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Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1998;30(2):159-163.
The Construction and Expression of Polycistronic Retroviral Vector Carrying Genes of TNF-alpha, IL-2 and NeoR.
Lai GH, Tang ZY, Chen SS.
Research Center for Human Gene Therapy, Shanghai Second Medical University, Shanghai 200025, China.
The coordinate expression of a marker gene and therapeutic genes in a single vector is markedly advantageous. The internal ribosome entry site (IRES) from encephalomyocarditis and that from polio viruses were used to construct a polycistronic retroviral vector pGCEN/TRI, where Neo-resistant gene was used as a marker gene and TNF-alpha cDNA, IL-2 cDNA as genes of interest, so that the LTR promoter derived the expression of a tricistronic mRNA. Amphotropic packaging cells PA317 transfected with pGCEN/TRI using LipofectAMINE was selected with G418 and the positive clones expressing the genes of interest to produce high-titer retrovirus (5x10(5) CFU/ml) were obtained. PCR confirmed the presence of proviral DNA in the positive producer cells and Northern blot analysis revealed a single, LTR promoted transcript. The transduced cells expressed TNF-alpha and IL-2 at different levels. This demonstrated that polycistronic retroviral vector containing IRES could efficiently express multiple therapeutic genes in the same target cell.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12174276&dopt=Abstract [PubMed - as supplied by publisher]
Sheng Wu Hua Xue Yu Sheng Wu Wu Li Xue Bao (Shanghai). 1998;30(2):164-168.
The Construction of an Adenovirus Vector Containing Cytosine Deaminase Gene and Its Application.
Xu DH, Ge K, Jian Q, Zheng ZC, Liu XY.
Shanghai Institute of Biochemistry, the Chinese Academy of Sciences, Shanghai 200031, China.
A replication-defective recombinant adenovirus vector containing CMV promoter and Escherichia coli cytosine deaminase (cd) gene (AdCMVCD) was constructed. It was shown by Southern blotting and RT-PCR that cd gene had been inserted into AdCMVCD and was expressed in the infected cells. The recombinant virus was purified by gradient centrifugation in CsCl and its titer was 1x10(15) pfu/L. HeLa and C6 cells infected with AdCMVCD(m.o.i=100) became sensitive to the prodrug 5FC, and the number of viable cells decreased to less than 20% of the control after treatment with 100 &mgr;mol/L 5FC. Significant bystander effect was also observed. When cells infected with AdCMVCD were mixed with wild type cells at a ratio of 3.3 : 96.7, more than 60% of the cells could be killed in the presence of 50 &mgr;mol/L 5FC. These results suggest that the AdCMVCD/5FC system may provide a new approach for gene therapy of cancers.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12174277&dopt=Abstract [PubMed - as supplied by publisher]
Blood. 2003 Mar 15;101(6):2167-74. Epub 2002 Nov 21.
IL-7 surface-engineered lentiviral vectors promote survival and efficient gene transfer in resting primary T lymphocytes.
Verhoeyen E, Dardalhon V, Ducrey-Rundquist O, Trono D, Taylor N, Cosset FL.
Laboratoire de Vectorologie Retrovirale et Therapie Genique, Ecole Normale Superieure de Lyon, Lyon, France.
Important gene therapy target cells such as resting human T cells are refractory to transduction with lentiviral vectors. Completion of reverse transcription, nuclear import, and subsequent integration of the lentiviral genome occur in these cells only if they have been activated. In T-cell-based gene therapy trials performed to date, cells have been activated via their cognate antigen receptor. To couple activation with gene transfer, we previously generated lentiviral vectors displaying an anti-CD3 scFv fragment that allowed up to 48% transduction of freshly isolated T cells. However, transduction of highly purified resting T cells with these anti-CD3-displaying lentiviral vectors was inefficient and shifted the T cells from the naive to the memory phenotype. Here, we describe interleukin-7 (IL-7)-displaying HIV-1-derived vectors. Like recombinant IL-7, these modified particles could promote the survival of primary T cells placed in culture without inducing a naive-to-memory phenotypic switch. Furthermore, a single exposure to the IL-7-displaying vectors resulted in efficient gene transfer in both resting memory adult T cells and naive cord blood T cells. With adult naive T cells, preactivation with recombinant IL-7 was necessary for efficient gene transfer. Altogether, these results suggest that IL-7-displaying vectors could constitute interesting tools for T-cell-targeted gene therapy.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12446448&dopt=Abstract
sunm.shcnc.ac.cn
The possibility of tumor-specific suicidal gene therapy of colorectalcarcinoma was investigated using carcinoembryonic antigen (CEA) -positive human colorectal carcinoma cell line LoVo and CEA-negative HeLa cell line as a model. After confirming cellular specificity of cea promoter by CAT assay, eukaryotic expression plasmid pCEACD was constructed in which the expresstion of E. coli cytosine deaminase (CD) gene was under the control of cea promoter. The expression of CD gene increased the sensitivity of LoVo cells to 5-fluorocytosine (5FC) by 750 fold, while the sensitivity of HeLa cells to 5FC was increased by only 7.5 fold. These results suggest that the expression of CD gene driven by cea promoter specifically killed CEA-positive colorectal carcinoma cells. Transmission electron microscopy and DNA fragmentation assay demonstrate that CD/5FC system induced apoptosis in LoVo cells.
online pharmacy ref. source: www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=12174289&dopt=Abstract [PubMed - as supplied by publisher]
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