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.

Morphological changes in different populations of bladder afferent neurons detected by herpes simplex virus (HSV) vectors with cell-type-specific promoters in mice with spinal cord injury

“…The replication-defective mCherry reporter expression vectors were designed with complete deletion of essential immediate early genes ICP4 and ICP27 (Miyagawa et al, 2015) from the KOS-37 BAC vector genome (Gierasch et al, 2006). The internal repeat region was also deleted resulting in the vector genome being stabilized by losing the ability to isomerize with the deletion of over 25 Kb of total sequence (Miyagawa et al, 2015).…”

“…The internal repeat region was also deleted resulting in the vector genome being stabilized by losing the ability to isomerize with the deletion of over 25 Kb of total sequence (Miyagawa et al, 2015). For ease of insertion of a wide variety of promoter-reporter gene constructs into the vector genome, a Gateway-Destination cassette was inserted into the remaining latency associated transcript (LAT) locus, replacing the LAT promoter elements (Fig.…”

“…1B). The 11 Kb BAC sequences were subsequently excised from all the isolates since the BAC sequences contribute to reduced vector growth (Miyagawa et al, 2015), on U2Os-4/27/Cre cells (Miyagawa et al, 2015) via Cre expression. Analysis of the BAC sequence removal was provided using X-gal staining (Goins et al, 2002) for detection of lac Z expression that is lost upon BAC excision.…”

“…All vector stocks were propagated on a human U2OS-ICP4/27 complementing cell line (Miyagawa et al, 2015). Twenty-four hours before infection, 1×10 8 U2OS-ICP4/27 cells were seeded as a 50% confluent monolayer to obtain a confluence of about 90–100% the day after in 10-layer cell factories (Nunc/Thermo-Fisher, Pittsburgh, PA).…”

Syncytial Mutations Do Not Impair the Specificity of Entry and Spread of a Glycoprotein D Receptor-Retargeted Herpes Simplex Virus

“…KGNEp-BhKt was established by cotransfection of Vero-EpCAM cells with pKGNEp-BhKt and pxCANCre (provided by Izumu Saito, University of Tokyo, Japan), followed by two rounds of limiting dilution on Vero-EpCAM cells. Confirmation of BAC deletion was carried out as described previously (49). Propagation, purification, and titration of viruses were performed essentially as described previously (45).…”