腺病毒编码人TRP2修饰DC诱发抗小鼠黑色素瘤免疫的研究

目的比较腺病毒载体(Ad)介导人和小鼠酪氨酸酶相关蛋白2(tyrosinase-relatedpro-tein2,TRP2)修饰小鼠骨髓来源的树突状细胞(BM-DC)诱发抗小鼠黑色素瘤免疫的差异。方法Ad编码人或小鼠TRP2(AdhTRP2或AdmTRP2)体外感染小鼠BM-DC并体内皮下免疫C57BL/6小鼠,7d后取出被免疫小鼠脾细胞行体内细胞毒性T淋巴细胞杀伤试验(invivoCTL)和细胞内IFN-γ染色(ICS)分析CTL的杀伤活性和IFN-γ的产生;或给免疫后小鼠皮下接种小鼠B16.F10黑色素瘤细胞,观察荷瘤小鼠的成活情况。结果invivoCTL和ICS分析显示,AdhTRP2/BM-DC免疫小鼠,其6hCTL杀伤率为(98.7±1.2)%,IFN-γ产生的CD8+T细胞占总CD8+T细胞的(1.25±0.21)%;而AdmTRP2/BM-DC免疫的小鼠,其6hCTL杀伤率和产生IFN-γ的CD8+T细胞比例分别为(28.6±6.3)%和(0.24±0.06)%。荷瘤试验表明,AdhTRP2/BM-DC免疫小鼠后1周皮下接种106B16.F10细胞,观察3个月100%的小鼠无瘤生长;而接种5×104B16.F10细胞至AdmTRP2/BM-DC免疫1周的小鼠,3个月后小鼠成活率仅为40%。结论Ad介导异种(人)TRP2较自身(小鼠)TRP2修饰的BM-DC更为有效地打破肿瘤免疫耐受、诱导强烈的抗黑色素瘤免疫反应,是一种高效的以DC为基础的肿瘤疫苗。     

Relationship of human adenoviruses 12, 18, and 31 as determined by hemagglutination inhibition.

Adenoviruses 12 and 31, but not Ad18, agglutinate rat blood cells at high titer, providing suitable blood cells be available and a prolonged contact period of virus with the erythrocytes is allowed. Purified virus particles show direct, and virus-free supernatants show direct and indirect, hemagglutination, ie, enhancement of HA by heterologous antiserum. Hemagglutination inhibition with rabbit antisera shows cross-reactions between Ad12 and Ad31 with titers 4--32 times lower than with homologous antigens; Ad18 antisera react with antigens from both of the other serotypes. No cross-reactions were seen with antisera from other adenoviruses. This suggests an antigenic relationship of the three viruses of subgroup IV in their fiber antigen gamma, in addition to the known relation in the hexon (epsilon), which is apparent in cross-neutralization.     

腺病毒介导的CD基因在人胰腺癌细胞中的靶向性表达

【目的】探讨腺病毒介导的大肠杆菌胞嘧啶脱氨酶 (E .coliCD)基因在胰腺癌细胞中靶向性表达的可能性及其作用。【方法】将含癌胚抗原 (carcinoembryonicantigenCEA)启动子的重组腺病毒感染人胰腺癌SW1990细胞 (CEA )、Capan 2细胞 (CEA-)和人宫颈癌Hela细胞 ,观察CD基因在 3种细胞中的表达及细胞对 5 FC的敏感性差异。【结果】腺病毒介导CD基因仅在SW1990细胞中呈专一性表达 ,转导CD基因的SW1990细胞对 5 FC的敏感性明显提高。【结论】含CEA启动子的CD基因疗法可能是胰腺癌靶向性基因治疗的可行途径。     

CD47-retargeted oncolytic adenovirus armed with melanoma differentiation-associated gene-7/interleukin-24 suppresses in vivo leukemia cell growth

Our previous studies have suggested that harboring a soluble coxsackie-adenovirus receptor-ligand (sCAR-ligand) fusion protein expression cassette in the viral genome may provide a universal method to redirect oncolytic adenoviruses to various membrane receptors on cancer cells resisting to serotype 5 adenovirus infection. We report here a novel oncolytic adenovirus vector redirected to CD47+ leukemia cells though carrying a sCAR-4N1 expression cassette in the viral genome, forming Ad.4N1, in which 4N1 represents the C-terminal CD47-binding domain of thrombospondin-1. The infection and cytotoxicity of Ad.4N1 in leukemia cells were determined to be mediated by the 4N1-CD47 interaction. Ad.4N1 was further engineered to harbor a gene encoding melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24), forming Ad.4N1-IL24, which replicated dramatically faster than Ad.4N1, and elicited significantly enhanced antileukemia effect in vitro and in a HL60/Luc xenograft mouse model. Our data suggest that Ad.4N1 could act as a novel oncolytic adenovirus vector for CD47+ leukemia targeting gene transfer, and Ad.4N1 harboring anticancer genes may provide novel antileukemia agents.     

溶瘤腺病毒抗肿瘤策略研究进展

溶瘤腺病毒是一类经过基因工程改造,能够选择性地在肿瘤细胞中复制并溶解、杀伤肿瘤细胞的腺病毒,但不影响正常细胞功能。至今,已尝试多种方式提高溶瘤腺病毒的抗肿瘤活性和安全性,包括改变腺病毒传导方式,利用特异性启动子和增强子驱动治疗基因的表达以及协同放化疗、免疫治疗等。     

Vector-based genetically modified vaccines: Exploiting Jenner’s legacy

The global vaccine market is diverse while facing a plethora of novel developments. Genetic modification (GM) techniques facilitate the design of ’smarter’ vaccines. For many of the major infectious diseases of humans, like AIDS and malaria, but also for most human neoplastic disorders, still no vaccines are available. It may be speculated that novel GM technologies will significantly contribute to their development. While a promising number of studies is conducted on GM vaccines and GM vaccine technologies, the contribution of GM technology to newly introduced vaccines on the market is disappointingly limited.In this study, the field of vector-based GM vaccines is explored. Data on currently available, actually applied, and newly developed vectors is retrieved from various sources, synthesised and analysed, in order to provide an overview on the use of vector-based technology in the field of GM vaccine development. While still there are only two vector-based vaccines on the human vaccine market, there is ample activity in the fields of patenting, preclinical research, and different stages of clinical research. Results of this study revealed that vector-based vaccines comprise a significant part of all GM vaccines in the pipeline. This study further highlights that poxviruses and adenoviruses are among the most prominent vectors in GM vaccine development.After the approval of the first vectored human vaccine, based on a flavivirus vector, vaccine vector technology, especially based on poxviruses and adenoviruses, holds great promise for future vaccine development. It may lead to cheaper methods for the production of safe vaccines against diseases for which no or less perfect vaccines exist today, thus catering for an unmet medical need. After the introduction of Jenner’s vaccinia virus as the first vaccine more than two centuries ago, which eventually led to the recent eradication of smallpox, this and other viruses may now be the basis for constructing vectors that may help us control other major scourges of mankind.     

Oncolytic Virus Therapy with HSV-1 for Hematological Malignancies

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Oncolytic viruses and DNA-repair machinery: overcoming chemoresistance of gliomas

The current standard of care for malignant gliomas is surgical resection and radiotherapy followed by extended adjuvant treatment with the alkylating agent temozolomide. Temozolomide causes DNA damage, which induces cell death. Through changes in the DNA-repair machinery, glioma cells develop resistance to temozolomide, compromising the therapeutic effect of the drug. Oncolytic viruses, such as herpes simplex viruses and adenoviruses, are being introduced into clinical trials as a new treatment for this malignancy. Biological studies have revealed that these viruses use mechanisms to either inactivate (adenovirus) or take advantage of (herpes simplex virus) the cellular DNA-repair machinery to achieve productive replication. Adenoviruses express proteins from the early genes to either downregulate the damage-repair enzyme, O⁶-methylguanine-DNA methyltransferase, or degrade poly (ADP-ribose) polymerase or the Mre11-Rad50-NBS1 complex, which detects DNA strand breaks. Temozolomide enhances herpes simplex virus oncolysis by upregulating the DNA repair-related genes growth arrest DNA damage 34 and ribonucleotide reductase. The interactions between viruses and the DNA-repair machinery suggest that a combined temozolomide and viral therapy will overcome the limitations of a single therapy by diminishing chemoresistance or enhancing oncolysis. This hypothesis has been supported by promising findings from preclinical and clinical studies.     

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.