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Xandra O Breakefield – One of the best experts on this subject based on the ideXlab platform.

  • HSV-1 Amplicon peptide display vector.
    Journal of virological methods, 2003
    Co-Authors: Matthew A. Spear, Curtis R. Brandt, Deborah E. Schuback, Kenichi Miyata, Paola Grandi, Fang Sun, Linda Yoo, Anh T. Nguyen, Xandra O Breakefield

    Abstract:

    There are significant uses for expressing foreign peptide epitopes in viral surface attachment proteins in terms of investigating viral targeting, biology, and immunology. HSV-1 attachment, followed by fusion and entry, is mediated in large part by the binding of viral surface glycoproteins to cell surface receptors, primarily through heparan sulfate (HS) glycosaminoglycan residues. We constructed a HSV-1 Amplicon plasmid (pCONGA) carrying the gC primary attachment protein gene with unique restriction sites flanking the HS binding domain (HSBD) (residues 33–176) to allow rapid, high efficiency substitution with foreign peptide domains. To test this system, a His tag with an additional unique restriction site (for selection and assay digests) was recombined into the pCONGA HSBD site to create pCONGAH. Infection of pCONGAH transfected Vero cells with HSV-1 helper virus (gCΔ2-3 or hrR3) produced His-modified gC as demonstrated by western blot analysis with co-localization of anti-gC and anti-His tag antibodies to a protein of appropriate molecular weight (50 kd). As CONGA and CONGAH Amplicons carry a GFP transgene and the gCΔ2-3 and hrR3 viruses carry a lacZ transgene, vector stocks produced from 1×105 Vero cells could be titered for competent vector on cell monolayers and were demonstrated to contain 2×105 Amplicon vector transducing units (t.u.)/ml and 1×107 virus t.u./ml. As the Amplicon plasmids also contain the neomycin resistance gene (neor), long term vector producer cell lines were created using G418 selection. This Amplicon system provides means to rapidly and efficiently generate HSV-1 Amplicon and viral vector expressing surface attachment proteins modified with different peptide epitopes for investigational and therapeutic uses, with the advantages of an Amplicon plasmid that can be used with interchangeable helper virus vectors, is designed specifically for easy manipulation, and carries GFP and neor transgenes for marker and selection functions.

  • improved helper virus free packaging system for hsv Amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca

    Abstract:

    Herpes simplex virus type 1 (HSV-1) Amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 “helper” functions, Amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of Amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 “stuffer” sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected Amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU Amplicon vectors), and (iii) the resulting Amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 Amplicon vector stocks further improves safety in gene transfer applications.

  • improved helper virus free packaging system for hsv Amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca

    Abstract:

    Abstract Herpes simplex virus type 1 (HSV-1) Amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis -acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 “helper” functions, Amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of Amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10 −4 –10 −6 . To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli , either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVΔpacΔ27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 “stuffer” sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected Amplicon DNA without generating replication-competent helper virus ( 8 TU Amplicon vectors), and (iii) the resulting Amplicon stocks have titers of up to 3–10 × 10 8 TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 Amplicon vector stocks further improves safety in gene transfer applications.

Cornel Fraefel – One of the best experts on this subject based on the ideXlab platform.

  • Preparation of Herpes Simplex Virus Type 1 (HSV-1)-Based Amplicon Vectors
    Herpes Simplex Virus, 2020
    Co-Authors: Cornel Fraefel, Alberto L. Epstein

    Abstract:

    Amplicon vectors, or Amplicons, are defective, helper-dependent, herpes simplex virus type 1 (HSV-1)-based vectors. The main interest of Amplicons as gene transfer tools stems from the fact that the genomes of these vectors do not carry protein-encoding viral sequences. Consequently, they are completely safe for the host and nontoxic for the infected cells. Moreover, the complete absence of virus genes provides a genomic space authorizing a very large payload, enough to accommodate foreign DNA sequences up to almost 150-kbp, the size of the HSV-1 genome. This transgene capacity can be used to deliver complete gene loci, including introns and exons, as well as long regulatory sequences conferring tissue-specific expression or stable maintenance of the transgene in proliferating cells. For many years the development of these vectors and their application in gene transfer experiments was hindered by the presence of contaminating toxic helper virus particles in the vector stocks. In recent years, however, two different methodologies have been developed that allow generating Amplicon stocks either completely free of helper particles or only faintly contaminated with fully defective helper particles. This chapter describes these two methodologies.

  • hsv 1 Amplicon vectors that direct the in situ production of foot and mouth disease virus antigens in mammalian cells can be used for genetic immunization
    Vaccine, 2010
    Co-Authors: Alejandra Dantuono, Andrea S Laimbacher, Jose La Torre, Virginia Tribulatti, Carina Romanutti, P Zamorano, Valeria Quattrocchi, Elisabeth M Schraner, Mathias Ackermann, Cornel Fraefel

    Abstract:

    Abstract HSV-1 Amplicon vectors encoding heterologous antigens were capable to mediate in situ generation of protein synthesis and to generate a specific immune response to the corresponding antigens. In this study, foot-and-mouth disease (FMD) virus antigens were used to generate a genetic vaccine prototype. The Amplicons were designed to provide a high safety profile as they do not express any HSV-1 genes when packaged using a helper virus-free system, and they are able to encapsidate several copies of the transgene or allow the simultaneous expression of different genes. Virus-like particles were produced after cell processing of the delivered DNA. Inoculation of mice with 5 × 105 transducing units of Amplicon vectors resulted in FMDV-specific humoral responses in the absence of adjuvants, which were dependent on the in situ de novo production of the vector-encoded antigens. Challenge of mice vaccinated with these Amplicons with a high dose of live virus, resulted in partial protection, with a significant reduction of viremia. This work highlights the potential use of a HSV-1 Amplicon vector platform for generation of safe genetic vaccines.

  • Gene delivery using helper virus-free HSV-1 Amplicon vectors.
    Current protocols in neuroscience, 2007
    Co-Authors: Cornel Fraefel

    Abstract:

    Herpes simplex virus type 1 (HSV-1)-based Amplicon vectors contain only a very small percentage of the 152-kbp viral genome. Consequently, replication and packaging of Amplicons depend on helper functions that are provided either by replication-defective mutants of HSV-1 or by replication-competent, but packaging-defective, HSV-1 genomes. Sets of cosmids that overlap and represent the entire HSV-1 genome can form, via homologous recombination, circular replication-competent viral genomes, which give rise to infectious virus progeny. However, if the DNA cleavage/packaging signals are deleted, reconstituted virus genomes are not packageable, but still provide all the helper functions required for the packaging of cotransfected Amplicon DNA. The resulting stocks of packaged Amplicon vectors are essentially free of contaminating helper virus. This unit describes the cotransfection of Amplicon and cosmid or bacterial artificial chromosome (BAC) DNA into 2-2 cells by cationic liposome-mediated transfection using and the harvesting of packaged vector particles. Support protocols provide methods for preparing cosmid DNA and determining the titers of Amplicon stocks.

Yoshinaga Saeki – One of the best experts on this subject based on the ideXlab platform.

  • gene transfer into hepatocytes mediated by herpes simplex virus epstein barr virus hybrid Amplicons
    Journal of Virological Methods, 2005
    Co-Authors: Lars Müller, Okay Saydam, Yoshinaga Saeki, Irma Heid, Cornel Fraefel

    Abstract:

    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.

  • An HSV Amplicon-based helper system for helper-dependent adenoviral vectors.
    Biochemical and biophysical research communications, 2003
    Co-Authors: Shuji Kubo, Yoshinaga Saeki, E. Antonio Chiocca, Kohnosuke Mitani

    Abstract:

    To produce a helper virus-free stock of helper-dependent adenoviral vectors (HDAdVs), we have developed a new helper system in which adenoviral genes for propagation of HDAdVs are delivered into producer cells by a herpes simplex virus-1 (HSV) Amplicon-adenovirus hybrid. The hybrid Amplicon was constructed to carry the E1 gene (HA-E1) or the entire adenoviral genome except E1 (HA-Ad). E1 expression from the HSV Amplicon successfully complemented propagation of an E1-deleted adenoviral vector in a human glioma cell line. HDAdVs were propagated in 293 cells infected with HA-Ad. In addition, HDAdVs were rescued and propagated in a glioma cell line superinfected with both HA-E1 and HA-Ad Amplicons, although relatively low titers of HSV Amplicon resulted in low propagation efficiency of HDAdVs. Since the HSV Amplicon can be easily and completely inactivated by chloroform extraction and/or heat treatment from the HDAdV stock, this helper system might be an alternative method to produce helper virus-free HDAdVs.

  • improved helper virus free packaging system for hsv Amplicon vectors using an icp27 deleted oversized hsv 1 dna in a bacterial artificial chromosome
    Molecular Therapy, 2001
    Co-Authors: Cornel Fraefel, Yoshinaga Saeki, Xandra O Breakefield, Tomotsugu Ichikawa, Antonio E Chiocca

    Abstract:

    Herpes simplex virus type 1 (HSV-1) Amplicons are prokaryotic plasmids containing one or more transcriptional units and two cis-acting HSV-1 sequences: a viral origin of DNA replication and a viral DNA cleavage/packaging signal. In the presence of HSV-1 “helper” functions, Amplicons are replicated and packaged into HSV-1 virions. Despite recent improvements in packaging methods, stocks of Amplicon vectors are still contaminated with replication-competent helper virus at a frequency of 10(-4)-10(-6). To overcome this problem, we report that: (i) genetic modifications of HSV-1 genomes can be routinely achieved in Escherichia coli, either by homologous or site-specific recombination, (ii) a novel HSV-1 bacterial artificial chromosome (fHSVDeltapacDelta27 0+), which has a deletion in the essential gene encoding ICP27 and an addition of ICP0 “stuffer” sequences to increase its size to 178 kb, supports the replication and packaging of cotransfected Amplicon DNA without generating replication-competent helper virus (<1 helper virus per 10(8) TU Amplicon vectors), and (iii) the resulting Amplicon stocks have titers of up to 3-10 x 10(8) TU/ml after concentration. Elimination of replication-competent helper virus from HSV-1 Amplicon vector stocks further improves safety in gene transfer applications.