重组溶瘤腺病毒RCAdC68-EGFP对结肠癌细胞增殖的影响

目的 本实验通过比较重组溶瘤腺病毒RCAdC68-EGFP与复制缺陷型重组腺病毒AdC68-EGFP对结肠癌细胞的杀伤作用,初步观察RCAdC68-EGFP对结肠癌细胞增殖的影响.方法 以不同病毒剂量(100、500、2500、12500vp/cell)感染结肠癌细胞HCT 116、NCI-H508、HT-29及人胚肾细胞HEK-293.感染后第3、5、7天用MTT法检测不同细胞的抑制率,结晶紫染色实验检测细胞的杀伤作用及相差显微镜观察细胞形态学变化和病变情况.结果 RCAdC68-EGFP对结肠癌细胞HCT 116、NCI-H508、HT-29的增殖抑制作用与病毒感染剂量及感染时间成正比,以500vp/cell病毒感染剂量感染细胞7天后,RCAdC68-EGFP对HCT 116、NCI-H508、HT-29、HEK293细胞的抑制率分别为86.70％、96.85％、99.70％、99.99％,AdC68-EGFP的细胞抑制率分别为6.57％、11.46％、9.11％、99.99％,RCAdC68-EGFP与AdC68-EGFP对细胞的增殖抑制作用有统计学差异(P＜0.01),而对HEK-293细胞增殖抑制作用无统计学差异(P＞0.05).结论 RCAdC68-EGFP能显著抑制HCT 116、NCI-H508、HT-29细胞的增殖,初步显示出RCAdC68-EGFP作为溶瘤病毒载体治疗肿瘤的潜能.     

溶瘤腺病毒靶向治疗脑胶质瘤的研究现状及进展

脑胶质瘤是常见的颅内恶性肿瘤之一,手术治疗及术后放、化疗仍是其主要的治疗手段,因其术后复发率高、放化疗副作用及耐药性等问题,无法达到令人满意的治疗效果。基因治疗作为目前发展迅速的精准治疗措施之一,递送载体是其发挥治疗作用的关键因素,溶瘤腺病毒因其特异性、高效性及安全性等优势,使其在多种肿瘤的基础研究及临床应用中被作为基因递送的载体。该文主要介绍了溶瘤腺病毒在脑胶质瘤中的研究现状,联合治疗的策略及临床应用最新进展,以期为溶瘤腺病毒在脑胶质瘤的靶向治疗中提供一定的理论依据。     

组织特异性启动子在溶瘤腺病毒中的应用

溶瘤腺病毒（Oncolytic Adenovirus,Ad）是肿瘤基因治疗的研究热点。利用肿瘤或组织特异性启动子（Tissue—specific promoter,TSP）控制腺病毒关键基因的表达、实现腺病毒在肿瘤细胞特异性复制是目前溶瘤腺病毒构建的主要策略。另外,负载治疗基因可以增强杀灭肿瘤细胞的效果,     

重组腺病毒载体靶向性策略的研究进展

近年来,研究者致力于突破基因治疗的瓶颈,极大推动了基因治疗的发展。载体系统是基因治疗的基础,截止2013年7月,在"Gene Therapy Clinical Trial Worldwide"网站登记注册的基因治疗方案中,重组腺病毒载体占476项(23.5%)。在6个亚群(A-F)、50多种血清型的腺病毒载体中,5型腺病毒(adenovirus type 5,Ad5)可特异性识     

溶瘤病毒应用于肿瘤治疗的研究进展

溶瘤病毒应用于肿瘤治疗是近 10年来兴起的一种肿瘤治疗的新方案 ,现已进行了临床试验研究 ,其结果令人鼓舞。本文详细介绍几种溶瘤病毒包括溶瘤腺病毒、溶瘤疱疹病毒、呼 (吸道 )肠 (道 )病毒、新城疫病毒等治疗肿瘤的最新进展 ,为溶瘤病毒应用于肿瘤治疗过程提供新的启示。     

腺病毒介导IFN—γ基因转染的瘤苗体内抗肿瘤转移作用

通过缺陷型腺病毒载体的介导将小鼠ＩＦＮ－γ基因转染入肿瘤细胞，经５０００ｒａｄ＾６０Ｃｏ照射后射备成瘤苗，观察其对实验性肺转移荷瘤小鼠的治疗效果，并对其治疗作用的免疫要现进行了研究。结果显示：经ＩＦＮ－γ基因转染瘤苗治疗后小鼠脾淋巴细胞诱导的ＣＴＬ，ＬＡＫ及腹腔巨噬细胞杀伤活性均明显升高。     

溶瘤病毒与肿瘤

溶瘤病毒治疗肿瘤是一种全新的肿瘤基因治疗方法,利用病毒自身特性发现并杀伤肿瘤细胞,近年来受到人们广泛关注.溶瘤病毒包括细小病毒、呼肠孤病毒、新城疫病毒、水疱性口炎病毒、麻疹病毒、腺病毒、疱疹病毒和甲型流感病毒等.目前已经有多个溶瘤病毒产品进入抗肿瘤临床试验阶段,且疗效较为显著.本文对该领域最新研究进展作一概述.     

特异性溶瘤腺病毒调控肺癌细胞表达PD-L1及诱导凋亡的研究

目的:探究特异性溶瘤腺病毒(Ad-Mock、Ad-T)对肺癌细胞上PD-L1表达的调控作用及对细胞凋亡的影响.方法:采用荧光定量PCR法及Western blot检测溶瘤腺病毒在24 h和48 h对NCI-H446、NCI-H226、A549细胞表达PD-L1的影响;通过结晶紫染色、WST-1、Hoechst与JC-1...     

溶瘤病毒的临床应用

新城疫病毒（newcastle disease virus，NDV）、单纯疱疹病毒-1（herpes simplex virus-1，HSV-1）、呼肠孤病毒（reovirus）、溶瘤腺病毒（oncolytic adenovirus）等是由嗜肿瘤特性而被用来改造成溶瘤病毒，它特异性识别并感染肿瘤细胞，最终导致细胞溶胀而摧毁肿瘤细胞，但无法在正常机体细胞内复制而不具有杀伤作用，理论上具有更高的抗肿瘤效应和更低的副作用。文章就以上溶瘤病毒的国内外临床应用现状及目前仍面临的主要问题进行综述。     

动物肠道溶瘤病毒体内外溶瘤作用的电镜研究

动物肠道溶瘤病毒体内外溶瘤作用的电镜研究凌虹，程志，李呼伦，张凤民，李绍贤，吴振锋，朱平，张莉（哈尔滨医科大学，黑龙江１５００８６）病毒治疗肿瘤是近年来较受重视的肿瘤疗法之一。我们选用鹿肠道病毒ＥＣＣＯ－１８株和牛肠道病毒ＭＺ－４６８株进行溶瘤作用的...     

Triple-controlled oncolytic adenovirus expressing melittin to exert inhibitory efficacy on hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a highly malignant disease, and its outcome of routine therapies is poor. Comprehensive treatment including gene therapy is an important way to improve patients’ prognosis and survival. In this study, we successfully constructed a triple-controlled cancer-selective oncolytic adenovirus, QG511-HA-Melittin, carrying melittin gene, in which the hybrid promoter, hypoxia-response element (HRE)-AFP promoter, was used to control viral E1a expression targeting AFP-positive cancer cells in hypoxia microenviroment, and the E1b-55 kDa gene was deleted in cancer cells with p53-deficiency. The cytological experiments found that the viral replication of QG511-HA-Melittin was increased to 12800-folds in Hep3B cells within 48 h, and 130-folds in SMMC-7721, but the virus did not replicate in L-02 cells. QG511-HA-Melittin had a strong inhibition effect on AFP-positive HCC cell proliferation, such as Hep3B and HepG2, whereas, there was low or no inhibition effect of QG511-HA-Melittin on AFP-negative cancer cells SMMC-7721 and normal cells L-02. In the in vivo experiment, compared with the blank control group, QG511-HA-Melittin can significantly inhibit the growth of HCC xenografts (P<0.05). The survival of mice in QG511-HA-Melittin group was much longer than that of the blank control group. Both in vitro and in vivo experiments manifested that QG511-HA-Melittin exerts an inhibitory effect on HCC cells, which may provide a new strategy for HCC biotherapy.     

Evaluation of E1A double mutant oncolytic adenovectors in anti-glioma gene therapy

Malignant glioma, in particular glioblastoma multiforme (GBM), represents one of the most devastating cancers currently known and existing treatment regimens do little to change patient prognosis. Conditionally replicating adenoviral vectors (CRAds) represent attractive experimental anti-cancer agents with potential for clinical application. However, early protein products of the wild type adenovirus backbone--such as E1A--limit CRAds' replicative specificity. In this study, we evaluated the oncolytic potency and specificity of CRAds in which p300/CPB and/or pRb binding capacities of E1A were ablated to reduce non-specific replicative cytolysis. In vitro cytopathic assays, quantitative PCR analysis, Western blot, and flow cytometry studies demonstrate the superior anti-glioma efficacy of a double-mutated CRAd, Ad2/24CMV, which harbors mutations that reduce E1A binding to p300/CPB and pRb. When compared to its single-mutated and wild type counterparts, Ad2/24CMV demonstrated attenuated replication and cytotoxicity in representative normal human brain while displaying enhanced replicative cytotoxicity in malignant glioma. These results have implications for the development of double-mutated CRAd vectors for enhanced GBM therapy.     

Imaging of viral thymidine kinase gene expression by replicating oncolytic adenovirus and prediction of therapeutic efficacy

Purpose

We have used a genetically attenuated adenoviral vector which expresses HSVtk to assess the possible additive role of suicidal gene therapy for enhanced oncolytic effect of the virus. Expression of TK was measured using a radiotracer-based molecular counting and imaging system.

Materials and Methods

Replication-competent recombinant adenoviral vector (Ad-ΔE1B19/55) was used in this study, whereas replication-incompetent adenovirus (Ad-ΔE1A) was generated as a control. Both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK comprise the HSVtk gene inserted into the E3 region of the viruses. YCC-2 cells were infected with the viruses and incubated with 2''-deoxy-2''-fluoro-β-D-arabinofuranosyl-5-iodouracil (I-131 FIAU) to measure amount of radioactivity. The cytotoxicity of the viruses was determined, and gamma ray imaging of HSVtk gene was performed. MTT assay was also performed after GCV treatment.

Results

On gamma counter-analyses, counts/minute (cpm)/µg of protein showed MOIs dependency with ΔE1B19/55-TK infection. On MTT assay, Ad-ΔE1B19/55-TK led to more efficient cell killing than Ad-ΔE1A-TK. On plate imaging by gamma camera, both Ad-ΔE1B19/55-TK and Ad-ΔE1A-TK infected cells showed increased I-131 FIAU uptake in a MOI dependent pattern, and with GCV treatment, cell viability of ΔE1B19/55-TK infection was remarkably reduced compared to that of Ad-ΔE1A-TK infection.

Conclusion

Replicating Ad-ΔE1B19/55-TK showed more efficient TK expression even in the presence of higher-cancer cell killing effects compared to non-replicating Ad-ΔE1A-TK. Therefore, GCV treatment still possessed an additive role to oncolytic effect of Ad-ΔE1B19/55-TK. The expression of TK by oncolytic viruses could rapidly be screened using a radiotracer-based counting and imaging technique.     

Using a magnetic field to redirect an oncolytic adenovirus complexed with iron oxide augments gene therapy efficacy

Adenovirus (Ad) is a widely used vector for cancer gene therapy but its therapeutic efficacy is limited by low coxsackievirus and adenovirus receptor (CAR) expression in tumors and non-specifically targeted infection. Ad infectivity and specificity can be markedly improved by creating Ad-magnetic nanoparticles cluster complexes and directing their migration with an external magnetic field (MGF). We electrostatically complexed GFP-expressing, replication-incompetent Ad (dAd) with PEGylated and cross-linked iron oxide nanoparticles (PCION), generating dAd-PCION complexes. The dAd-PCION showed increased transduction efficiency, independent of CAR expression, in the absence or presence of an MGF. Cancer cell killing and intracellular oncolytic Ad (HmT)-PCION replication significantly increased with MGF exposure. Site-directed, magnetically-targeted delivery of the HmT-PCION elicited significantly greater therapeutic efficacy versus treatment with naked HmT or HmT-PCION without MGF in CAR-negative MCF7 tumors. Immunohistochemical tumor analysis showed increased oncolytic Ad replication in tumors following infection by HmT-PCION using an MGF. Whole-body bioluminescence imaging of tumor-bearing mice showed a 450-fold increased tumor-to-liver ratio for HmT-PCION with, versus without, MGF. These results demonstrate the feasibility and potential of external MGF-responsive PCION-coated oncolytic Ads as smart hybrid vectors for cancer gene therapy.     

Dual tumor targeting with pH-sensitive and bioreducible polymer-complexed oncolytic adenovirus

Oncolytic adenoviruses (Ads) have shown great promise in cancer gene therapy but their efficacy has been compromised by potent immunological, biochemical, and specific tumor-targeting limitations. To take full advantage of the innate cancer-specific killing potency of oncolytic Ads but also exploit the subtleties of the tumor microenvironment, we have generated a pH-sensitive and bio-reducible polymer (PPCBA)-coated oncolytic Ad. Ad-PPCBA complexes showed higher cellular uptake at pH 6.0 than pH 7.4 in both high and low coxsackie and adenovirus receptor-(CAR)-expressing cells, thereby demonstrating Ad-PPCBA's ability to target the low pH hypoxic tumor microenvironment and overcome CAR dependence for target cell uptake. Endocytic mechanism studies indicated that Ad-PPCBA internalization is mediated by macropinocytosis instead of the CAR-dependent endocytic pathway that internalizes naked Ad. VEGF-specific shRNA-expressing oncolytic Ad complexed with PPCBA (RdB/shVEGF-PPCBA) elicited much more potent suppression of U87 human brain cancer cell VEGF gene expression in vitro, and human breast cancer MCF7 cell/Matrigel plug vascularization in a mouse model, when cancer cells had been previously infected at pH 6.0 versus pH 7.4. Moreover, intratumorally and intravenously injected RdB/shVEGF-PPCBA nanocomplexes elicited significantly higher therapeutic efficacy than naked virus in U87-tumor mouse xenograft models, reducing IL-6, ALT, and AST serum levels. These data demonstrated PPCBA's biocompatibility and capability to shield the Ad surface to prevent innate immune response against Ad after both intratumoral and systemic administration. Taken together, these results demonstrate that smart, tumor-specific, oncolytic Ad-PPCBA complexes can be exploited to treat both primary and metastatic tumors.     

Chimeric oncolytic Ad5/3 virus replicates and lyses ovarian cancer cells through desmoglein-2 cell entry receptor

Despite new therapies, the estimated 229 875 women living with ovarian cancer have a 5-year survival rate of 47.6%. This cavity-localized cancer lends itself to local administration of modalities, such as the oncolytic adenovirus (Ad) Ad5/3-D24-granulocyte-macrophage colony-stimulating factor virus (ONCOS-102). Its repeated administration to a patient with chemotherapy-refractory ovarian cancer induced CD8+ antitumor immune responses with the overall survival reaching 40 months. Here we probe the dominant receptor used by ONCOS-102 in four established epithelial ovarian cancer cell lines. Ad3 can use the desmoglein-2 (DSG2) and CD46 receptors on susceptible cells. DSG2 was nearly absent in A2780 cells but was expressed in more than 90% of OAW42, OVCAR3, and OV-90 cells. After 96 hours, ONCOS-102 treatment showed significant oncolytic activity (≧50%) in OAW42, OVCAR3, and OV-90 cells, but minimal activity in A2780 cells, suggesting DSG2 as the dominant receptor for ONCOS-102. Furthermore, retrospective analyses of phase I clinical trial of ONCOS-102 treatment of 12 patients with varied tumors indicated a correlation between viral genomes in blood and DSG2 RNA expression. These data support the role of DSG2 expression on cancer cells in virus infectivity and the continued development of ONCOS-102 for ovarian cancer treatment.     

Targeting eradication of chronic myeloid leukemia using chimeric oncolytic adenovirus to drive IL-24 expression

Chronic myeloid leukemia (CML) is a clonal disorder in which cells of the myeloid lineage undergo massive clonal expansion as well as resistance to conventional chemotherapy. Gene therapy hold a great promise for treatment of malignancies based on the transfer of genetic material to the tissues. In this study, we explore whether chimeric oncolytic adenovirus-mediated transfer of human interleukin-24 (IL-24) gene induce the enhanced antitumor potency. Our results showed that chimeric oncolytic adenovirus carrying hIL-24 (AdCN205-11-IL-24) could produce high levels of hIL-24 in CML cancer cells, as compared with constructed double-regulated oncolytic adenovirus expressing hIL-24 (AdCN205-IL-24). AdCN205-11-IL-24 could specifically induce cytotoxocity to CML cancer cells, but little or no effect on normal cell lines. AdCN205-11-IL-24 exhibited remarkable anti-tumor activities and induce higher antitumor activity to CML cancer cells by inducing apoptosis in vitro. Our study may provides a potent and safe tool for CML gene therapy.     

VG161 activates systemic antitumor immunity in pancreatic cancer models as a novel oncolytic herpesvirus expressing multiple immunomodulatory transgenes

The VG161 represents the first recombinant oncolytic herpes simplex virus type 1 carrying multiple synergistic antitumor immuno-modulating factors. Here, we report its antitumor mechanisms and thus provide firm theoretical foundation for the upcoming clinical application in pancreatic cancer. Generally, the VG161-mediated antitumor outcomes were analyzed by a collaboration of techniques, namely the single-cell sequencing, airflow-assisted desorption electrospray ionization-mass spectrometry imaging (AFADSI-MSI) and nanostring techniques. In vitro, the efficacy of VG161 together with immune checkpoint inhibitors (ICIs) has been successfully shown to grant a long-term antitumor effect by altering tumor immunity and remodeling tumor microenvironment (TME) metabolisms. Cellular functional pathways and cell subtypes detected from patient samples before and after the treatment had undergone distinctive changes including upregulated CD8+ T and natural killer cells. More importantly, significant antitumor signals have emerged since the administration of VG161 injection. In conclusion, VG161 can systematically activate acquired and innate immunity in pancreatic models, as well as improve the tumor immune microenvironment, indicative of strong antitumor potential. The more robusting antitumor outcome for VG161 monotherapy or in combination with other therapies on pancreatic cancer is worth of being explored in further clinical trials.