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460. Gene Correction of Embryonic Stem Cells by the Methylation Resistance Retroviral Vector GCDNsap
RNA VIRUS VECTORS II production in rat HSC. Methods: A rat HSC cell line (SBC-10) provided by Dr. H. Tsukamoto, Keck School of Medicine, Los Angles, CA, was transduced with either LG (control retrovirus) or LEIG (retrovirus coding for lefty A) in the presence of TGF-β (10 ng/ml). Four days after the transduction, lefty A expression in the retroviral vector-transduced cells was determined by RT-PCR. Levels of procollagen type I mRNA and TGF-β1 mRNA in the transduced SBC-10 cells were measured by quantitative RT-PCR using β-actin as a house-keeping gene. Results: Enhanced lefty A gene expression was confirmed in LEIG-transduced SBC-10 cells four days after the retroviral transduction. Relative expression of the procollagen type I (α1) gene in LEIG-transduced SBC-10 cells was reduced by more than 50% of untransduced or LG-transduced cells (44 ± 8 vs. 100 ± 2, 116 ± 13%, p < 0.01). In addition, the TGF-β1 gene expression in LEIG-transduced SBC-10 cells was also decreased significantly (73 ± 5% vs. 100%, p < 0.05) in comparison with untransduced cells. Conclusions: Effective lefty A gene delivery to HSC may offer a novel and effective therapeutic approach for the inhibition of hepatic stellate cell activation and hepatic fibrogenesis.
460. Gene Correction of Embryonic Stem Cells by the Methylation Resistance Retroviral Vector GCDNsap Masafumi Onodera,1 Sanae Hamanaka,1 Takeru Shimizu,1 Toshiro Nagasawa,1 Hiromitsu Nakauchi.2 1 Hematology, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2 Laboratory of Stem Cell, University of Tokyo, Tyoko, Japan. [Purpose] Embryonic stem (ES) cells are so pluripotent that they can differentiate into various types of cells by being cultured in proper conditions while they self-renew in keep their immaturity in vitro, which makes them one of ideal target cell populations in regenerative medicine. However, the mechanism of their differentiation is still unclear and control of the differentiation of them into a certain type of cells remains challenge. Furthermore, genetical modification of ES cells by viral vectors is not easy because the transgene expression driven by viral vectors is often shut off in immature cells by the mechanism of gene silencing that is de novo methylaiton and presence of negative transcription factors. In this study, we have constructed a new retroviral vector GCDNsap that is resistant to gene silencing and evaluated the ability of the vector to maintain the transgene expression in ES cells. [Materials and methods] GCDNsap has the primer biding site and the LTR derived from dl587rev and the PCMV, respectively. Especially, the negative control region (NCR) in the LTR is removed not to bind to the repression transcription factor YY1. Enhanced green florescent protein (EGFP) is used as a marker. To generate the viruses, the GCDNsapEGFP was transduced to 293gpg in which expression of the vesicular stomatitis virus G protein (VSV-G) as the viral envelope is controlled by the tetracycline inducible system (Tet off system) and the supernatant was collected 48 and 72 hours after removing Tet followed by centrifugation and resuspension with FSC free medium. For ES marking, 100μl of the viral sup was added to the culture of mouse ES cell E14. [Results and Discussion] The virus titer was approximately 2.3x10 8 cells IU/ml on HeLa cells. Replication competent retrovirus (RCR) was not detected in the culture. 2. One round transduction brought about high expression of EGFP in over 70% of ES cells and the high expression lasted more than 8 weeks. 3. When the EGFP transduced ES cells were transplanted into sub-lethally irradiated non obese diabetic/ severe combined immunodeficiency (NOD/SCID) mice intraperitoneally, they proliferated to form teratomas in the mice. Interestingly, some of them still highly expressed EGFP after differentiation in vivo, suggesting that the GCDNsap with the strong resistance to gene silencing is a useful gene transfer vehicle to ES cells. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
461. The Development of an Experimental System To Investigate the Role of Host Cell Factors in Retrovirus Transduction Delfi Krishna,1 Leslie Coburn,1 Jinsong Sheng,2 Don H. Rubin,2 Thomas W. Hodge,3 Joseph M. Le Doux.4 1 School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA; 2Vanderbilt Ingram Cancer Center, Nashville, TN; 3 Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA; 4School of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA. We have begun to develop two independent experimental approaches for examining the role of host cell factors in retrovirus transduction. In one approach, we have constructed a model system for studying the role of receptor trafficking on targeted retrovirus transduction. We stably transfected HeLa cells to express one of three different chimeras of the IL2 receptor alpha chain (TAC, TACCD16, and TAC-DKQTLL), each of which is internalized via a distinct intracellular trafficking pathway. In parallel, we constructed a targeted retrovirus to bind to these receptors by fusing a singlechain antibody variable fragment against TAC (ScFv anti-TAC)) to the N-terminus of the amphotropic envelope protein. We found that the level of gene transfer was significantly lower in HeLa cells that expressed TAC-DKQTLL, a receptor that is rapidly internalized and transported to the lysosomes, than in HeLa cells that expressed TAC, TAC-CD16, or in the parent cell line. These findings show that the intracellular trafficking itinerary of the targeted receptor can significantly affect the efficiency of gene transfer. We have also begun to use a retrovirus gene trap to identify host cell factors that are critical for retrovirus transduction. A library of gene trapped rat intestinal epithelial cells was constructed, then infected with reovirus to select for cells resistant to infection. Recombinant amphotropic retroviruses were then used to transduce clonal populations of reovirus-resistant cells, as well as the reovirus-susceptible parent cell line as a control. Interestingly, some of the reovirus-resistant cell lines were resistant to retrovirus transduction. These results suggest that retroviruses and reoviruses may use related mechanisms to infect cells even though they are members of different RNA virus families. In summary, we have begun to develop two novel experimental approaches for elucidating the role of host cell factors in retrovirus transduction, information that may prove useful for the development of more effective gene transfer vectors.
462. A Stable RD114 Retroviral Packaging Cell Line Efficiently Transduces Human Hematopoietic Cells Maureen M. Ward,1 Rose Sattler,1 Anthony Bell,1 Laxmi Baxi,2 Donna Skerrett,3 Arthur Bank.1 1 Genetics & Development, Columbia University, NYC, NY, United States; 2Obstetrics and Gynecology, Columbia University, NYC, NY, United States; 3Department of Pathology, Columbia University, NYC, NY, United States. Oncoretroviruses pseudotyped with the envelope of the feline endogenous virus (RD114) have been shown to transduce human hematopoietic cells (HSC) using transient packaging systems and are able to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient packaging systems because of the larger volumes of viral supernatant and more reproducible viral production that can be attained. We have constructed a new packaging cell line pseudotyping RD114 to an oncoretroviral gag and pol-containing murine cell line (GP101). Viral titers from this cell line measured on Hela cells were 10-fold higher than a similar amphotropic envelope-containing packaging cell line using a NeoR containing vector construct. Viral particles from the RD114 cell line were concentrated up to 100-fold (104-105 S181
460. Gene Correction of Embryonic Stem Cells by the Methylation Resistance Retroviral Vector GCDNsap Masafumi Onodera,1 Sanae Hamanaka,1 Takeru Shimizu,1 Toshiro Nagasawa,1 Hiromitsu Nakauchi.2 1 Hematology, University of Tsukuba, Tsukuba, Ibaraki, Japan; 2 Laboratory of Stem Cell, University of Tokyo, Tyoko, Japan. [Purpose] Embryonic stem (ES) cells are so pluripotent that they can differentiate into various types of cells by being cultured in proper conditions while they self-renew in keep their immaturity in vitro, which makes them one of ideal target cell populations in regenerative medicine. However, the mechanism of their differentiation is still unclear and control of the differentiation of them into a certain type of cells remains challenge. Furthermore, genetical modification of ES cells by viral vectors is not easy because the transgene expression driven by viral vectors is often shut off in immature cells by the mechanism of gene silencing that is de novo methylaiton and presence of negative transcription factors. In this study, we have constructed a new retroviral vector GCDNsap that is resistant to gene silencing and evaluated the ability of the vector to maintain the transgene expression in ES cells. [Materials and methods] GCDNsap has the primer biding site and the LTR derived from dl587rev and the PCMV, respectively. Especially, the negative control region (NCR) in the LTR is removed not to bind to the repression transcription factor YY1. Enhanced green florescent protein (EGFP) is used as a marker. To generate the viruses, the GCDNsapEGFP was transduced to 293gpg in which expression of the vesicular stomatitis virus G protein (VSV-G) as the viral envelope is controlled by the tetracycline inducible system (Tet off system) and the supernatant was collected 48 and 72 hours after removing Tet followed by centrifugation and resuspension with FSC free medium. For ES marking, 100μl of the viral sup was added to the culture of mouse ES cell E14. [Results and Discussion] The virus titer was approximately 2.3x10 8 cells IU/ml on HeLa cells. Replication competent retrovirus (RCR) was not detected in the culture. 2. One round transduction brought about high expression of EGFP in over 70% of ES cells and the high expression lasted more than 8 weeks. 3. When the EGFP transduced ES cells were transplanted into sub-lethally irradiated non obese diabetic/ severe combined immunodeficiency (NOD/SCID) mice intraperitoneally, they proliferated to form teratomas in the mice. Interestingly, some of them still highly expressed EGFP after differentiation in vivo, suggesting that the GCDNsap with the strong resistance to gene silencing is a useful gene transfer vehicle to ES cells. Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
461. The Development of an Experimental System To Investigate the Role of Host Cell Factors in Retrovirus Transduction Delfi Krishna,1 Leslie Coburn,1 Jinsong Sheng,2 Don H. Rubin,2 Thomas W. Hodge,3 Joseph M. Le Doux.4 1 School of Chemical Engineering, Georgia Institute of Technology, Atlanta, GA; 2Vanderbilt Ingram Cancer Center, Nashville, TN; 3 Division of HIV/AIDS Prevention, Centers for Disease Control and Prevention, Atlanta, GA; 4School of Biomedical Engineering, Georgia Institute of Technology, Atlanta, GA. We have begun to develop two independent experimental approaches for examining the role of host cell factors in retrovirus transduction. In one approach, we have constructed a model system for studying the role of receptor trafficking on targeted retrovirus transduction. We stably transfected HeLa cells to express one of three different chimeras of the IL2 receptor alpha chain (TAC, TACCD16, and TAC-DKQTLL), each of which is internalized via a distinct intracellular trafficking pathway. In parallel, we constructed a targeted retrovirus to bind to these receptors by fusing a singlechain antibody variable fragment against TAC (ScFv anti-TAC)) to the N-terminus of the amphotropic envelope protein. We found that the level of gene transfer was significantly lower in HeLa cells that expressed TAC-DKQTLL, a receptor that is rapidly internalized and transported to the lysosomes, than in HeLa cells that expressed TAC, TAC-CD16, or in the parent cell line. These findings show that the intracellular trafficking itinerary of the targeted receptor can significantly affect the efficiency of gene transfer. We have also begun to use a retrovirus gene trap to identify host cell factors that are critical for retrovirus transduction. A library of gene trapped rat intestinal epithelial cells was constructed, then infected with reovirus to select for cells resistant to infection. Recombinant amphotropic retroviruses were then used to transduce clonal populations of reovirus-resistant cells, as well as the reovirus-susceptible parent cell line as a control. Interestingly, some of the reovirus-resistant cell lines were resistant to retrovirus transduction. These results suggest that retroviruses and reoviruses may use related mechanisms to infect cells even though they are members of different RNA virus families. In summary, we have begun to develop two novel experimental approaches for elucidating the role of host cell factors in retrovirus transduction, information that may prove useful for the development of more effective gene transfer vectors.
462. A Stable RD114 Retroviral Packaging Cell Line Efficiently Transduces Human Hematopoietic Cells Maureen M. Ward,1 Rose Sattler,1 Anthony Bell,1 Laxmi Baxi,2 Donna Skerrett,3 Arthur Bank.1 1 Genetics & Development, Columbia University, NYC, NY, United States; 2Obstetrics and Gynecology, Columbia University, NYC, NY, United States; 3Department of Pathology, Columbia University, NYC, NY, United States. Oncoretroviruses pseudotyped with the envelope of the feline endogenous virus (RD114) have been shown to transduce human hematopoietic cells (HSC) using transient packaging systems and are able to be concentrated to high titers by centrifugation. Stable packaging systems have potential advantages over transient packaging systems because of the larger volumes of viral supernatant and more reproducible viral production that can be attained. We have constructed a new packaging cell line pseudotyping RD114 to an oncoretroviral gag and pol-containing murine cell line (GP101). Viral titers from this cell line measured on Hela cells were 10-fold higher than a similar amphotropic envelope-containing packaging cell line using a NeoR containing vector construct. Viral particles from the RD114 cell line were concentrated up to 100-fold (104-105 S181