Herpes simplex viral-vector design for efficient transduction of nonneuronal cells without cytotoxicity

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 112; no. 13; pp. E1632 - E1641
• Main Authors: Miyagawa, Yoshitaka, Marino, Pietro, Verlengia, Gianluca, Uchida, Hiroaki, Goins, William F., Yokota, Shinichiro, Geller, David A., Yoshida, Osamu, Mester, Joseph, Cohen, Justus B., Glorioso, Joseph C.
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 31.03.2015; National Acad Sciences
• Series: PNAS Plus
• Subjects: Amino Acid Motifs; Animals; Biological Sciences; Cell Line, Tumor; Cells; Cercopithecus aethiops; Cytotoxicity; Design; Dystrophin - genetics; Gene expression; Gene Expression Regulation; Gene Silencing; gene therapy; Genes, Reporter; Genetic Therapy - methods; Genetic Vectors; Genome; Green Fluorescent Proteins - metabolism; herpes simplex; Herpes simplex virus; Herpes viruses; Humans; Immediate-Early Proteins - metabolism; Lentivirus - metabolism; Mice; Muscle Cells - cytology; Muscles - cytology; Neurons; PNAS Plus; polypeptides; Promoter Regions, Genetic; Rats; Rodents; Signal transduction; Simplexvirus - metabolism; transcription (genetics); Transduction, Genetic; transgenes; Vero Cells; viruses; ICP0; gene therapy; dystrophin; insulator; HSV vector
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1423556112

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 genetherapy vectors.