期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：13
页码：E1632-E1641
DOI：10.1073/pnas.1423556112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceGene therapy has made significant strides in the treatment and even cure of single-gene defects. However, the maturation of this field will require more sophisticated vehicles capable of cell-selective delivery of large genetic payloads whose regulated expression will restore or enhance cellular functionality. High-capacity herpes simplex virus vectors have the potential to meet these challenges but have been limited by the need to preserve one particularly cytotoxic viral product, infected cell polypeptide 0, to maintain transgene transcriptional activity. Our study describes a vector design that solves this conundrum, thereby promising the near-term availability of viral vectors that can efficiently deliver large or multiple regulated transgenes to a diversity of cells without attendant cytotoxicity. 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.
