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adenovirus-mediated expression of both antisense ODC and AdoMetDC inhibited colorectal cancer cell growth in vitro

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Acta Pharmacologica Sinica 2006年第3期27卷 353-359页

作者： Bing ZHANG~2 Xian-xi LIU~(2,5) Yan ZHANG~2 Chun-ying JIANG~3 Qing-shan TENG~4 Hai-yan HU~2 Wei WANG~2 Lei GONG~2 ~2Institute of Biochemistry and Molecular Biology,School of Medicine.Shandong University,Jinan 250012,China ~3Department of Coloproctology,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan 250014,China,~4Department of Neurosurgery.Cleveland Clinic Foundation,Cleveland,OH 44195,USA Institute of Biochemistry and Molecular Biology School of Medicine Shandong University Jinan China Department of Colo-proctology Affiliated Hospital of Shandong University of Traditional Chinese Medicine Jinan China Department of Neurosurgery Cleveland Clinic Foundation Cleveland USA

Aim:To construct a recombinant adenovirus that can simultaneously expressboth antisense ornithine decarboxylase(ODC)and adenosylmethionine decar-boxylase(AdoMetDC)and detect its inhibitory effect on the intracellular polyaminepool and colorectal cancer cell growth.Methods:A 205-bp cDNA of AdoMetDCwas reverse-inserted into recombinant pAdTrack-ODCas vectors and recombinedwith pAdEasy-1 vectors in AdEasy-1 cells.Positive clones were selected andtransfected into the packaging cell HEK293 after they were linearized by PacI.Green fluorescent protein expression was used to monitor the process of adenovi-rus packaging.The ODC and AdoMetDC protein levels were identified by west-ern blotting,and intracellular polyamine content was detected by reverse-phasehigh performance liquid chromatography.A viable cell count was used to deter-mine the growth of HT-29 cells with or without exogenous polyamine.Results:Sequencing confirmed that AdoMetDC cDNA was successfully ligated into thepAdTrack-ODCas vector.GFP expression in 293 cells during virus packing andamplification was observed by fluorescence microscopy.Western blotting dem-onstrated that both ODC and AdoMetDC were downregulated by Ad-ODC-AdoMetDCas,and consequently 3 kinds of polyamine(putrescine,spermidineand spermine)were reduced to very low levels.HT-29 cell growth was signifi-cantly inhibited as compared with control conditions,and growth arrest was notreversed by exogenous putrescine.Conclusion:The successfully constructedrecombinant adenovirus,Ad-ODC-AdoMetDCas,blocked polyamine synthesisand has therapeutic potential for treating colorectal cancer in vitro.

关键词： polyamine ornithine decarboxylase adenosylmethionine decarboxylase adenovirus colorectal cancer

adenovirus-mediated gene delivery:Potential applications for gene and cell-based therapies in the new era of personalized medicine

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Genes & Diseases 2017年第2期4卷 43-63页

作者： Cody S.Lee Elliot S.Bishop Ruyi Zhang Xinyi Yu Evan M.Farina Shujuan Yan Chen Zhao Zongyue Zeng Yi Shu Xingye Wu Jiayan Lei Yasha Li Wenwen Zhang Chao Yang Ke Wu Ying Wu Sherwin Ho Aravind Athiviraham Michael J.Lee Jennifer Moriatis Wolf Russell R.Reid Tong-Chuan He The University of Chicago Pritzker School of Medicine ChicagoIL 60637USA Laboratory of Craniofacial Biology and Development Section of Plastic and Reconstructive SurgeryDepartment of SurgeryThe University of Chicago Medical CenterChicagoIL 60637USA Molecular Oncology Laboratory Department of Orthopaedic Surgery and Rehabilitation MedicineThe University of Chicago Medical CenterChicagoIL 60637USA Ministry of Education Key Laboratory of Diagnostic Medicine and the Affiliated Hospitals of Chongqing Medical UniversityChongqing 400016China Department of Laboratory Medicine and Clinical Diagnostics The Affiliated Yantai HospitalBinzhou Medical UniversityYantai 264100China Department of Immunology and Microbiology Beijing University of Chinese MedicineBeijing 100029China

With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies.Despite numerous setbacks,efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases.It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies.There has been a long-lasting interest in using viral vectors,especially adenoviral vectors,to deliver therapeutic genes for the past two decades.Among all currently available viral vectors,adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types.The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development.In fact,among over 2000 gene therapy clinical trials approved worldwide since 1989,a significant portion of the trials have utilized adenoviral vectors.This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors,including adenoviral biology,approaches to engineering adenoviral vectors,and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment,vaccination and regenerative medicine.Current challenges and future directions regarding the use of adenoviral vectors are also discussed.It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.

关键词： adenovirus Adenoviral vector Cell therapy Gene transfer Gene therapy Oncolytic virus Regenerative medicine Vaccine development

Trained innate immunity and human adenovirus infection as a novel mechanism linked to the severe acute hepatitis of unknown origin in children during the COVID‐19 pandemic

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Portal Hypertension & Cirrhosis 2022年第2期1卷 145-148页

作者： Lingyao Du Jiayi Wang Lang Bai Hong Tang Center of Infectious Diseases West China Hospital of Sichuan UniversityChengduSichuanChina

1|OUTBREAK OF SEVERE ACUTE HEPATITIS OF UNKNOWN ORIGIN IN CHILDREN Severe acute hepatitis of unknown origin in children was first reported by the United Kingdom to the World Health Organization on April 5,2022.Subsequently,650 probable cases were reported worldwide by May 26,2022,1 and four patients were analyzed after reviewing the clinical records of a children's hospital in Alabama since October 1,2021.2 Although most patients recovered,the disease progressed to acute liver failure in several patients,who further required liver transplantation;thus,additional research is required to determine the disease pathogenesis.

关键词： adenovirus child innate immunity severe acute hepatitis unknown etiology

adenovirus-mediated NDRG2 inhibits the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro

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Asian Pacific Journal of Tropical Medicine 2014年第11期7卷 873-878页

作者： Sheng Qiang Zhen-Fang Du Min Huang Zhangjiagang Hospital of Traditional Chinese Medicine

Objective:To investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of human renal cell carcinoma cell line OS-RC-2 in vitro.Methods:NDRG2 was harvested by RT-PCR,confirmed by DNA sequencing,and then cloned into the eukaryotic expression vector pIRES2-EGFP,which encodes green fluorescent protein(GFP),to construct p1RES2-EGFP-NDRG2 plasmid.OS-RC-2 cells with NDRG2 negative expression were transfected with p1RES2-EGFP-NDRG2 plasmid.The growth of transfected OS-RC-2 cells was observed under light and fluorescence microscopes.After colony-forming cell assays,cell proliferation detection and MTT assays,the growth curves of cells in each group were plotted to investigate the inhibitory effects of adenovirus-mediated NDRG2 on the proliferation of OS-RC-2 cells.Cell cycle was determined by flow cytometry.Confocal laser scanning microscopy showed that NDRG2 protein was specifically located on subcellular organelle.Results:A eukaryotic expression vector p1RES2-EGFP-NDRG2 was successfully constructed.After NDRG2 transfection,the growth of OS-RC-2 cells was inhibited.Flow cytometry showed that cells were arrested in S phase but the peak of cell apoptosis was not present,and confocal laser scanning microscopy showed that NDRG2 protein was located in mitochondrion.Conclusions:NDRG2 can significantly inhibit the proliferation of OS-RC-2 cells in vitro and its protein is specifically expressed in the mitochondrion.

关键词： NDRC2 OS-RC-2 adenovirus Proliferation:Inhibition

adenovirus-mediated Transfer of p53 and p16Inhibiting Proliferating Activity of Human Bladder Cancer Cell EJin vitro and in vivo

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2002年第4期22卷 324-326页

作者：朱朝辉邢诗安林晨曾甫清鲁功成付明张雪艳梁萧吴旻 DepartmentofUrology CentralLaboratory XieheHospital NationalLaboratoryofMolecularOncology

To evaluate the effects of adenovirus (Ad) - mediated transfer of p5 3and p16 on hum an bladder cancer cells EJ,EJwere transfected with Ad- p5 3and Ad- p16 . Cell growth,m orphologi- cal change,cell cycle,apoptosis were measured using MTT assay,flow cytom etry,cloning form a- tion,im munocytochemical assays.Ad- p16 or Ad- p5 3alone could inhibit the proliferating activity of EJcells in vitro.Ad- p5 3could induce apoptosis of partial EJcells.G1arrest was observed72 h after infection with Ad- p16 ,but apoptosis was not obvious.The transfer of Ad- p16 and Ad- p5 3 could significantly inhibit the growth of EJcells,decrease the cloning formation rate and induce apoptosis of large num ber of EJcells. The occurrence time of subcutaneous tumor was delayed and the tum or volume in 4 weeks was dim inished by using Ad- p5 3com bined with Ad- p16 and the dif- ference was significant com pared with using Ad- p5 3or Ad- p16 alone.It was suggested that the transfer of wild- type p5 3and p16 could significantly inhibit the growth of human bladder cancer in vitro and in vivo.

关键词： p5 3 p16 bladder cancer adenovirus

adenovirus-mediated short hairpin RNA interference against p75 neurotrophin receptor in pheochromocytoma cells

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Neural Regeneration Research 2011年第7期6卷 517-522页

作者： Dongxu Feng Haopeng Li Siyue Xu YU Liu Xiaofei Hou Second Department of Orthopaedics Second Affiliated Hospital College of Medicine Xi'an Jiaotong University Xi'an 710004 ShaanxiProvince China First Affiliated Hospital Baotou Medical College Baotao 014010 Inner Mongolia Autonomous Region China

Previous studies have confirmed that motor neuron apoptosis in the anterior horn of the lumbosacral spinal cord is positively correlated with p75 neurotrophin receptor （p75NTR） expression in rat models of cauda equina syndrome. This study used adenovirus to carry a short hairpin RNA （shRNA） for p75NTR gene silencing, to reduce p75NTR expression in the damaged phase and to decrease motor neuron apoptosis. Three p75 siRNA template oligonucleotide segments （shRNA） were designed, and cloned into the 1.0 CMV shuttle vector. HEK293 cells were cotransfected with shuttle vector （carrying shRNA） and an adenovirus vector framework expressing enhanced green fluorescent protein. Thus, this study successfully obtained adenovirus carrying p75shRNA. The obtained viruses were named Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3. The recombinant adenoviruses were separately used to infect cultured pheochromocytoma cells （PC12）. Forty-eight hours later, p75NTR mRNA and total protein were analyzed from the PC12 cells. Compared with the negative controls, RNA interference rates were separately 98.49 ± 0.68%, 95.08 ± 1.79% and 96.60 ± 1.14% at the mRNA level, and 72.89 ± 2.17%, 58.83 ± 1.15% and 59.88 ± 0.44% at the protein level in the Ad.shRNA1, Ad.shRNA2, and Ad.shRNA3 groups, respectively. Thus, recombinant adenovirus shRNA-mediated gene silencing successfully suppressed p75NTR expression.

关键词： p75 neurotrophin receptor RNA interference adenovirus rat pheochromocytoma cells human embryonic kidney 293 cells apoptosis cauda equina syndrome

Effect of recombinant adenovirus vector mediated human interleukin-24 gene transfection on pancreatic carcinoma growth

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Chinese Medical Journal 2008年第20期121卷 2031-2036页

作者： PAN Xin-ting ZHU Qing-yun LI De-chun YANG Ji-cheng ZHANG Zi-xiang ZHU Xing-guo ZHAO Hua Department of General Surgery First Hospital Affiliated to Soochow University Suzhou Jiangsu 215006 China Department of Medical Imaging Hospital Affiliated to Guiyang Medical College Guiyang Guizhou 550000 China Cell and Molecular Biology Institute College of Medicine Soochow University Suzhou Jiangsu 215132 China

Background Pancreatic cancer is a highly malignant tumor affecting an ever increasing number of patients with a mean 5-year survival rate below 4%. Therefore, gene therapy for cancer has become a potential novel therapeutic modality. In this study we sought to determine the inhibitory effects of adenovirus-mediated human interleukin-24 （AdhIL-24） on pancreatic cancer.Methods Human interleukin-24 gene was cloned into replication-defective adenovirus specific for patu8988 tumor cells by virus recombination technology. Reverse transcription-polymerase chain reaction and Western blotting analysis were used to determine the expression of human interleukin-24 mRNA in patu8988 cells in vitro. Induction of apoptosis by overexpression of human interleukin-24 in patu8988 cells was determined by flow cytometry. In vivo efficacy of adenoviral delivery of human interleukin-24 was assessed in nude mice （n=10 for each group） bearing patu8988 pancreatic cancer cell lines by determining inhibition of tumor growth, endothelial growth factor and CD34 expression, and intratumoral microvessel density （MVD）.Results The recombinant adenovirus vector AdVGFP/IL-24 was constructed with a packaged recombinant retrovirus titer of 1.0×10^10 pfu/ml and successfully expressed of both mRNA and protein in patu8988 cells. The AdVGFP/IL-24 induced apoptosis of patu8988 tumor cells in vitro and significantly inhibited tumor growth in vivo （P 〈0.05）. The intratumoral MVD decreased significantly in the treated tumors （P 〈0.05）.Conclusion The recombinant adenovirus AdGFP/IL-24 can effectively express biologically active human interleukin-24, which results in inhibition of pancreatic cancer growth.

关键词： interleukin-24 adenovirus pancreatic carcinoma gene therapy angiogenesis apoptosis

Construction of recombinant adenovirus co-expression vector carrying the human transforming growth factor-β1 and vascular endothelial growth factor genes and its effect on anterior cruciate ligament fibroblasts

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Chinese Medical Journal 2008年第15期121卷 1426-1432页

作者： WEI Xue-lei LIN Lin HOU Yu FU Xin ZHANG Ji-ying MAO Ze-bin YU Chang-long Institute of Sports Medicine Peking University Third Hospital Beijing 100083 China Department of Biochemistry and Molecular Biology Peking University School of Basic Medical Sciences Beijing 100083 China

Background Remodeling of the anterior cruciate ligament （ACL） graft usually takes longer than expected. Gene therapy offers a radical different approach to remodeling of the graft. In this study, the internal ribosome entry site （IRES） sequence was used to construct a new recombinant adenovirus which permits co-expression of transforming growth factor-β1 （TGFβ1） and vascular endothelial growth factor 165 （VEGF165） genes （named Ad-VEGF165-IRES-TGFβ1）. We investigated the effects of the new adenovirus on the migration of and matrix synthesis by ACL fibroblasts. Methods Adenoviral vector containing TGFβ1 and VEGF165 genes was constructed. ACL fibroblasts were obtained from New Zealand white rabbits. After ACL fibroblasts were exposed to Ad-VEGF165-IRES-TGFβ1, the expression of VEGF165 and TGFβ1 proteins were assessed by enzyme-linked immunosorbent assay （ELISA） and Western blotting analysis. Bioassay of VEGF165 and TGFβ1 proteins were assessed by Western blotting analysis. Proliferation and migration of ACL fibroblasts were assessed by in vitro wound closure assay. Gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers were assessed by real-time PCR. Results The results showed the successful construction of a recombinant co-expression adenovirus vector containing TGFβ1 and VEGF165 genes. Co-expression of TGFβ1 and VEGF165 can induce relatively rapid and continuous proliferation of ACL fibroblasts and high gene expression of collagen type Ⅰ, collagen type Ⅲ, and fibronectin mRNA among matrix markers. Conclusion Co-expression of TGFβ1 and VEGF165 genes has more powerful and efficient effects on the migration of and matrix synthesis by ACL fibroblasts.

关键词： adenovirus fibroblasts transforming growth factor-β1 vascular endothelial growth factor 165

Combinational adenovirus-mediated gene therapy and dendritic cell vaccine in combating well-established tumors

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Cell Research 2006年第3期16卷 241-259页

作者： Terence Moyana Jim Xiang Department of Pathology and Laboratory Medicine University of Ottawa Ontario K1H 8L6 Canada Research Unit Health Research Division Saskatchewan Cancer Agency Departments of Oncology.Microbiology and Immunology.University of Saskatchewan 20 Campus Drive Saskatoon Saskatchewan STN 4H4

Recent developments in tumor immunology and biotechnology have made cancer gene therapy and immunotherapyfeasible.The current efforts for cancer gene therapy mainly focus on using immunogenes,chemogenes and tumor sup-pressor genes.Central to all these therapies is the development of efficient vectors for gene therapy.By far,adenovirus(AdV)-mediated gene therapy is one of the most promising approaches,as has confirmed by studies relating to animaltumor models and clinical trials.Dendritic cells (DCs) are highly efficient,specialized antigen-presenting cells,and DC-based tumor vaccines are regarded as having much potential in cancer immunotherapy.Vaccination with DCs pulsedwith tumor peptides,Iysates,or RNA,or loaded with apoptotic/necrotic tumor cells,or engineered to express certaincytokines or chemokines could induce significant antitumor cytotoxic T lymphocyte (CTL) responses and antitumorimmunity.Although both AdV-mediated gene therapy and DC vaccine can both stimulate antitumor immune responses,their therapeutic efficiency has been limited to generation of prophylactic antitumor immunity against re-challenge withthe parental tumor cells or to growth inhibition of small tumors.However,this approach has been unsuccessful in com-bating welt-established tumors in animal models.Therefore,a major strategic goal of current cancer immunotherapy hasbecome the development of novel therapeutic strategies that can combat welt-established tumors,thus resembling realclinical practice since a good proportion of cancer patients generally present with significant disease.In this paper,wereview the recent progress in AdV-mediated cancer gene therapy and DC-based cancer vaccines,and discuss combinedimmunotherapy including gene therapy and DC vaccines.We underscore the fact that combined therapy may have someadvantages in combating well-established tumors vis-a-vis either modality administered as a monotherapy.

关键词： gene therapy adenovirus dendritic cells vaccine cytotoxic T lymphocytes antitumor immunity