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Effect of hydroxyurea and etoposide on transduction of human bone marrow mesenchymal stem and progenitor cell by adeno-associated virus vectors

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Acta Pharmacologica Sinica 2004年第2期25卷 70-76页

作者： Xiao-dong JU, Si-quan LOU2, Wei-guo WANG, Jian-qiang PENG3, Hua TIAN Department of Orthopaedics, Peking University Third Hospital, Beijing 100083 3Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100262, China Department of Orthopaedics Peking University Third Hospital 北京 100083 Institute of Virology Chinese Academy of Preventive Medicine 北京 100262

AIM: To study the effect of hydroxyurea and etoposide on transduction of human marrow mesenchymal and progenitor stem cells by adeno-associated virus (AAV). METHODS: Isolated human bone marrow mesenchymal stem and progenitor cells (hMSCs) were cultured in DMEM containing 10 % FBS or 5 % FBS and dexamethasone 1 μmol/L respectively. After being treated with hydroxyurea and etoposide, hMSCs were transduced by AAV-LUC. After two days luciferase activity (relative light unites per second or RLU/s) were tested, which indirectly reflected the relative transduction efficiency of different groups, and virus DNA was isolated by Hirt extraction for Southern hybridization. RESULTS: Transduction luciferase activity and transduction efficiency in cultures treated with hydroxyurea and etoposide were significantly higher than that in control cultures. Dividing cells had about 20-fold higher transduction efficiencycompared with control cells. Transduction efficiency in stationary cells was about 50 times higher than that in control cells. Southern analysis showed that hydroxyurea and etoposide enhanced second-strand DNA synthesis by rAAV. CONCLUSION: Hydroxyurea and etoposide could increase transduction efficiency of hMSCs by AAV vectors, and stationary cells were more sensitive to these drugs than dividing cells.

关键词： simian adenoviruses hydroxyurea etoposide mesoderm stem cells gene transfer techniques

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Efficient delivery of human clotting factor IX after injection of lentiviral vectors in utero

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Acta Pharmacologica Sinica 2004年第6期25卷 87-91页

作者： Xiao-guang CHEN1, Huan-zhang ZHU1, Ju-li GONG, Feng LI, Jing-lun XUE2 State Key Laboratory of genetic Engineering, Institute of genetics, Schoolof Life Sciences, Fudan University, Shanghai 200433,China Institute of genetics School of Life Sciences Fudan University State Key Laboratory of Genetic Engineering 上海 200433

AIM: To explore gene transfer feasibility for human clotting factor IX (hFIX) mediated by recombinant lentivirus in utero. METHODS: ICR mice fetus at 17-19 d gestation were received lentiviral vectors carrying hFIX cDNA under the control of liver specific promoter by intrahepatic injection. The expression and distribution of hFIX cDNA and possible immune responses against the hFIX were assessed by ELISA, PCR, RT-PCR, and immunohistochemistry, respectively. RESULTS: The serum hFIX protein were detected at different time points in all newborn mice, the highest level of hFIX was 50 μg/L and lasted for more than 30 d. Anti-hFIX antibody was not detected. hFIX cDNA was detected in liver, spleen, and heart. The expression of hFIX cDNA was only detected in liver. Besides, no germ line transmission was found at DNA and RNA levels, and no side effect associated with gene transfer was detected. CONCLUSION: The efficient delivery of hFIX can be achieved by prenatal gene transfer. It thus shows the feasibility of gene therapy for hemophilia in utero.

关键词： lentiviral vector factor IX gene transfer techniques in utero

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Tumor cell-specific gene transfer with retroviral vectors displaying single-chain antibody

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Chinese Medical Journal 2002年第7期115卷 1064-1069,153页

作者：汤宇澄李煜钱关祥 Department of Biochemistry and Molecular Biology the Researeh Center for Human Gene TherapyShanghai Second Medical UniversityShanghai 200025China

OBJECTIVE: To specifically deliver the therapeutic gene to cancer cells and construct target retroviral vectors by inserting the single-chain variable antibody fragment into the retroviral envelope. METHODS: Single-chain antibody expression vector pET -20bScfv was constructed. Binding activity of the genetically engineered single-chain variable antibody fragment was verified by enzyme-linked immunosorbent assay (ELISA) and Western blot. At the same time, by means of polymerase chain reaction (PCR)-directed mutagenesis, the appropriate cloning site EcoT22/Sal was generated at the N-terminus of receptor-binding SU domain in the MoMLV env polypeptide. Then the single- chain antibody gene, encoding a functional antibody, was inserted into the cloning site. The Scfv-env fusion gene fragment was subcloned into CMV expression vector. The Lac-Z retrovirus that co-displayed the Scfv-env chimeric protein and wild-type env protein was produced by transfection of Psi 2 cells with retroviral plasmid and the fusion gene expressing *** confirm the specificity of the recombinant retrovirus, infection assays and competitive inhibition assays were performed. RESULTS: The results of ELISA and Western blot showed that the genetically engineered single-chain variable antibody fragment could bind to the SHG44 cells surface membrane antigen. Virus-binding assay, viral infection and competitive inhibition assays confirmed that the harvested virus efficiently bound to and infected SHG44 cancer cells expressing the relative membrane antigen specially via the recognition of the target antigen. CONCLUSION: These results imply that insertion of Scfv into the retroviral envelope can specifically deliver the interested gene into specific antigen-producing cancer cells.

关键词： gene Therapy gene transfer techniques genetic Vectors Hela Cells Humans Immunoglobulin Fragments Neoplasms Research Support, Non-U.S. Gov't Retroviridae

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Current gene therapy for stomach carcinoma

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World Journal of Gastroenterology 2001年第6期7卷 752-759页

作者： Chang-Tai Xu~1 Lian-Tian Huang~1 Bo-Rong Pan~2 1 Editorial Department,the Journal of Fourth Military Medical University2 Oncology Center,Xijing Hospital,Fourth Military Medical University,169 Changle Xilu,Xi’an 710032,Shaanxi Province,China Editorial Department the Journal of Fourth Military Medical University Xi'an Shaanxi Province China. xuct2000@***

Gastric cancer is common in China [1-42],and its early diagnosis and treatment in advanced stage are difficult [31-50].In recent years ,gene study in cancer is a hotspot ,and great progress has been achieved [41-80] .... 详细信息

关键词： gene Therapy Carcinoma gene transfer techniques Humans Stomach Neoplasms

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Efficacy and safety of therapeutic angiogenesis from direct myocardial administration of an adenoviral vector expressing vascular endothelial growth factor 165

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Chinese Medical Journal 2002年第5期115卷 643-648,145页

作者：张端珍盖鲁粤范瑞云董蔚文应峰北京市解放军总医院心内科北京100853

OBJECTIVE: To investigate whether direct administration of adenoviral vectors (Ad) containing the complementary deoxyribonucleic acid (cDNA) of vascular endothelial growth factor 165 (Ad-VEGF165) induces porcine coronary collateral vessel formation, improves regional myocardial perfusion and function and is safe. METHODS: Three weeks after miniature swine underwent left thoracotomy and placement of an Ameroid constrictor on the left circumflex coronary artery (LCX), Ad-VEGF165 (n = 6) or the control, Ad expressing beta-galactosidase cDNA (Ad-Gal, n = 6), was directly administered into the ischemic myocardium in the circumflex distribution. All animals were sacrificed 4 wk after the second surgery. Myocardial perfusion and function were assessed by electrocardiogram-gated single photon emission computed tomography (GSPECT) imaging. Ex vivo coronary angiography was performed to examine collateral vessels. Toxicity was assessed by blood analyses on the day just before (day 0) and on day 1, 3, 7, 28 after vector delivery and by vascular, myocardial and liver histology after sacrifice. RESULTS: GSPECT imaging 4 wk after administration of Ad-VEGF165 demonstrated significant reduction in ischemic area (P

关键词： Adenoviridae Animals Collateral Circulation Coronary Angiography Coronary Vessels DNA, Complementary Electrocardiography Endothelial Growth Factors Female gene Therapy gene transfer techniques genetic Vectors Lymphokines Male Myocardial Ischemia Neovascularization, Physiologic Swine Swine, Miniature Tomography, Emission-Computed, Single-Photon Treatment Outcome Vascular Endothelial Growth Factor A Vascular Endothelial Growth Factors

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gene functional research using polyethylenimine-mediated in vivo gene transfection into mouse spermatogenic cells

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Asian Journal of Andrology 2006年第1期8卷 53-59页

作者： Li Lu Min Lin Min Xu Zuo-Min Zhou Jia-Hao Sha Laboratory of Reproductive Medicine Department of Histology and Embryology Nanjing Medical University Nanjing210029 China

Aim： To study polyethylenimine （PEI）-mediated in vivo gene transfection into testis cells and preliminary functional research of spermatogenic cell-specific gene NYD-SP12 using this method. Methods： PEI/DNA complexes were introduced into the seminiferous tubules of mouse testes using intratesticular injection. Transfection efficiency and speciality were analyzed on the third day of transfection with fluorescent microscopy and hematoxylin staining. The long-lasting expression of the GFP-NYD-SP12 fusion protein and its subcelluar localization in spermatogenic cells at different stages were analyzed with fluorescent microscopy and propidium iodide staining. Results： With the mediation of PEI, the GFP-NYD-SP12 fusion gene was efficiently transferred and expressed in the germ cells （especially in primary spermatocytes）. Transfection into Sertoli cells was not observed. The subcellular localization of the GFP-NYD-SP2 fusion protein showed dynamic shifts in spermatogenic cells at different stages during spermatogenesis. Conclusion： PEI can efficiently mediate gene transfer into spermatocytes. Thus, it might be useful for the functional research of spermatogenic-cell specific genes such as the NYD-SP12 gene. In our gtudy, the NYD-SP12 protein was visualized and was involved in the formation of acrosome during spermatogenesis. Our research will continue into the detailed function of NYD-SP12 in spermatocytes. （Asian J Androl 2006 Jan; 8： 53-59）

关键词： gene transfer techniques polyethylenimine NYD-SP12 gene spermatogenic cells spermatogenesis

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Anti-tumor effect of human endostatin mediated by retroviral gene transfer in nude mice

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Chinese Medical Journal 2002年第11期115卷 1664-1669,151页

作者：王轩刘福坤李希黎介寿徐根兴 Research Institute of general Surgery Nanjing General HospitalClinical School of MedicineNanjing UniversityNanjing 210002China Department of Life Sciences Nanjing UniversityNanjing 210002China Department of Molecular Biology Nanjing Military Medical SchoolNanjing 210002China

OBJECTIVE: To explore the inhibitory effect of human endostatin gene mediated by retroviral vector on the growth of human liver carcinoma. METHODS: A recombinant retroviral plasmid containing human endostatin gene and signal peptide was engineered and transferred into PA317 cells to produce retrovirus. Human liver carcinoma cells (SMMC7721) were infected with the above retrovirus to build a stable endostatin-transfected liver carcinoma cell line (SMMC-endo). The control liver carcinoma cell line (SMMC-pLncx) was developed in a similar way except that the plasmid was replaced by an empty retroviral vector. Immunohistochemistry and Western blot were used to test the expression and secretion of human endostatin. The biological activity of the expressed human endostatin was assessed by endothelial cell proliferation assay. The growth rates of SMMC-endo and control SMMC-pLncx cells in vivo and in vitro were also observed. RESULTS: The expression and secretion of human endostatin by endostatin-transfected SMMC-endo cells were confirmed by immunohistochemistry and Western blot. Compared with the control group, concentrated supernatant of SMMC-endo cells remarkably inhibited the proliferation of human umbilical vein endothelial cells by 48%, significantly higher than the inhibition by the control (10.2%; P

关键词： gene Therapy Animals Cell Division Cell Line Collagen Endostatins Endothelium, Vascular gene transfer techniques Mice Mice, Nude Neoplasms, Experimental Peptide Fragments Retroviridae Transfection

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Protection of retinal ganglion cells against glaucomatous neuropathy by neurotrophin-producing,genetically modified neural progenitor cells in a rat model

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Chinese Medical Journal 2002年第9期115卷 1394-1400,155-156页

作者：王宁利曾明兵阮奕文吴河坪陈静嫦范志刚郑湖玲中山大学中山眼科中心广州510060 广州医学院附二院眼科广州510260 中山大学解剖学教研室广州510089

OBJECTIVE: To investigate in vivo survival of retinal ganglion cells (RGCs) after partial blockage of optic nerve (ON) axoplasmic flow by sub-retinal space or vitreous cavity injection of brain-derived neural factor (BDNF) produced by genetically modified neural progenitor cells (NPCs). METHODS: Adult Sprague-Dawley (SD) rat RGCs were labeled with granular blue (GB) applied to their main targets in the brain. Seven days later, the left ON was intra-obitally crushed with a 40 g power forceps to partially block ON axoplasmic flow. Animals were randomized to three groups. The left eye of each rat received a sham injection, NPCs injection or an injection of genetically modified neural progenitors producing BDNF (BDNF-NPCs). Seven, 15 and 30 days after ON crush, retinas were examined under a fluorescence microscope. By calculating and comparing the average RGCs densities and RGC apoptosis density, RGC survival was estimated and the neuro-protective effect of transplanted cells was evaluated. RESULTS: Seven, 15 and 30 days after crush, in the intra-vitreous injection group, mean RGC densities had decreased to 1885 +/- 68, 1562 +/- 20, 1380 +/- 7 and 1837 +/- 46, 1561 +/- 58, 1370 +/- 16, respectively with sham injection or neural progenitors injection. However, RGCs density in the groups treated with intra-vitreous injection of BDNF-NPC was 2101 +/- 15, 1809 +/- 19 and 1625 +/- 34. Similar results were found in groups after sub-retinal injection. Higher densities were observed in groups treated with BDNF-NPCs. There were statistically significant differences among groups through nonparametric tests followed by the Mann-Whitely test. RGC apoptosis density in BDNF-NPC at each follow-up time was less than in other groups. CONCLUSIONS: A continuous supply of neurotrophic factors by the injection of genetically modified neural progenitors presents a highly effective approach to counteract optic neuropathy and RGC degeneration after partial ON axoplasmic flow blockage.

关键词： gene Therapy Animals Apoptosis Axonal Transport Brain-Derived Neurotrophic Factor Cell Survival gene transfer techniques Glaucoma Male Rats Rats, Sprague-Dawley Research Support, Non-U.S. Gov't Retinal Ganglion Cells Stem Cells Vitreous Body

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Differentiation of embryonic stem cells into insulin-producing cells promoted by Nkx2.2 gene transfer

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World Journal of Gastroenterology 2005年第27期11卷 4161-4166页

作者： Akira Shiroi Shigehiko Ueda Yukiteru Ouji Ko Saito Kei Moriya Yuko Sugie Hiroshi Fukui Shigeaki Ishizaka Masahide Yoshikawa Division of Developmental Biology Department of Gastroenterology and Hepatology

AIM： To investigate the ability of a genetically altered embryonic stem （ES） cell line to generate insulin-producing cells in vitro following transfer of the Nkx2.2 ***： Hamster Nkx2.2 genes were transferred into mouse ES cells. Parental and Nkx2.2-transfected ES cells were initiated toward differentiation in embryoid body （EB） culture for 5 d and the resulting EBs were transferred to an attached culture system. Dithizone （DTZ）, a zincchelating agent known to selectively stain pancreatic beta cells, was used to detect insulin-producing *** outgrowths were incubated in DTZ solution （final concentration, 100μg/mL） for 15 rain before being examined microscopically. gene expression of the endocrine pancreatic markers was also analyzed by RT-PCR. In addition, insulin production was determined immunohistochemically and its secretion was examined using an ***： DTZ-stained cellular clusters appeared after approximately 14 d in the culture of Nkx2.2-transfected ES cells （Nkx-ES cells）, which was as much as 2 wk earlier, than those in the culture of parental ES cells （wt-ES）. The frequency of DTZ-positive cells among total cultured cells on day 28 accounted for approximately 1.0% and 0.1% of the Nkx-ES- and wt-ES-derived EB outgrowths, respectively. The DTZ-positive cellular clusters were found to be immunoreactive to insulin, while the gene expressions of pancreatic-duodenal homeobox 1 （PDX1）, proinsulin 1 and proinsulin 2 were observed in the cultures that contained DTZ-positive cellular *** secretion was also confirmed by ELISA, whereas glucose-dependent secretion was not ***： Nkx2.2-transfected ES cells showed an ability to differentiate into insulin-producing cells.

关键词： Animals Cell Differentiation Cell Line Cricetinae gene transfer techniques Homeodomain Proteins Insulin Islets of Langerhans Mice Mice, Inbred Strains Stem Cells Transcription Factors

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Adenovirus induced acute hepatitis in non-human primates after liver-directed gene therapy

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Chinese Medical Journal 2002年第5期115卷 726-731,153页

作者：鲁慧英 Deborah Sullivan Michael A Gerbera Srikanta Dash 辽宁省大连旅顺406医院传染科辽宁116041 杜兰大学医学院病理和实验医学系美国路易斯安那州新奥尔良70112

OBJECTIVE: To define the mechanism of acute hepatitis in non-human primates after liver directed gene therapy. METHODS: Differences in immune response exhibited by 8 rhesus monkeys receiving adenovirus (Ad) or lipofectamine-mediated gene transfer by various routes, the time course, and the nature of the specific immune responses to both adenoviral vectors and transgene products were studied using HE staining (H&E) and immunohistochemical staining. RESULTS: The monkeys developed mild to moderate acute hepatitis 1 to 3 weeks after intravenous or intrabiliary injection of first generation replication-defective adenoviruses carrying the Escherichia coli lacZ gene. This was accompanied by adenovirus-mediated T-cell proliferation and neutralizing antibodies to the adenovirus. Increased numbers of CD3(+), CD4(+) and CD8(+) T-lymphocytes were detected in the diseased livers, while B-lymphocytes were absent. Hepatocytes demonstrated increased expression of beta 2-microglobulins (beta 2-MG) and HLA-DR antigens in the plasma membranes. The development of acute hepatitis and the accompanying immune abnormalities were delayed in immunosuppressed monkeys until after the discontinuation of immunosuppressive therapy. The monkeys infused with Ad. CMVluc showed more significant and longer durations of hepatitis than the monkeys infused with adenoviruses carrying the lacZ gene. Lipofectamine-mediated gene transfer was inefficient. There was neither lacZ expression nor significant immune response in the liver of monkeys infused with lipofectamine via the portal vein or the common bile duct. CONCLUSION: Immune response to the hepatocytes in liver directed gene therapy is MHC class I restricted and T-cell mediated. Both adenoviral vectors and foreign genes are related to the liver damage. Mild to moderate hepatic inflammation seen with the E-1 deleted vector is reversible. Immunosuppression regimens may prolong transgene expression and delay the development of acute adenoviral hepatitis.

关键词： Acute Disease Adenoviridae Adenoviridae Infections Animals Antigens, CD3 Antigens, CD4 Antigens, CD8 DNA, Recombinant gene transfer techniques HLA-DR Antigens Hepatitis, Animal Immunohistochemistry Liver Macaca mulatta beta 2-Microglobulin

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