The Experts below are selected from a list of 1038 Experts worldwide ranked by ideXlab platform
X O Breakefield - One of the best experts on this subject based on the ideXlab platform.
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Integration of active human β-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector
Gene Therapy, 2007Co-Authors: A Oehmig, M L Cortés, K F Perry, M Sena-esteves, Cornel Fraefel, X O BreakefieldAbstract:Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal β -galactosidase ( BGAL ) gene of 100 kb. Temporary expression and functional activity of β -galactosidase ( β -gal) could be detected in human β -gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of β -gal was achieved in Gli36 cells infected with rep -plus, but not rep -minus, HSV/AAV hybrid vectors. None of five clones isolated after rep -minus hybrid vector infection showed elevated β -gal activity or site-specific integration. In contrast, 80% of the rep -plus clones possessed β -gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep -plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and β -gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.
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Integration of active human β -galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector
Gene Therapy, 2007Co-Authors: A Oehmig, M L Cortés, K F Perry, M Sena-esteves, Cornel Fraefel, X O BreakefieldAbstract:Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal β-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of β-galactosidase (β-gal) could be detected in human β-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of β-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated β-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed β-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and β-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.
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targeted transgene integration into transgenic mouse fibroblasts carrying the full length human AAVS1 locus mediated by hsv aav rep hybrid amplicon vector
Gene Therapy, 2003Co-Authors: Joanna C Bakowska, M V Di Maria, Sara M Camp, Y Wang, Paul D Allen, X O BreakefieldAbstract:Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep + hybrid amplicon vector
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Targeted transgene integration into transgenic mouse fibroblasts carrying the full-length human AAVS1 locus mediated by HSV/AAV rep^+ hybrid amplicon vector
Gene Therapy, 2003Co-Authors: Joanna C Bakowska, M V Di Maria, Sara M Camp, Y Wang, Paul D Allen, X O BreakefieldAbstract:Herpes simplex virus type 1/adeno-associated virus (HSV/AAV) rep^+ hybrid amplicon vectors containing AAV inverted terminal repeats (ITRs) and rep gene sequences can mediate site-specific integration into the human genome. In this study, we have generated and characterized the first transgenic mice that bear the full-length (8.2 kb) human AAVS1 locus. Immortalized mouse embryonic fibroblasts from this mouse line were transduced with the rep^+, rep^− (containing only ITRs flanking the transgene) hybrid amplicon vectors, and the standard amplicon vector to determine stable integration frequency and the site of integration. Transduction of transgenic fibroblasts resulted in a 10-fold higher stable integration frequency with rep^+ hybrid amplicon vector than with rep^− or standard amplicon vectors. Southern blot analysis of genomic DNA from transgenic cells stably transduced with the rep^+ hybrid amplicon vector revealed site-specific integration of transgenes at the AAVS1 locus in 50% of clones. Some site-specific and random integration events were limited to the ITR-flanked transgene cassette. In contrast, transduction of transgenic mouse cells with the rep^− or standard amplicon vectors resulted in random integrations of the entire rep^− hybrid amplicon or amplicon DNA that were incorporated into the host genome as a concatenate of various sizes. These results demonstrate for the first time that the genome of transgenic mice bearing the human AAVS1 locus serves as a platform for site-specific integration of AAV ITR-flanked transgene cassettes within the hybrid amplicon vector in the presence of Rep.
Shu Wang - One of the best experts on this subject based on the ideXlab platform.
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targeted transgene insertion into the AAVS1 locus driven by baculoviral vector mediated zinc finger nuclease expression in human induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Can Chen, Shouhui Du, Rui Zhe Phang, Mohammad Shahbazi, Shu WangAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
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efficient recombinase mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
Nucleic Acids Research, 2011Co-Authors: Chrishan J A Ramachandra, Mohammad Shahbazi, Timothy Weixin Kwang, Yukti Choudhury, Jing Yang, Shu WangAbstract:Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
Kenneth I. Berns - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a bipartite recombinant adeno-associated viral vector for site-specific integration.
Human Gene Therapy, 2007Co-Authors: C. Zhang, N.g. Cortez, Kenneth I. BernsAbstract:Adeno-associated virus type 2 (AAV2) is the only virus known to integrate into a specific locus in the human genome. The locus, AAVS1, is on the q arm of chromosome 19 at position 13.4. AAV is currently a popular vector for human gene therapy. However, current vectors do not contain two important elements needed for site-specific integration, that is, the rep gene or the P5 promoter, although they do integrate with low frequency at random locations in the human genome. We have designed a bipartite vector that does insert the transgene into AAVS1. One component, rAAVSVAV2, contains the rep gene, driven by the simian virus 40 early promoter rather than the P5 promoter. Thus, the integration enhancer element (IEE) within P5, which greatly enhances site-specific integration, has been deleted. The other component, rAAVP5UF11, contains the P5 IEE plus the transgene with associated regulatory elements. We have created clones of transduced HeLa cells, most of which appear to have the transgene inserted in AAVS1. ...
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adeno associated virus site specific integration and AAVS1 disruption
Journal of Virology, 2004Co-Authors: Henry Hamilton, Kenneth I. Berns, Janette Gomos, Erik FalckpedersenAbstract:Adeno-associated virus (AAV) is a single-stranded DNA virus with a unique biphasic lifestyle consisting of both a productive and a latent phase. Typically, the productive phase requires coinfection with a helper virus, for instance adenovirus, while the latent phase dominates in healthy cells. In the latent state, AAV is found integrated site specifically into the host genome at chromosome 19q13.4 qtr (AAVS1), the only animal virus known to integrate in a defined location. In this study we investigated the latent phase of serotype 2 AAV, focusing on three areas: AAV infection, rescue, and integration efficiency as a function of viral multiplicity of infection (MOI); efficiency of site-specific integration; and disruption of the AAVS1 locus. As expected, increasing the AAV MOI resulted in an increase in the percentage of cells infected, with 80% of cells infected at an MOI of 10. Additional MOI only marginally effected a further increase in percentage of infected cells. In contrast to infection, we found very low levels of integration at MOIs of less than 10. At an MOI of 10, at which 80% of cells are infected, less than 5% of clonal cell lines contained integrated AAV DNA. At an MOI of 100 or greater, however, 35 to 40% of clonal cell lines contained integrated AAV DNA. Integration and the ability to rescue viral genomes were highly correlated. Analysis of integrated AAV indicated that essentially all integrants were AAVS1 site specific. Although maximal integration efficiency approached 40% of clonal cell lines (essentially 50% of infected cells), over 80% of cell lines contained a genomic disruption at the AAVS1 integration locus on chromosome 19 (≈100% of infected cells). Rep expression by itself and in the presence of a plasmid integration substrate was able to mediate this disruption of the AAVS1 site. We further characterized the disruption event and demonstrated that it resulted in amplification of the AAVS1 locus. The data are consistent with a revised model of AAV integration that includes preliminary expansion of a defined region in AAVS1.
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adeno associated virus aav site specific integration formation of aav AAVS1 junctions in an in vitro system
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Julie Dyall, Paul Szabo, Kenneth I. BernsAbstract:An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.
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Adeno-associated virus (AAV) site-specific integration: Formation of AAV–AAVS1 junctions in an in vitro system
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Julie Dyall, Paul Szabo, Kenneth I. BernsAbstract:An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo.
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The recombination signals for adeno-associated virus site-specific integration
Proceedings of the National Academy of Sciences of the United States of America, 1996Co-Authors: R M Linden, E Winocour, Kenneth I. BernsAbstract:The adeno-associated virus (AAV) genome integrates site specifically into a defined region of human chromosome 19 (termed AAVS1). Using a functional assay for AAV integration into AAVS1 DNA propagated as an episome, we obtained evidence that a 33-nucleotide AAVS1 DNA sequence contains the minimum signal required for targeted integration. The recombination signal comprises a DNA-binding motif for the AAV regulatory Rep protein [Rep binding site (RBS)] separated by an eight-nucleotide spacer from a sequence that can act as a substrate for Rep endonucleolytic activity [terminal resolution site (TRS)]. Mutations in either the AAVS1-encoded RBS or TRS elements abort targeted integration. Since both the RBS and TRS elements are present in the viral origin of replication and are required for AAV replication, targeted integration into chromosome 19 AAVS1 DNA may involve a replicative type of recombination that is discussed. An additional chromosome 19 element, which is responsible for DNA rearrangements in episomes propagating AAVS1 DNA, was identified and shown not to be required for AAV episomal integration, despite its location adjacent to the recombination signal.
Axel Schambach - One of the best experts on this subject based on the ideXlab platform.
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differential transgene silencing of myeloid specific promoters in the AAVS1 safe harbor locus of induced pluripotent stem cell derived myeloid cells
Human Gene Therapy, 2020Co-Authors: Denise Klatt, Dirk Hoffmann, Erica Cheng, Giorgia Santilli, Adrian J Thrasher, Christian Brendel, Axel SchambachAbstract:Targeted integration into a genomic safe harbor, such as the AAVS1 locus on chromosome 19, promises predictable transgene expression and reduces the risk of insertional mutagenesis in the host geno...
Chrishan J A Ramachandra - One of the best experts on this subject based on the ideXlab platform.
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targeted transgene insertion into the AAVS1 locus driven by baculoviral vector mediated zinc finger nuclease expression in human induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Can Chen, Shouhui Du, Rui Zhe Phang, Mohammad Shahbazi, Shu WangAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
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Targeted transgene insertion into the AAVS1 locus driven by baculoviral vector‐mediated zinc finger nuclease expression in human‐induced pluripotent stem cells
Journal of Gene Medicine, 2013Co-Authors: Chrishan J A Ramachandra, Can Chen, Shouhui Du, Rui Zhe Phang, Mohammad ShahbaziAbstract:Background The AAVS1 locus is viewed as a ‘safe harbor’ for transgene insertion into human genome. In the present study, we report a new method for AAVS1 targeting in human-induced pluripotent stem cells (hiPSCs). Methods We have developed two baculoviral transduction systems: one to deliver zinc finger nuclease (ZFN) and a DNA donor template for site-specific gene insertion and another to mediate Cre recombinase-mediated cassette exchange system to replace the inserted transgene with a new transgene. Results Our ZFN system provided the targeted integration efficiency of a Neo-EGFP cassette of 93.8% in G418-selected, stable hiPSC colonies. Southern blotting analysis of 20 AASV1 targeted colonies revealed no random integration events. Among 24 colonies examined for mono- or biallelic AASV1 targeting, 25% of them were biallelically modified. The selected hiPSCs displayed persistent enhanced green fluorescent protein expression and continued the expression of stem cell pluripotency markers. The hiPSCs maintained the ability to differentiate into three germ lineages in derived embryoid bodies and transgene expression was retained in the differentiated cells. After pre-including the loxP-docking sites into the Neo-EGFP cassette, we demonstrated that a baculovirus-Cre/loxP system could be used to facilitate the replacement of the Neo-EGFP cassette with another transgene cassette at the AAVS1 locus. Conclusions Given high targeting efficiency, stability in expression of inserted transgene and flexibility in transgene exchange, the approach reported in the present study holds potential for generating genetically-modified human pluripotent stem cells suitable for developmental biology research, drug development, regenerative medicine and gene therapy. Copyright © 2013 John Wiley & Sons, Ltd.
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efficient recombinase mediated cassette exchange at the AAVS1 locus in human embryonic stem cells using baculoviral vectors
Nucleic Acids Research, 2011Co-Authors: Chrishan J A Ramachandra, Mohammad Shahbazi, Timothy Weixin Kwang, Yukti Choudhury, Jing Yang, Shu WangAbstract:Insertion of a transgene into a defined genomic locus in human embryonic stem cells (hESCs) is crucial in preventing random integration-induced insertional mutagenesis, and can possibly enable persistent transgene expression during hESC expansion and in their differentiated progenies. Here, we employed homologous recombination in hESCs to introduce heterospecific loxP sites into the AAVS1 locus, a site with an open chromatin structure that allows averting transgene silencing phenomena. We then performed Cre recombinase mediated cassette exchange using baculoviral vectors to insert a transgene into the modified AAVS1 locus. Targeting efficiency in the master hESC line with the loxP-docking sites was up to 100%. Expression of the inserted transgene lasted for at least 20 passages during hESC expansion and was retained in differentiated cells derived from the genetically modified hESCs. Thus, this study demonstrates the feasibility of genetic manipulation at the AAVS1 locus with homologous recombination and using viral transduction in hESCs to facilitate recombinase-mediated cassette exchange. The method developed will be useful for repeated gene targeting at a defined locus of the hESC genome.
