title: Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity.
abstract: The design of highly defective herpes simplex virus (HSV) vectors for transgene expression in nonneuronal cells in the absence of toxic viral-gene activity has been elusive. Here, we report that elements of the latency locus protect a nonviral promoter against silencing in primary human cells in the absence of any viral-gene expression. We identified a CTCF motif cluster 5' to the latency promoter and a known long-term regulatory region as important elements for vigorous transgene expression from a vector that is functionally deleted for all five immediate-early genes and the 15-kb internal repeat region. We inserted a 16.5-kb expression cassette for full-length mouse dystrophin and report robust and durable expression in dystrophin-deficient muscle cells in vitro. Given the broad cell tropism of HSV, our design provides a nontoxic vector that can accommodate large transgene constructs for transduction of a wide variety of cells without vector integration, thereby filling an important void in the current arsenal of gene-therapy vectors.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219;
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Miyagawa
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219; Section of Pharmacology, Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy;
authors.forename: Pietro
authors.initials: P
authors.lastname: Marino
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219; Section of Pharmacology, Department of Medical Sciences, University of Ferrara, 44121 Ferrara, Italy;
authors.forename: Gianluca
authors.initials: G
authors.lastname: Verlengia
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219;
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Uchida
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219;
authors.forename: William F
authors.initials: WF
authors.lastname: Goins
authors.affiliation: Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Shinichiro
authors.initials: S
authors.lastname: Yokota
authors.affiliation: Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261; Liver Cancer Center, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213; and.
authors.forename: David A
authors.initials: DA
authors.lastname: Geller
authors.affiliation: Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261;
authors.forename: Osamu
authors.initials: O
authors.lastname: Yoshida
authors.affiliation: Department of Biological Sciences, Northern Kentucky University, Highland Heights, KY 41099.
authors.forename: Joseph
authors.initials: J
authors.lastname: Mester
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219;
authors.forename: Justus B
authors.initials: JB
authors.lastname: Cohen
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, Pittsburgh, PA 15219; glorioso@pitt.edu.
authors.forename: Joseph C
authors.initials: JC
authors.lastname: Glorioso
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D045744:Cell Line, Tumor; D002522:Chlorocebus aethiops; D016189:Dystrophin / Q000235:genetics; D005786:Gene Expression Regulation; D020868:Gene Silencing; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016678:Genome; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism; D051379:Mice; D032342:Muscle Cells / Q000166:cytology*; D009132:Muscles / Q000166:cytology; D009474:Neurons; D011401:Promoter Regions, Genetic; D051381:Rats; D018139:Simplexvirus / Q000378:metabolism*; D014161:Transduction, Genetic; D014709:Vero Cells
chemical_list: D016189:Dystrophin; D017874:Immediate-Early Proteins; C048925:herpes simplex virus, type 1 protein ICP4; D049452:Green Fluorescent Proteins
keywords: HSV vector; ICP0; dystrophin; gene therapy; insulator