFβ) gene has any protective effect on blast hearing impairment. Methods:Deafness was induced by blast exposure (172.0 dB) in 30 healthy guinea pigs. On day 7 of blast exposure, Ad-hNGFβ was infused into the perilymphatic space of 20 animals as the study group (hNGFβ group), and artificial perilymph fluid (APF) was infused into the perilymphatic space of the other 10 animals as the control group. At weeks 1, 4 and 8 after blast exposure, the animals were sacrificed and the cochleae were removed for immunohistochemical and HE stainings. Results: Expression of Ad-hNGFβ protein was detected in each turn of the cochlea at the 1st week, with almost equal intensity in all turns. At the 4th week, the reactive intensity of the expression of Ad-hNGFβ protein decreased. At the 8th week, no expression was detectable. The results of HE staining showed that the amount of spiral ganglions in hNGFβ group was significantly greater than that of the control group at week 4 (P<0.01). Conclusion: Ad-hNGFβ can be expressed at a high level and for a relatively long period in the blast impaired cochlea, suggesting that Ad-hNGFβ has a protective effect on cochlear spiral ganglion cells after blast exposure and the efficient gene transfer into cochlea had been achieved without toxicity.

Oncolytic adenovirus-mediated MDA-7/IL-24 overexpression enhances antitumor activity in hepatocellular carcinoma cell lines

BACKGROUND: Melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24) is a novel tumor suppressor gene, which has suppressor activity in a broad spectrum of human cancer cells. We investigated the effect of the replication-competent oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24, both expressing human MDA-7/IL-24 on the hepatocellular carcinoma cell lines HepG2, Hep3B, SMMC-7721, HCCLM3, and the normal liver cell line L02. METHODS: Hepatocellular carcinoma cell lines and the normal liver cell line were infected with SG600-IL24 and Ad.IL-24. The mRNA and protein expression of MDA-7/IL-24 in infected cells was confirmed by RT-PCR, ELISA, and Western blotting. MTT assay was used to investigate the proliferation effect. Hoechst staining and Annexin-V and PI staining were performed to study the MDA-7/IL-24 gene expressed in HCC cell lines and the normal liver cell line. Flow cytometry was used to analyse the cell cycle. RESULTS: RT-PCR, ELISA and Western blotting confirmed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT and apoptosis detection indicated that SG600-IL24 induced growth suppression, promoted apoptosis, and blocked cancer cell lines in the G2/M phase in hepatocellular carcinoma cell lines but not in the normal liver cell line. CONCLUSIONS: SG600-IL24 selectively induces growth suppression and apoptosis in hepatocellular carcinoma cell lines in vitro but not in the normal liver cell line L02. Compared with Ad.IL-24, SG600-IL24 dramatically enhances antitumor activity in hepatocellular carcinoma cell lines. (Hepatobiliary Pancreat Dis Int 2010; 9:615-621)

Oncolytic adenovirus SG600-IL24 selectively kills hepatocellular carcinoma cell lines

AIM: To investigate the effect of oncolytic adenovirus SG600-IL24 and replication-incompetent adenovirus Ad.IL-24 on hepatocellular carcinoma (HCC) cell lines and normal liver cell line. METHODS: HCC cell lines (HepG2, Hep3B and MHCC97L) and normal liver cell line (L02) with a different p53 status were infected with SG600-IL24 and Ad.IL-24, respectively. Melanoma differentiation-associated (MDA)-7/interleukin (IL)-24 mRNA and protein expressions in infected cells were detected by reverse transcription-polymerase chain reaction (RT-PCR), enzymelinked immunosorbent assay (ELISA), and Western blotting, respectively. Apoptosis of HCC cells and normal liver cells was detected by cytometric assay with Hoechst33258 staining. 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) assay was used to investigate proliferation of HCC cells and normal liver cells, and cell cycle was assayed by flow cytometry. RESULTS: RT-PCR, ELISA and Western blotting showed that the exogenous MDA-7/IL-24 gene was highly expressed in cells infected with SG600-IL24. MTT indicated that SG600-IL24 could suppress the growth of HepG2, Hep3B, MHCC97L, with an inhibition rate of 75% ± 2.5%, 85% ± 2.0%, 72% ± 1.8%, respectively (P < 0.01), promote the apoptosis of HepG2, Hep3B, MHCC97L, with an apoptosis rate of 56.59% ± 4.0%, 78.36% ± 3.5%, 43.39% ± 2.5%, respectively (P < 0.01), and block the HCC cell lines in the G2/M phase with a blocking rate of 35.4% ± 4.2%, 47.3% ± 6.2%, 42% ± 5.0%, respectively (P < 0.01) but not the normal liver cell line in a p53-independent manner. CONCLUSION: SG600-IL24 can selectively suppress the proliferation and apoptosis of HCC cell lines in vitro but not normal liver cell line L02 in a p53-independent manner. Compared with Ad.IL-24, SG600-IL24 can significantly enhance the antitumor activity in HCC cell lines.

Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biologica processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus （Ad-BIMS） was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTI- and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

Twenty years of Gendicine?rAd-p53 cancer gene therapy:The first-in-class human cancer gene therapy in the era of personalized oncology

Genetic mutations in TP53 contribute to human malignancies through various means.To date,there have been a variety of therapeutic strategies targeting p53,including gene therapy to restore normal p53 function,mutant p53 rescue,inhibiting the MDM2-p53 interaction,p53-based vaccines,and a number of other approaches.This review focuses on the functions of TP53 and discusses the aberrant roles of mutant p53 in various types of cancer.Recombinant human p53 adenovirus,trademarked as Gendicine,which is the first anti-tumor gene therapy drug,has made tremendous progress in cancer gene therapy.We herein discuss the biological mechanisms by which Gendicine exerts its effects and describe the clinical re-sponses reported in clinical trials.Notably,the clinical studies suggest that the combination of Gendicine with chemotherapy and/or radiotherapy may produce more pronounced efficacy in slowing tumor growth and progression than gene therapy/chemotherapy alone.Finally,we summarize the methods of administration of recombinant human p53 adenovirus for different cancer types to provide a reference for future clinical trials.

Effects of adenoviral vector-mediated transduction of human p53,B7-1 and GM-CSF genes on liver cancer cells

The potential efficacy and clinical feasibility of gene therapy for liver cancer were tested through therecombinant adenovirus-mediated (Ad-multigenes ) co-transfer of human wild-type p53, B7-l co-stimulation(CD8o) and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes into human hepatocellular carcinoma cell lines. The treated cells underwent apoptosis with specific DNA fragmentation and became more sensitiveto cisplatin, a chemotherapeutic drug. Their growth was partly inhibited. Efficient proliferation and generation ofCTLs and cytokine production were induced in mixed lymphocytes through tumor cell reaction (MLTR) using peripheral blood T lymphocytes from donors as effector cells and Ad-multigenes or Ad-p53-transfected human hepatocellular carcinoma cells (HepG2 or BEL7402) as stimulator cells. Ad-multigenes-transfected rat carcinosarcomaWalker 256 cells were inoculated subcutaneously into normal rats. Fourteen days later, the activity of spleen cellsin rats inoculated with Ad-multigenes-transduced Walker 256 cells was higher than that in Ad-p53-transducedones. These findings suggest that adenovirus-mediated multigenes p53, B7-1 and GM-CSF can induce apoptosis ofliver cancer cells and initiate a potent antitumor immune response against them.

Construction of recombinant adenoviral vector Ad-CMV-hTGFβ1 for reversion of intervertebral disc degeneration by gene transfer

To provide a highly efficient adenov iral vector Ad CMV hTGFβ1 for the study of gene therapy for reversion of the intervertebral disc degeneration.Methods: A newly developed recombinant adenoviral vector constr uction system was used in the study. The cDNA of hTGFβ1 was first subcloned into a shuttle plasmid pShuttle CMV. The resultant plasmid was linearized by d ig esting with restriction endonuclease PmeI, and subsequently transformed into E .coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy 1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected in to 293 cells. Recombinant adenoviruses were generated within 2 weeks. Results: The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8 % agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The exp ression of hTGFβ1 was verified by immunohistochemical staining. Conclusions: The successful generation of the adenoviral vector Ad CMV hTGFβ1 and the confirmation of the interest gene expression make it p ossible for the experimental study of the reversion of the intervertebral disc d egeneration by gene therapy.

人骨形成蛋白-2重组腺病毒表达载体的构建

目的 构建人骨形成蛋白 - 2 (BMP- 2 )基因的重组腺病毒表达载体 ,为应用 BMP- 2基因治疗的研究奠定基础。方法 首先构建携带 BMP- 2基因的腺病毒穿梭质粒 p Ad CMV- BMP- 2 ,在脂质体介导下 ,与腺病毒基因组质粒 p JM17共转染 2 93细胞 ,经同源重组获得重组腺病毒 Ad- BMP- 2。结果 经限制性酶切和 PCR扩增鉴定 ,证实为复制缺陷型 BMP- 2重组腺病毒。结论 成功构建了 BMP- 2腺病毒表达载体。

AdTK治疗肺癌的实验研究

构建含HSV-TK基因的重组腺病毒AdTK,在两种肺癌细胞和鼠肺癌模型中对AdTK/GCV系统进行抑瘤实验研究。方法:采用同源重组法构建AdTK,用MTT法测定AdTK/GCV系统对H460、PG49人肺癌细胞的生长抑制率,建立T739鼠肺癌模型,观察AdTK/GCV系统对瘤体大小及鼠存活期的影响。结果:成功构建AdTK,AdTK转染H460、PG49细胞后对细胞抑制率随AdTK、GCV及药物作用时间的增加而增高,有显著性差异,小鼠体内实验中观察到AdTK/GCV系统对鼠肺癌有明显生长抑制作用,且明显延长了小鼠的存活期。结论:本文建立的AdTK/GCV系统在体内外对肺癌细胞均有明显抑制作用,为AdTK用于基因治疗奠定了基础。

以Ad-Easy载体系统快速构建PTEN重组腺病毒表达载体

目的目的应用Ad-Easy腺病毒载体系统构建携带抑癌基因PTEN的重组腺病毒表达载体。方法用插入有抑癌基因PTEN和绿色荧光蛋白的穿梭载体pAdTrack-CMV转化已经含有腺病毒骨架载体pAdEasy1的E.coliBJ5183(Ad-1cells)进行同源重组,获得重组腺病毒质粒,经PacⅠ线性化后,转染293细胞。结果得到重组腺病毒,转染重组腺病毒DNA的293细胞出现细胞病变,经PCR对传代的Ad-PTEN分析证实得到目的基因,荧光显微镜能观察到293细胞内有绿色荧光。结论腺病毒Ad-Easy系统是一种较为简便、快速的构建重组腺病毒的好方法,且生成的病毒滴度高、转导效率好,为进一步研究PTEN基因应用于癌症基因治疗提供实验基础。

超声引导下瘤体内注射重组人p53腺病毒治疗晚期肝癌

目的评价超声引导下瘤体内注射重组人p53腺病毒治疗晚期难治性肝癌的临床疗效及超声导入基因治疗的临床应用价值。方法对57例晚期肝癌患者行超声引导下瘤体内注射重组人p53腺病毒(rAd-p53),每次剂量1×1012VP或2×1012VP,每周1次,4次1疗程,治疗后选择完成1个疗程及以上,并符合基因治疗入选标准者38例,采用彩色多普勒超声(彩超)及病理学检查评估其临床疗效。结果彩超结果示:肝脏肿块均有不同程度缩小,其中4例患者肿块缩小明显(>50%);16例患者肿块周边和内部彩色血流信号减少或消失。大部分患者病情稳定,症状好转,一般状况改善,Karuafsky评分明显提高[术前(55.79±11.30)vs术后(61.05±21.64),P<0.05]。11例患者行肝穿刺活检组织病理学检查,7例可见肿瘤组织纤维化伴淋巴细胞浸润及肿瘤组织明显变性坏死。治疗的不良反应主要为一过性自限性发热。结论超声引导瘤内注射既能控制肝癌基因治疗的靶向性,又能对基因药物疗效进行影像学评估,具有较高的临床应用价值。

人TH基因重组腺病毒的构建及生物学活性研究

将人酪氨酸羟化酶cDNA表达盒克隆于质粒型腺病毒载体p△Elsp1A，得到重组质粒pAd－TH。随后用脂质体法将重组质粒pAd－TH和拯救型腺病毒质粒pBHG11一起共转染293细胞，通过体内同源重组生成重组腺病毒AdCMVth，THcDNA重组进入腺病毒E1区并受CMV启动子控制。采用形态学、病毒核酸酶切和PCR／RT－PCR等方法进行鉴定正确。重组腺病毒滴度达到1010pfu／ml。初步结果表明，该重组腺病毒感染MN9D细胞后可使细胞内多巴胺水平增加1倍，显示出明显的TH生物学活性。提示TH重组腺病毒AdCMVth可作为高效的基因转移载体用于帕金森氏病基因治疗。

经犬肝动/静脉输入反义c-myc重组腺病毒的安全性研究

目的:经Beagle犬肝脏血管注射重组腺病毒介导反义c—myc基因(Ad-ASmye)载体,观测其体内分布及毒副作用,以评价Ad-ASmye治疗肝癌的临床前期安全性。方法:选择4只体重8-10 kg健康Beagle犬,全麻后开腹,经肝动脉或门静脉注射Ad-ASmyc,注射Ad-ASmye前及注射后第3,7,14,21天取肝组织及抽取静脉血化验,PCR检测Ad-ASmyc在各器官组织中的分布,常规切片观察各器官病理变化,ELISA方法检测血中抗腺病毒抗体的产生。结果:经Beagle犬肝脏血管注射后,Ad-ASmyc可持续转导正常肝细胞达3周,实验犬一般情况好,血、肝、肾功能无明显异常,在肝脏、脾脏、肾脏、胃、心脏、皮肤中可检测到重组腺病毒的分布,镜下可见Ad-ASmyc剂量依赖性的肝组织轻微的炎症反应,注射后7 d血中有抗腺病毒载体的抗体产生,14 d达高峰,21 d开始下降。结论:经Beagle犬肝动脉或门静脉途径注射Ad—ASmyc均可转导至肝细胞,Ad-AS-myc对实验犬的毒副作用较轻。

双自杀基因融合基因重组腺病毒的制备与鉴定

目的 :制备含胞嘧啶脱氨酶 (CD)基因、胸苷激酶 (TK )基因的融合基因重组腺病毒 ,为恶性肿瘤的基因治疗研究作准备。方法 :构建含有 CD、TK融合基因的重组粘粒 ,将其与腺病毒 DNA末端肽复合物混合后以磷酸钙共沉淀法转染人胚肾2 93细胞株细胞 ,获取重组腺病毒 ,并进行鉴定。结果 :酶切结果及 PCR结果显示 ,重组粘粒中 CD、TK融合基因插入正确 ,经鉴定所获重组腺病毒带有融合基因 ,并且无具有复制能力的腺病毒存在。 结论 :所获重组腺病毒为 E1、E3缺陷型 ,含有所需的双自杀基因 。

细菌内同源重组快速构建和制备表达hSDF-1α的重组腺病毒

目的:利用细菌内同源重组快速构建携带hSDF-1αcDNA重组腺病毒质粒和制备表达hSDF-1α重组腺病毒.方法:制备感受态BJ5183,将pAdEasy-1转化BJ5183,制备含pAdEasy-1的BJ5183感受态,将线性化的pShuttle-EGFP-hS-DF-1α转化含pAdEasy-1的BJ5183感受态菌.采用细菌中同源重组法构建重组腺病毒质粒pAd-EGFP-hSDF-1α.pAd-EG-FP-hSDF-1α用PacI线性化后,再用LipoVec介导其转染至AD293细胞内包装扩增出重组腺病毒颗粒,采用CsCl密度梯度离心法纯化重组腺病毒Ad-EGFP-hSDF-1α.采用荧光显微镜下观察LipoVec介导的重组腺病毒质粒的转染效果及病毒包装情况.结果:pShuttle-EGFP-hSDF-1α成功地转化了含pAdEasy-1的BJ5183,并在其内发生了同源重组.荧光显微镜下观察证实了pAd-EGFP-hSDF-1α转染AD293后,产生了重组腺病毒Ad-EGFP-hSDF-1α.病毒滴度为4.1×1015pfu/L.结论:用细菌内同源重组法可快速、高效制备表达hSDF-1α的高滴度重组腺病毒.为研究hSDF-1α在动员骨髓源干细胞迁移到组织损伤部位修复组织损伤的研究奠定了基础.

腺病毒介导的大肠杆菌CD自杀基因体内外对肿瘤杀伤作用的研究

表达大肠杆菌胞嘧啶脱胺酶(CD)基因的重组腺病毒AdCD体外转染小鼠黑色素瘤细胞B16F10,结果显示转染了CD基因的B16F10细胞对5-氟胞嘧啶(5FC)的敏感性显著提高.将经AdCD/5FC系统处理的B16F10细胞上清倍比稀释后.加至野生型B16F10细胞中,发现当上清仅占6.25%时即可对野生型B16F10细胞发挥明显的杀伤作用,提示AdCD/5FC介导的旁观者效应可能是通过5FC经CD酶代谢产生的毒性产物扩散而实现的.本实验还观察了CD基因体内转染后的杀伤效果,荷瘤小鼠经注射AdCD并连续10天给予5FC治疗后,与PBS、对照病毒AdLacZ/5FC治疗小鼠比较,小鼠肿瘤生长明显受到抑制,小鼠存活期明显延长.

重组p16腺病毒的构建及其对人白血病细胞的抑制作用

为了探讨腺病毒载体用于基因治疗的可行性及野生型 p1 6基因的抗肿瘤特性 ,构建了复制缺陷型重组 p1 6腺病毒 .首先将 p1 6全长 c DNA插入穿梭质粒 p Ad CMV产生重组质粒 p Ad-CMV- p1 6,然后通过脂质体介导与 p JM1 7共转染 2 93细胞 ,经同源重组产生 E1区缺失的重组腺病毒空斑 .用纯化后的腺病毒感染人白血病细胞株 HL- 60后 ,PCR及 Western blot分析显示在感染细胞中有外源性 p1 6 c DNA存在和 p1 6蛋白表达 ;被感染的 HL- 60细胞的生长受到明显抑制 ,而未感染细胞及对照腺病毒感染的细胞没有受到抑制 .结果表明 ,腺病毒作为一种新型基因转移载体 ,可有效地介导肿瘤抑制基因 p1 6的表达 ,在肿瘤基因治疗方面具有很大的应用前景 .