Long-Term Expression of a Foreign Gene from a Unique Position in the Latent Herpes Simplex Virus Genome

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Abstract
As a result of its capacity to establish and maintain a life-long latent infection in the nervous system, herpes simplex virus (HSV) has been promoted as an ideal vector for introducing DNA into mature, differentiated, post-mitotic neurons. Although delivery of foreign genes into neurons using HSV vectors has been well established, the potential of these vectors for scientific inquiry or therapeutic use has been hampered by the lack of efficient long-term expression of these foreign genes. In the few instances where expression from the latent genome has been reported, expression appears to be minimal and levels of mRNA present have not been established. Here we describe HSV viral vectors that express a foreign gene during latency in dorsal root ganglia (DRG). More particularly, we have constructed a vector that, by histochemical assays for the protein, expresses the beta-galactosidase (beta-Gal) gene for at least 18 months post infection. We have further characterized the expression of beta-Gal transcripts by quantitative reverse transcription polymerase chain reaction (RT-PCR) and determined that there are 32,000 copies of beta-Gal transcripts per 0.5 microgram of total RNA at 18 months post infection. The vector makes use of the mouse Moloney leukemia virus (MMLV) long terminal repeat (LTR) promoter located directly upstream from the latency-associated transcripts (LAT) promoter region and expresses mRNA from the DNA strand opposite to that expressing the LAT. Finally, the vector was constructed using a system that allows other promoter/gene constructs to be easily inserted into the viral genome. It may have utility in studying the effects of cellular or viral gene expression on establishment, maintenance or reactivation from latency or for the delivery and expression of therapeutic proteins employed in gene therapy of the nervous system.