The Experts below are selected from a list of 30975 Experts worldwide ranked by ideXlab platform
Arnold J. Berk - One of the best experts on this subject based on the ideXlab platform.
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robust in vivo transduction of a genetically stable epstein barr virus episome to hepatocytes in mice by a Hybrid viral Vector
Journal of Virology, 2009Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:To make a safe, long-lasting gene delivery vehicle, we developed a Hybrid Vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd Vector is recombined to form a circular plasmid. This episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this Hybrid approach is highly efficient at delivering EBV episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of Vector-transduced mice demonstrated that luciferase transgene expression from the Hybrid was robust and compared well to a traditional HDAd Vector. Quantitative PCR analysis confirmed that the EBV episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV Hybrid to a variety of disease targets and the potential benefits of this approach are discussed.
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14 high efficiency and long term persistence in vivo from a helper dependent adenovirus epstein barr virus Hybrid Vector
Molecular Therapy, 2006Co-Authors: Sean D. Gallaher, Jose S Gil, Arnold J. BerkAbstract:Top of pageAbstract Current strategies for gene replacement therapy are constrained by a number of factors. Adenovirus-based Vectors can confer high transduction efficiency, but may persist poorly in mitotic cells. Integrating Vectors, e.g. retrovirus Vectors, can cause insertional mutagenesis/oncogenesis. Epstien-Barr virus episomes can persist extra-chromosomally, but delivery is inefficient. Our goal is to create a novel gene delivery vehicle that leverages the transduction efficiency of adenovirus with the non-integrating, but long-term persistence of an EBV episome. In our Hybrid Vector system, an EBV-based episome bearing a therapeutic or reporter transgene is delivered to target cells via a Helper Dependent Adenovirus (HDA or |[ldquo]|gutted|[rdquo]|) Vector. The episome contains an expressions cassette for the EBV Nuclear Antigen-1 (EBNA-1) protein, and its target binding site. It has been shown that these elements can tether an episome to host cell chromosomes, thus conferring long-term extra-chromosomal maintenance. A human origin in the episome allows for replication during S-phase. These sequences are present in linear form flanked by Cre recombinase targets in the HDA backbone. Upon transduction of target cells, Cre expressed from a second HDA Vector excises and circularizes the episome sequence. This recombination places a promoter upstream of the transgene and EBNA-1. Previously, we have shown that our HDA-EBV Hybrid produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression. In the present study, we examine the benefit of using the Hybrid Vector for gene expression in mouse hepatocytes in vivo. The Renilla luciferase (RL) reporter was chosen as a transgene because its activity can be assayed repeatedly and non-invasively in mouse. In this Vector, the RL transgene is only expressed following Cre-mediated circularization of the episome. Thus, the magnitude, location, and duration of bioluminescence indicates the efficiency of episome delivery and its persistence. Currently, 28 mice have been injected i.v. with either 5|[times]|109 genomes of an HDA-EBV expressing RL or various controls. An HDA Vector with the same RL expression cassette but lacking the EBV elements was included in the control group. Transduction by the HDA-EBV Vector was found to be highly efficient when assayed by confocal fluorescence microscopy on harvested liver tissue. Bioluminescence studies on the HDA-EBV reporter indicate that RL activity is detectable in the mouse as early as three days post injection and is localized to the liver. Luminosity then increases up to 100,000 times by day 30 before dropping about ten-fold by day 60. Continuing analysis shows that expression remains at this high level for at least four months. These results demonstrate how efficiently HDA Vectors can be used to deliver EBV episomes to mouse hepatocytes in vivo. We propose that this Vector system is ideally suited for applications such as FIX gene replacement for hemophilia where long-term hepatic expression of a therapeutic transgene is desirable.
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848 helper dependent adenovirus epstein barr virus Hybrid Vector for long term persistance in hepatocytes
Molecular Therapy, 2006Co-Authors: Jose S Gil, Sean D. Gallaher, Arnold J. BerkAbstract:Helper Dependent Adenoviral (HDA) Vectors are an effective method for in vivo gene transfer. The lack of viral genes and replication competent virus greatly diminishes the immune response and cytotoxicity of transduced cells. They are relatively easy to grow to high titers, appropriate for in vivo gene therapy. Though, like typical Adenoviral Vectors, HDAs have no mechanism to persist in dividing cells.
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874. Long-Term Transgene Expression In Vivo from a Helper Dependent Adenovirus|[mdash]|Epstein-Barr Virus Hybrid Vector
Molecular Therapy, 2005Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) Vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal episome in replicating B cells. Maintenance is mediated by the virally-expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the episome and tethers it to metaphase chromosomes for segregation into daughter cells during mitosis. In our Hybrid binary system, an HDA Vector (HDA-EBV) is used to deliver a linearized, EBV-based episome to target cells. Co-infection with a second HDA Vector expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a promoter upstream of the transgene. The episome also contains a human origin and the EBNA-1 binding site for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA-EBV Vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression1.
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773. Non-Invasive In Vivo Detection of Episomes Delivered to Mouse Hepatocytes by a Helper Dependent Adenovirus- Epstein-Barr Virus Hybrid Vector System
Molecular Therapy, 2004Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) Vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal episome in replicating B cells. Maintenance is mediated by the virally expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the Family of Repeats (FR) region of the EBV episome, and tethers it to metaphase chromosomes for segregation during telophase. In our Hybrid binary system, an HDA Vector (HDA.EBV) is used to deliver a linear EBV-based episome to target cells. Co-infection with a second HDA expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a CMV promoter upstream of the transgene. The episome also contains a human origin and the FR for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA.EBV Vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular episomes in vitro and that EBNA-1 and the FR significantly prolong transgene expression (in publication).
Sean D. Gallaher - One of the best experts on this subject based on the ideXlab platform.
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robust in vivo transduction of a genetically stable epstein barr virus episome to hepatocytes in mice by a Hybrid viral Vector
Journal of Virology, 2009Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:To make a safe, long-lasting gene delivery vehicle, we developed a Hybrid Vector that leverages the relative strengths of adenovirus and Epstein-Barr virus (EBV). A fully gene-deleted helper-dependent adenovirus (HDAd) is used as the delivery vehicle for its scalability and high transduction efficiency. Upon delivery, a portion of the HDAd Vector is recombined to form a circular plasmid. This episome includes two elements from EBV: an EBV nuclear antigen 1 (EBNA1) expression cassette and an EBNA1 binding region. Along with a human replication origin, these elements provide considerable genetic stability to the episome in replicating cells while avoiding insertional mutagenesis. Here, we demonstrate that this Hybrid approach is highly efficient at delivering EBV episomes to target cells in vivo. We achieved nearly 100% transduction of hepatocytes after a single intravenous injection in mice. This is a substantial improvement over the transduction efficiency of previously available physical and viral methods. Bioluminescent imaging of Vector-transduced mice demonstrated that luciferase transgene expression from the Hybrid was robust and compared well to a traditional HDAd Vector. Quantitative PCR analysis confirmed that the EBV episome was stable at approximately 30 copies per cell for up to 50 weeks and that it remained circular and extrachromosomal. Approaches for adapting the HDAd-EBV Hybrid to a variety of disease targets and the potential benefits of this approach are discussed.
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14 high efficiency and long term persistence in vivo from a helper dependent adenovirus epstein barr virus Hybrid Vector
Molecular Therapy, 2006Co-Authors: Sean D. Gallaher, Jose S Gil, Arnold J. BerkAbstract:Top of pageAbstract Current strategies for gene replacement therapy are constrained by a number of factors. Adenovirus-based Vectors can confer high transduction efficiency, but may persist poorly in mitotic cells. Integrating Vectors, e.g. retrovirus Vectors, can cause insertional mutagenesis/oncogenesis. Epstien-Barr virus episomes can persist extra-chromosomally, but delivery is inefficient. Our goal is to create a novel gene delivery vehicle that leverages the transduction efficiency of adenovirus with the non-integrating, but long-term persistence of an EBV episome. In our Hybrid Vector system, an EBV-based episome bearing a therapeutic or reporter transgene is delivered to target cells via a Helper Dependent Adenovirus (HDA or |[ldquo]|gutted|[rdquo]|) Vector. The episome contains an expressions cassette for the EBV Nuclear Antigen-1 (EBNA-1) protein, and its target binding site. It has been shown that these elements can tether an episome to host cell chromosomes, thus conferring long-term extra-chromosomal maintenance. A human origin in the episome allows for replication during S-phase. These sequences are present in linear form flanked by Cre recombinase targets in the HDA backbone. Upon transduction of target cells, Cre expressed from a second HDA Vector excises and circularizes the episome sequence. This recombination places a promoter upstream of the transgene and EBNA-1. Previously, we have shown that our HDA-EBV Hybrid produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression. In the present study, we examine the benefit of using the Hybrid Vector for gene expression in mouse hepatocytes in vivo. The Renilla luciferase (RL) reporter was chosen as a transgene because its activity can be assayed repeatedly and non-invasively in mouse. In this Vector, the RL transgene is only expressed following Cre-mediated circularization of the episome. Thus, the magnitude, location, and duration of bioluminescence indicates the efficiency of episome delivery and its persistence. Currently, 28 mice have been injected i.v. with either 5|[times]|109 genomes of an HDA-EBV expressing RL or various controls. An HDA Vector with the same RL expression cassette but lacking the EBV elements was included in the control group. Transduction by the HDA-EBV Vector was found to be highly efficient when assayed by confocal fluorescence microscopy on harvested liver tissue. Bioluminescence studies on the HDA-EBV reporter indicate that RL activity is detectable in the mouse as early as three days post injection and is localized to the liver. Luminosity then increases up to 100,000 times by day 30 before dropping about ten-fold by day 60. Continuing analysis shows that expression remains at this high level for at least four months. These results demonstrate how efficiently HDA Vectors can be used to deliver EBV episomes to mouse hepatocytes in vivo. We propose that this Vector system is ideally suited for applications such as FIX gene replacement for hemophilia where long-term hepatic expression of a therapeutic transgene is desirable.
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848 helper dependent adenovirus epstein barr virus Hybrid Vector for long term persistance in hepatocytes
Molecular Therapy, 2006Co-Authors: Jose S Gil, Sean D. Gallaher, Arnold J. BerkAbstract:Helper Dependent Adenoviral (HDA) Vectors are an effective method for in vivo gene transfer. The lack of viral genes and replication competent virus greatly diminishes the immune response and cytotoxicity of transduced cells. They are relatively easy to grow to high titers, appropriate for in vivo gene therapy. Though, like typical Adenoviral Vectors, HDAs have no mechanism to persist in dividing cells.
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874. Long-Term Transgene Expression In Vivo from a Helper Dependent Adenovirus|[mdash]|Epstein-Barr Virus Hybrid Vector
Molecular Therapy, 2005Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) Vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal episome in replicating B cells. Maintenance is mediated by the virally-expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the episome and tethers it to metaphase chromosomes for segregation into daughter cells during mitosis. In our Hybrid binary system, an HDA Vector (HDA-EBV) is used to deliver a linearized, EBV-based episome to target cells. Co-infection with a second HDA Vector expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a promoter upstream of the transgene. The episome also contains a human origin and the EBNA-1 binding site for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA-EBV Vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular episomes in vitro and that the EBV elements significantly prolong transgene expression1.
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773. Non-Invasive In Vivo Detection of Episomes Delivered to Mouse Hepatocytes by a Helper Dependent Adenovirus- Epstein-Barr Virus Hybrid Vector System
Molecular Therapy, 2004Co-Authors: Sean D. Gallaher, Oliver Dorigo, Arnold J. BerkAbstract:Helper Dependent Adenovirus (HDA) Vectors deleted in all viral genes persist poorly in vivo due to viral genome loss during mitosis. We have addressed this limitation by using elements from Epstein-Barr Virus (EBV), the genome of which persists as an extra-chromosomal episome in replicating B cells. Maintenance is mediated by the virally expressed Epstein-Barr Nuclear Antigen 1 (EBNA-1) protein that binds the Family of Repeats (FR) region of the EBV episome, and tethers it to metaphase chromosomes for segregation during telophase. In our Hybrid binary system, an HDA Vector (HDA.EBV) is used to deliver a linear EBV-based episome to target cells. Co-infection with a second HDA expressing Cre recombinase (HDA.Cre) leads to the excision and circularization of the loxP-flanked episome sequence, and places a CMV promoter upstream of the transgene. The episome also contains a human origin and the FR for replication and segregation in mitotic cells. We have shown that co-infection with HDA.Cre and an HDA.EBV Vector carrying either a Cyan Fluorescence Protein (CFP) reporter gene or Puromycin AcetylTransferase (PAC) drug resistance gene produces circular episomes in vitro and that EBNA-1 and the FR significantly prolong transgene expression (in publication).
Cornel Fraefel - One of the best experts on this subject based on the ideXlab platform.
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herpes simplex virus type 1 adeno associated virus Hybrid Vectors mediate site specific integration at the adeno associated virus preintegration site aavs1 on human chromosome 19
Journal of Virology, 2002Co-Authors: Thomas Heister, Irma Heid, Mathias Ackermann, Cornel FraefelAbstract:Herpes simplex virus type 1 (HSV-1)-based amplicon Vectors have a large transgene capacity and can efficiently infect many different cell types. One disadvantage of HSV-1 Vectors is their instability of transgene expression. By contrast, Vectors based on adeno-associated virus (AAV) can either persist in an episomal form or integrate into the host cell genome, thereby supporting long-term gene expression. AAV expresses four rep genes, rep68, -78, -40, and -52. Of those, rep68 or rep78 are sufficient to mediate site-specific integration of the AAV DNA into the host cell genome. The major disadvantage of AAV Vectors is the small transgene capacity (∼4.6 kb). In this study, we constructed HSV/AAV Hybrid Vectors that contained, in addition to the standard HSV-1 amplicon elements, AAV rep68, rep78, both rep68 and -78, or all four rep genes and a reporter gene that was flanked by the AAV inverted terminal repeats (ITRs). Southern blots of Hirt DNA from cells transfected with the Hybrid Vectors and HSV-1 helper DNA demonstrated that both the AAV elements and the HSV-1 elements were functional in the context of the Hybrid Vector. All Hybrid Vectors could be packaged into HSV-1 virions, although those containing rep sequences had lower titers than Vectors that did not. Site-specific integration at AAVS1 on human chromosome 19 was directly demonstrated by PCR and sequence analysis of ITR-AAVS1 junctions in Hybrid Vector-transduced 293 cells. Cell clones that stably expressed the transgene for at least 12 months could easily be isolated without chemical selection. In the majority of these clones, the transgene cassette was integrated at AAVS1, and no sequences outside the ITR cassette, rep in particular, were present as determined by PCR, ITR rescue/replication assays, and Southern analysis. Some of the clones contained random integrations of the transgene cassette alone or together with sequences outside the ITR cassette. These data indicate that the long-term transgene expression observed following transduction with HSV/AAV Hybrid Vectors is, at least in part, supported by chromosomal integration of the transgene cassette, both randomly and site specifically.
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gene transfer to the nigrostriatal system by Hybrid herpes simplex virus adeno associated virus amplicon Vectors
Human Gene Therapy, 1999Co-Authors: Lauren Costantini, Cornel Fraefel, Xandra O Breakefield, David R Jacoby, Samuel J Wang, Ole IsacsonAbstract:To improve gene transfer to CNS neurons, critical elements of herpes simplex virus 1 (HSV-1) amplicons and recombinant adeno-associated virus (AAV) Vectors were combined to construct a Hybrid amplicon Vector, and then packaged via a helper virus-free system. We tested the HSV/AAV Hybrid amplicon Vectors for transduction efficiency and stability of transgene expression (green fluorescent protein) in primary neuronal cultures from rat fetal ventral mesencephalon, in comparison with traditional HSV amplicon, AAV, or adenovirus (Ad) Vectors at the same multiplicity of infection. The HSA/AAV Hybrid Vectors transduced the highest number of primary neurons in culture 2 days after infection. As compared with all other Vectors tested, only Hybrid Vectors containing the AAV rep gene maintained the 2-day level of transgene expression over 12 days in culture. This rep-containing Hybrid Vector was then tested for efficiency and safety in the brain. One month after injection into adult rat striatum (1 X 106 transducing...
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gene transfer into hepatocytes mediated by helper virus free hsv aav Hybrid Vectors
Molecular Medicine, 1997Co-Authors: Cornel Fraefel, Xandra O Breakefield, David R Jacoby, Christopher R Lage, Harold Hilderbrand, Janice Y Chou, Frederick W Alt, Joseph A MajzoubAbstract:Vectors based on herpes simplex virus type 1 (HSV-1) can efficiently transduce hepatocytes in the mouse liver, and Vector genomes can persist for at least 2 months. However, 24 hr after gene transfer, the number of cells that express the transgene decreases rapidly and no transduced cells are detectable after 7 days. In this study, we examined the capability of a helper virus-free HSV/AAV Hybrid amplicon Vector to extend transgene expression in hepatocytes in vivo. HSV-1 amplicon or HSV/AAV Hybrid amplicon Vectors that express reporter genes from different transcriptional regulatory sequences were packaged into HSV-1 virions using a helper virus-free packaging system. To determine relative transduction efficiencies, Vector stocks were titered on four different cell lines, including hamster kidney (BHK21) and human lung (Hs913T) fibroblasts, and mouse (G6Pase−/−) and human (NPLC) hepatocytes. After in vivo injection of Vector stocks into mouse liver, tissue sections were examined for reporter gene expression and cellular inflammatory response. Blood samples were collected to measure serum transaminase levels as a biochemical index of liver toxicity. Expression of a reporter gene from liver-specific promoter sequences was consistently more effective in hepatic cells compared with fibroblasts, whereas the opposite was true when using an HSV-1 immediate-early promoter. Expression in hepatocytes in vivo was markedly longer from HSV/AAV Hybrid Vector compared with traditional HSV-1 amplicon Vector: the number of transduced cells (∼2% of all hepatocytes) remained stable over 7 days after injection of HSV/AAV Hybrid Vector, whereas no transduced cells were detected 7 days after gene transfer with standard HSV-1 amplicon Vector. The rapid decline in reporter gene expression from standard amplicons was not solely caused by a B or T lymphocyte-mediated immune response, as it also occurred in RAG2−/− mice. Hepatocyte toxicity and cellular inflammatory effects associated with HSV/AAV Hybrid Vector-mediated gene transfer were minimal, and readministration of Vector stock proved equally effective in naive mice and in animals that received a first Vector dose 4 weeks earlier. HSV/AAV Hybrid amplicon Vectors support gene expression in vivo for considerably longer than do traditional HSV-1 amplicon Vectors. Moreover, expression from these Vectors does not provoke an overt inflammatory or immune response, allowing efficacious expression following repeated in vivo dosing. These characteristics suggest that such Vectors may hold future promise for hepatic gene replacement therapy.
Toshiro Hara - One of the best experts on this subject based on the ideXlab platform.
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btk gene targeting by homologous recombination using a helper dependent adenovirus adeno associated virus Hybrid Vector
Gene Therapy, 2016Co-Authors: Hidetaka Yamamoto, Masataka Ishimura, Kohnosuke Mitani, Hidetoshi Takada, Koichi Kusuhara, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Masayuki Ochiai, Toshiro HaraAbstract:BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus Hybrid Vector
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btk gene targeting by homologous recombination using a helper dependent adenovirus adeno associated virus Hybrid Vector
Gene Therapy, 2016Co-Authors: Hidetaka Yamamoto, Masataka Ishimura, Kohnosuke Mitani, Hidetoshi Takada, Koichi Kusuhara, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Masayuki Ochiai, Toshiro HaraAbstract:X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting Vector (HD-Ad.AAV BTK Vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK Vector via homologous recombination in hematopoietic stem cells.
Kohnosuke Mitani - One of the best experts on this subject based on the ideXlab platform.
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btk gene targeting by homologous recombination using a helper dependent adenovirus adeno associated virus Hybrid Vector
Gene Therapy, 2016Co-Authors: Hidetaka Yamamoto, Masataka Ishimura, Kohnosuke Mitani, Hidetoshi Takada, Koichi Kusuhara, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Masayuki Ochiai, Toshiro HaraAbstract:BTK gene targeting by homologous recombination using a helper-dependent adenovirus/adeno-associated virus Hybrid Vector
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btk gene targeting by homologous recombination using a helper dependent adenovirus adeno associated virus Hybrid Vector
Gene Therapy, 2016Co-Authors: Hidetaka Yamamoto, Masataka Ishimura, Kohnosuke Mitani, Hidetoshi Takada, Koichi Kusuhara, Yoshimichi Nakatsu, Teruhisa Tsuzuki, Masayuki Ochiai, Toshiro HaraAbstract:X-linked agammaglobulinemia (XLA) is one of the most common humoral immunodeficiencies, which is caused by mutations in Bruton's tyrosine kinase (BTK) gene. To examine the possibility of using gene therapy for XLA, we constructed a helper-dependent adenovirus/adeno-associated virus BTK targeting Vector (HD-Ad.AAV BTK Vector) composed of a genomic sequence containing BTK exons 6-19 and a green fluorescence protein-hygromycin cassette driven by a cytomegalovirus promoter. We first used NALM-6, a human male pre-B acute lymphoblastic leukemia cell line, as a recipient to measure the efficiency of gene targeting by homologous recombination. We identified 10 clones with the homologous recombination of the BTK gene among 107 hygromycin-resistant stable clones isolated from two independent experiments. We next used cord blood CD34⁺ cells as the recipient cells for the gene targeting. We isolated colonies grown in medium containing cytokines and hygromycin. We found that the targeting of the BTK gene occurred in four of the 755 hygromycin-resistant colonies. Importantly, the gene targeting was also observed in CD19⁺ lymphoid progenitor cells that were differentiated from the homologous recombinant CD34⁺ cells during growth in selection media. Our study shows the potential for the BTK gene therapy using the HD-Ad.AAV BTK Vector via homologous recombination in hematopoietic stem cells.
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a novel helper dependent adenovirus retrovirus Hybrid Vector stable transduction by a two stage mechanism
Molecular Therapy, 2002Co-Authors: Harris S Soifer, Kohnosuke Mitani, Collin Higo, Christopher R Logg, Lily J Jih, Toshiaki Shichinohe, Erik Harboeschmidt, Noriyuki KasaharaAbstract:We have developed a novel Vector system that uses a helper-dependent adenoviral Vector as a carrier to deliver a fully functional retrovirus Vector. The helper-dependent adenovirus (HDAd) can accommodate large inserts, provide high titers, and infect nondividing as well as dividing cells. However, adenoviral DNA is rarely integrated into the host cell genome, and its episomal expression is transient. Therefore we inserted a replication-competent, ecotropic retrovirus Vector containing the green fluorescent protein (GFP) reporter gene as a second-stage component. The well-characterized host species tropism of each Vector component provided a stringent biological assay system that demonstrates the two-stage transduction mechanism of the Hybrid Vector, because the adenovirus stage can efficiently transduce human cells but cannot replicate in murine cells, and conversely, the ecotropic retrovirus stage cannot enter human cells but can efficiently proliferate in murine cells, resulting in permanent integration and progressive spread of reporter gene expression.
