Deletion of the Virion Host Shut-off Gene Enhances Neuronal-Selective Transgene Expression from an HSV Vector Lacking Functional IE Genes

Abstract

The ability of herpes simplex virus (HSV) to establish lifelong latency in neurons suggests that HSV-derived vectors hold promise for gene delivery to the nervous system. However, vector toxicity and transgene silencing have created significant barriers to vector applications to the brain. Recently, we described a vector defective for all immediate-early gene expression and deleted for the joint region between the two unique genome segments that proved capable of extended transgene expression in non-neuronal cells. Sustained expression required the proximity of boundary elements from the latency locus. As confirmed here, we have also found that a transgene cassette introduced into the ICP4 locus is highly active in neurons but silent in primary fibroblasts. Remarkably, we observed that removal of the virion host shutoff (vhs) gene further improved transgene expression in neurons without inducing expression of viral genes. In rat hippocampus, the vhs-deleted vector showed robust transgene expression exclusively in neurons for at least 1 month without evidence of toxicity or inflammation. This HSV vector design holds promise for gene delivery to the brain, including durable expression of large or complex transgene cassettes.

Cellular Antisilencing Elements Support Transgene Expression from Herpes Simplex Virus Vectors in the Absence of Immediate Early Gene Expression

“…In contrast, very little, if any, EGFP signal was detected at any time point. These results indicated that A2UCOE can enhance transient, independent expression of a second transgene in the central nervous system to complement the previously described durable expression of a first transgene positioned in the deleted ICP4 locus (31,32). It remains to be determined what mechanisms can account for the rapid or immediate silencing of the LAT-based EGFP cassette of these vectors in mouse hippocampus.…”

“…We hypothesize that the unexpected long-term reporter gene expression from the terminal ICP4 locus of our defective vectors in central nervous system neurons in vivo (Fig. 9C) (31,32) and the simultaneous lack of reporter expression from the LAT locus reported here are consequences of the disruption of CTCF interactions and the loss of LAT and ICP0 functions. However, it is uncertain whether A2UCOE activity is influenced by the same mechanisms.…”

Herpes Simplex Virus Vectors for Gene Transfer to the Central Nervous System

“…Our studies demonstrated prolonged, non-toxic transgene expression from a foreign promoter inserted directly downstream from the LAT promoter/enhancer region in non-neuronal cells. Interestingly, although expression from the LAT region was transient in rDRG cultures and largely undetectable in mouse hippocampus [ 104 , 105 ], reporter expression from the deleted ICP4 locus without adjacent LAT regulatory elements persisted for months in mouse hippocampus [ 106 ]. Furthermore, we observed that this expression could be further enhanced by deletion of the virion host shutoff gene (vhs or UL41) [ 104 ].…”

“…Interestingly, although expression from the LAT region was transient in rDRG cultures and largely undetectable in mouse hippocampus [ 104 , 105 ], reporter expression from the deleted ICP4 locus without adjacent LAT regulatory elements persisted for months in mouse hippocampus [ 106 ]. Furthermore, we observed that this expression could be further enhanced by deletion of the virion host shutoff gene (vhs or UL41) [ 104 ]. Studies by Harkness et al [ 107 ], using their IE-inactive d 109 vector, showed that genes near or within the long viral inverted repeat regions remained more active than genes in the unique regions of the largely quiescent genome in cultured trigeminal ganglion neurons.…”

“…The vector genome is deleted for the immediate-early genes ICP4, ICP27 and ICP0, the promoter region of the ICP47 gene, the VP16 binding site in the promoter of the ICP22 gene causing regulation as an early gene (βICP22), a large portion of the LAT gene, and the complete joint region. Additional deletion of the UL41 gene has been reported to enhance transgene expression in a cell type-dependent manner [ 104 ]. UL37 and UL38 embed the bacterial artificial chromosome (BAC) genes.…”