The Experts below are selected from a list of 204 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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848. Helper Dependent AdenoVirus-Epstein- Barr Virus Hybrid Vector for Long Term Persistance in Hepatocytes
Molecular Therapy, 2020Co-Authors: 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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14. High Efficiency and Long-Term Persistence In Vivo from a Helper Dependent AdenoVirus/Epstein-Barr Virus Hybrid Vector
Molecular Therapy, 2020Co-Authors: Sean D. Gallaher, 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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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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development of a novel helper dependent adenoVirus epstein barr Virus Hybrid system for the stable transformation of mammalian cells
Journal of Virology, 2004Co-Authors: Oliver Dorigo, Sean D. Gallaher, Maria G Castro, Pedro R Lowenstein, Michele P Calos, Arnold J BerkAbstract:Epstein-Barr Virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenoVirus (HDA)-EBV Hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV Hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.
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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).
Kahwhye Peng - One of the best experts on this subject based on the ideXlab platform.
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faster replication and higher expression levels of viral glycoproteins give the vesicular stomatitis Virus measles Virus Hybrid vsv fh a growth advantage over measles Virus
Journal of Virology, 2014Co-Authors: Camilo Ayalabreton, Luke O J Russell, Stephen J Russell, Kahwhye PengAbstract:VSV-FH is a Hybrid vesicular stomatitis Virus (VSV) with a deletion of its G glycoprotein and encoding the measles Virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic Virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways. IMPORTANCE We studied the cytotoxic activity of a vesicular stomatitis/measles Hybrid Virus (VSV-FH), which is superior to that of measles Virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and a more potent cytopathic effect in permissive cells. Importantly, VSV-FH, similar to MV, can discriminate between low- and high-expressing CD46 cells, a phenotype important for cancer therapy as the Virus will be able to preferentially infect cancer cells that overexpress CD46 over low-CD46-expressing normal cells.
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Faster Replication and Higher Expression Levels of Viral Glycoproteins Give the Vesicular Stomatitis Virus/Measles Virus Hybrid VSV-FH a Growth Advantage over Measles Virus
Journal of Virology, 2014Co-Authors: Camilo Ayala-breton, Luke O J Russell, Stephen J Russell, Kahwhye PengAbstract:VSV-FH is a Hybrid vesicular stomatitis Virus (VSV) with a deletion of its G glycoprotein and encoding the measles Virus (MV) fusion (F) and hemagglutinin (H) envelope glycoproteins. VSV-FH infects cells expressing MV receptors and is fusogenic and effective against myeloma xenografts in mice. We evaluated the fusogenic activities of MV and VSV-FH in relationship to the density of receptor on the target cell surface and the kinetics of F and H expression in infected cells. Using a panel of cells expressing increasing numbers of the MV receptor CD46, we evaluated syncytium size in MV- or VSV-FH-infected cells. VSV-FH is not fusogenic at low CD46 density but requires less CD46 for syncytium formation than MV. The size of each syncytium is larger in VSV-FH-infected cells at a specific CD46 density. While syncytium size reached a plateau and did not increase further in MV-infected CHO cells expressing ≥4,620 CD46 copies/cell, there was a corresponding increase in syncytium size with increases in CD46 levels in VSV-FH-infected CD46-expressing CHO (CHO-CD46) cells. Further analysis in VSV-FH-infected cell lines shows earlier and higher expression of F and H mRNAs and protein. However, VSV-FH cytotoxic activity was reduced by pretreatment of the cells with type I interferon. In contrast, the cytopathic effects are not affected in MV-infected cells. In summary, VSV-FH has significant advantages over MV as an oncolytic Virus due to its higher viral yield, faster replication kinetics, and larger fusogenic capabilities but should be used in cancer types with defective interferon signaling pathways. IMPORTANCE We studied the cytotoxic activity of a vesicular stomatitis/measles Hybrid Virus (VSV-FH), which is superior to that of measles Virus (MV), in different cancer cell lines. We determined that viral RNA and protein were produced faster and in higher quantities in VSV-FH-infected cells. This resulted in the formation of larger syncytia, higher production of infectious particles, and a more potent cytopathic effect in permissive cells. Importantly, VSV-FH, similar to MV, can discriminate between low- and high-expressing CD46 cells, a phenotype important for cancer therapy as the Virus will be able to preferentially infect cancer cells that overexpress CD46 over low-CD46-expressing normal cells.
Xandra O. Breakefield - One of the best experts on this subject based on the ideXlab platform.
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Review Critical Issues in Gene Therapy for Neurologic Disease
2020Co-Authors: Gary Hsich, Miguel Sena-esteves, Xandra O. BreakefieldAbstract:Gene therapy for the nervous system is a newly emerging field with special issues related to modes of delivery, potential toxicity, and realistic expectations for treatment of this vital and highly complex tissue. This review focuses on the potential for gene delivery to the brain, as well as possible risks and benefits of these procedures. This includes discussion of appropriate vectors, such as adeno-associated Virus, lentiVirus, gutless adenoVirus, and herpes simplex Virus Hybrid amplicons, and cell vehicles, such as neuroprogenitor cells. Routes of delivery for focal and global diseases are enumerated, including use of migratory cells, facilitation of vascular delivery across the blood‐ brain barrier, cerebrospinal fluid delivery, and convection injection. Attention is given to examples of diseases falling into different etiologic types: metabolic deficiency states, including Canavan disease and lysosomal storage disorders; and degenerative conditions, including Parkinson’ s disease and other neurodegenerative conditions.
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Potentiated gene delivery to tumors using herpes simplex Virus/Epstein-Barr Virus/RV tribrid amplicon vectors.
Human Gene Therapy, 2003Co-Authors: Jürgen A. Hampl, Miguel Sena-esteves, Sara M. Camp, Wojciech K. Mydlarz, Monika Hampl, Tomotsuga Ichikawa, E. Antonio Chiocca, David N. Louis, Xandra O. BreakefieldAbstract:The development and use of gene transfer techniques creates an opportunity to achieve better treatment modalities for numerous disease entities. Promising results for treatment in tumor cells in culture and in small animal models have been reported. Nevertheless, the lack of widespread vector distribution throughout tumor tissue is one of the current limitations for successful clinical application of gene therapy paradigms. The use of migratory tumor cells themselves as vector delivery vehicles may allow wider vector distribution in tumors. In addition, continuous release of retroVirus vectors on-site could generate a high local virion concentration over an extended time period with consequent increases in transduction efficiency. In this paper, we present in culture and in vivo data of a herpes simplex Virus-Epstein-Barr Virus Hybrid amplicon vector containing retroVirus vector components (tribrid vector) that allows conversion of tumor cells into retroviral producer cells. With this method, we were able...
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generation of stable retroVirus packaging cell lines after transduction with herpes simplex Virus Hybrid amplicon vectors
Journal of Gene Medicine, 2002Co-Authors: Miguel Senaesteves, Sara M. Camp, Jürgen A. Hampl, Xandra O. BreakefieldAbstract:Background A number of properties have relegated the use of Moloney murine leukemia Virus (Mo-MLV)-based retroVirus vectors primarily to ex vivo protocols. Direct implantation of retroVirus producer cells can bypass some of the limitations, and in situ vector production may result in a large number of gene transfer events. However, the fibroblast nature of most retroVirus packaging cells does not provide for an effective distribution of vector producing foci in vivo, especially in the brain. Effective development of new retroVirus producer cells with enhanced biologic properties may require the testing of a large number of different cell types, and a quick and efficient method to generate them is needed. Methods Moloney murine leukemia Virus (Mo-MLV) gag-pol and env genes and retroVirus vector sequences carrying lacZ were cloned into different minimal HSV/AAV Hybrid amplicons. Helper Virus-free amplicon vectors were used to co-infect glioma cells in culture. Titers and stability of retroVirus vector production were assessed. Results Simultaneous infection of two glioma lines, Gli-36 (human) and J3T (dog), with both types of amplicon vectors, generated stable packaging populations that produced retroVirus titers of 0.5–1.2×105 and 3.1–7.1×103 tu/ml, respectively. Alternatively, when cells were first infected with retroVirus vectors followed by infection with HyRMOVAmpho amplicon vector, stable retroVirus packaging populations were obtained from Gli-36 and J3T cells producing retroVirus titers comparable to those obtained with a traditional retroVirus packaging cell line, ΨCRIPlacZ. Conclusions This amplicon vector system should facilitate generation of new types of retroVirus producer cells. Conversion of cells with migratory or tumor/tissue homing properties could result in expansion of the spatial distribution or targeting capacity, respectively, of gene delivery by retroVirus vectors in vivo. Copyright © 2002 John Wiley & Sons, Ltd.
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Critical issues in gene therapy for neurologic disease.
Human Gene Therapy, 2002Co-Authors: Gary Hsich, Miguel Sena-esteves, Xandra O. BreakefieldAbstract:Gene therapy for the nervous system is a newly emerging field with special issues related to modes of delivery, potential toxicity, and realistic expectations for treatment of this vital and highly complex tissue. This review focuses on the potential for gene delivery to the brain, as well as possible risks and benefits of these procedures. This includes discussion of appropriate vectors, such as adeno-associated Virus, lentiVirus, gutless adenoVirus, and herpes simplex Virus Hybrid amplicons, and cell vehicles, such as neuroprogenitor cells. Routes of delivery for focal and global diseases are enumerated, including use of migratory cells, facilitation of vascular delivery across the blood–brain barrier, cerebrospinal fluid delivery, and convection injection. Attention is given to examples of diseases falling into different etiologic types: metabolic deficiency states, including Canavan disease and lysosomal storage disorders; and degenerative conditions, including Parkinson's disease and other neurodegen...
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single step conversion of cells to retroVirus vector producers with herpes simplex Virus epstein barr Virus Hybrid amplicons
Journal of Virology, 1999Co-Authors: Miguel Senaesteves, Yoshinaga Saeki, Sara M. Camp, Antonio E Chiocca, Xandra O. BreakefieldAbstract:We report here on the development and characterization of a novel herpes simplex Virus type 1 (HSV-1) amplicon-based vector system which takes advantage of the host range and retention properties of HSV–Epstein-Barr Virus (EBV) Hybrid amplicons to efficiently convert cells to retroVirus vector producer cells after single-step transduction. The retroVirus genes gag-pol and env (GPE) and retroviral vector sequences were modified to minimize sequence overlap and cloned into an HSV-EBV Hybrid amplicon. RetroVirus expression cassettes were used to generate the HSV-EBV-retroVirus Hybrid vectors, HERE and HERA, which code for the ecotropic and the amphotropic envelopes, respectively. RetroVirus vector sequences encoding lacZ were cloned downstream from the GPE expression unit. Transfection of 293T/17 cells with amplicon plasmids yielded retroVirus titers between 106 and 107 transducing units/ml, while infection of the same cells with amplicon vectors generated maximum titers 1 order of magnitude lower. RetroVirus titers were dependent on the extent of transduction by amplicon vectors for the same cell line, but different cell lines displayed varying capacities to produce retroVirus vectors even at the same transduction efficiencies. Infection of human and dog primary gliomas with this system resulted in the production of retroVirus vectors for more than 1 week and the long-term retention and increase in transgene activity over time in these cell populations. Although the efficiency of this system still has to be determined in vivo, many applications are foreseeable for this approach to gene delivery.
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.
The Open Virology Journal, 2010Co-Authors: Anna Paula De Oliveira, Cornel FraefelAbstract:Herpes simplex Virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size up to 150 kbp and, therefore, allow extensive combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other Viruses to create Hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other Viruses that either direct integration of transgene sequences into the host genome or allow episomal maintenance of the vector. Thus, the advantages of the HSV-1 amplicon system, including large transgene capacity, broad host range, strong nuclear localization, and availability of helper Virus-free packaging systems are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA. Adeno-associated Virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The AAV rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that contain the rep gene and a transgene cassette flanked by AAV ITRs. These HSV/AAV Hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression.
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Herpes Simplex Virus Type 1/Adeno-Associated Virus Hybrid Vectors~!2009-12-12~!2010-01-13~!2010-06-17~!
The Open Virology Journal, 2010Co-Authors: Anna Paula De Oliveira, Cornel FraefelAbstract:Herpes simplex Virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size up to 150 kbp and, therefore, allow extensive combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other Viruses to create Hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other Viruses that either direct integration of transgene sequences into the host genome or allow episomal maintenance of the vector. Thus, the advantages of the HSV-1 amplicon system, including large transgene capacity, broad host range, strong nuclear localization, and availability of helper Virus-free packaging systems are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA. Adeno-associated Virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The AAV rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that contain the rep gene and a transgene cassette flanked by AAV ITRs. These HSV/AAV Hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression.
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gene transfer into hepatocytes mediated by herpes simplex Virus epstein barr Virus Hybrid amplicons
Journal of Virological Methods, 2005Co-Authors: Lars Muller, Irma Heid, Okay Saydam, Yoshinaga Saeki, Cornel FraefelAbstract:Abstract Gene transfer into hepatocytes is highly desirable for the long-term goal of replacing deficient proteins and correcting metabolic disorders. Vectors based on herpes simplex Virus type-1 (HSV-1) have been demonstrated to mediate efficient gene transfer into hepatocytes both in vitro and in vivo. Large transgene capacity and extrachromosomal persistence make HSV-1/EBV Hybrid amplicon vectors an attractive candidate for hepatic gene replacement therapy. To assess liver-directed gene transfer, we constructed (i) a conventional HSV-1 amplicon vector encoding a secreted reporter protein (secreted alkaline phosphatase, SEAP) under the control of the HSV-1 immediate-early 4/5 promoter; (ii) a HSV-1 amplicon encoding SEAP under the control of the artificial CAG promoter (the chicken β-actin promoter and cytomegaloVirus (CMV) immediate-early enhancer); and (iii) a HSV-1/EBV Hybrid amplicon, also encoding SEAP under the control of the CAG promoter. While all three vector constructs yielded high SEAP concentrations in vitro and in vivo, use of HSV-1/EBV Hybrid amplicon vectors significantly prolonged the duration of gene expression. Using conventional amplicon vectors in cultured hepatocytes, SEAP was detected for two weeks, whereas SEAP was detected for at least six weeks when HSV-1/EBV amplicons were used. Intraparenchymal injection into the liver of SICD mice yielded high (up to 77 ng of SEAP per milliliter serum) and sustained (greater than three weeks) expression of SEAP. Serum transaminases (ALT/AST) were measured at different time points to monitor for hepatocellular damage. While initially elevated four times above baseline, the transaminase levels returned to normal after three to seven days. These results demonstrate the usefulness of HSV-1-based amplicons and SEAP for the evaluation of gene replacement strategies in the liver.
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Herpes simplex Virus type 1/adeno-associated Virus Hybrid vectors
Gene Therapy and Regulation, 2003Co-Authors: Cornel Fraefel, Thomas Heister, Mathias AckermannAbstract:Herpes simplex Virus type 1 (HSV-1) amplicons can accommodate foreign DNA of any size between 1 kbp and 150 kbp and, therefore, give room for almost unlimited combinations of genetic elements. Genomic sequences as well as cDNA, large transcriptional regulatory sequences for cell type-specific expression, multiple transgenes, and genetic elements from other Viruses to create Hybrid vectors may be inserted in a modular fashion. Hybrid amplicons use genetic elements from HSV-1 that allow replication and packaging of the vector DNA into HSV-1 virions, and genetic elements from other Viruses that either direct integration of transgene sequences into the host genome or allow the vector to replicate autonomously as an episome. Thus, the advantages of the HSV-1 amplicon system, such as large transgene capacity, broad host range, strong nuclear localization, and availability of a helper Virus-free packaging system are retained and combined with those of heterologous viral elements that confer genetic stability to the vector DNA within transduced cells. In this respect, adeno-associated Virus (AAV) has the unique capability of integrating its genome into a specific site, designated AAVS1, on human chromosome 19. The viral rep gene and the inverted terminal repeats (ITRs) that flank the AAV genome are sufficient for this process. HSV-1 amplicons have thus been designed that incorporate the AAV rep gene on the backbone and the AAV ITRs on the bondaries of the transgene cassette. These HSV/AAV Hybrid vectors direct site-specific integration of transgene sequences into AAVS1 and support long-term transgene expression. Optimization of these Hybrid vectors is discussed in terms of elimination of both background random-integration and rep interference on vector packaging.
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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.
James M Wilson - One of the best experts on this subject based on the ideXlab platform.
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adenoVirus adeno associated Virus Hybrid for large scale recombinant adeno associated Virus production
Human Gene Therapy, 2009Co-Authors: Hongwei Zhang, James M WilsonAbstract:Abstract Recombinant adeno-associated Virus (rAAV) holds promise for applications in gene therapy. Advances in clinical studies of rAAV-based gene therapeutics have generated an encouraging momentum in the field of gene therapy; however, one of the major obstacles to the eventual clinical success of rAAV-mediated gene therapy is the need for large-scale production of clinical-grade vectors. The transfection-based rAAV production method is well suited for preclinical studies in small animal models, but it is difficult to support large-scale clinical studies with this method. In the past decade, several scalable rAAV production methods have emerged from extensive efforts to develop large-scale manufacturing processes. Among those, the recombinant adenoVirus–AAV infection method has some unique features in vector quality and yield. This minireview provides an overview of this scaleable rAAV production platform, describing its basic components and biological mechanisms and process.
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AdenoVirus–Adeno-Associated Virus Hybrid for Large-Scale Recombinant Adeno-Associated Virus Production
Human Gene Therapy, 2009Co-Authors: Hongwei Zhang, James M WilsonAbstract:Abstract Recombinant adeno-associated Virus (rAAV) holds promise for applications in gene therapy. Advances in clinical studies of rAAV-based gene therapeutics have generated an encouraging momentum in the field of gene therapy; however, one of the major obstacles to the eventual clinical success of rAAV-mediated gene therapy is the need for large-scale production of clinical-grade vectors. The transfection-based rAAV production method is well suited for preclinical studies in small animal models, but it is difficult to support large-scale clinical studies with this method. In the past decade, several scalable rAAV production methods have emerged from extensive efforts to develop large-scale manufacturing processes. Among those, the recombinant adenoVirus–AAV infection method has some unique features in vector quality and yield. This minireview provides an overview of this scaleable rAAV production platform, describing its basic components and biological mechanisms and process.
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a novel adenoVirus adeno associated Virus Hybrid vector that displays efficient rescue and delivery of the aav genome
Human Gene Therapy, 1996Co-Authors: Krishna J Fisher, Mark W Kelley, John F Burda, James M WilsonAbstract:ABSTRACT AdenoVirus and adeno-associated Virus (AAV) are eukaryotic DNA Viruses being developed as vectors for human gene therapy. The strengths of each system have been exploited in a novel vector that is based on an ade noVirus–AAV Hybrid Virus incorporated into a plasmid-based molecular conjugate. Efficient rescue and replication of the recombinant AAV genome in this Hybrid required transient expression of rep. This feature was incorporated into the transducing particle by conjugating a rep expression plasmid to the Hybrid Virus through a polylysine bridge. The resulting particle is an attractive vehicle for gene therapy because it is easily manufactured and capable of efficiently transducing cells with the end result being rescue and replication of the recombinant AAV genome. This particle is also useful in the production of recombinant AAV resulting in yields 10-fold greater than that achieved with transfection-based protocols.
