322. Benign Herpes Simplex Virus Vector Design for Efficient Delivery of Large or Multiple Transgenes To a Diversity of Cells

Abstract

Viral vectors derived from herpes simplex virus (HSV) have the potential to revolutionize gene therapy due to their ability to accommodate large and multiple therapeutic transgenes. However, current HSV gene therapy vectors express toxic levels of an immediate-early (IE) protein, ICP0, whose function is required for robust and sustained transgene expression. Here we report the development of a new generation of HSV vectors that are IE-gene independent and non-toxic, yet capable of persistent transgene expression in a variety of human primary non-neuronal cell types. We identified a CTCF motif cluster upstream of the latency promoter and a known long-term regulatory region as key elements for the protection of transgene expression cassettes from global silencing of the viral genome in the absence of all viral IE gene products. Using this new HSV vector system, we have observed vigorous expression of full-length dystrophin cDNA (14 kb) for several weeks in a dystrophin-deficient muscle cell line. We further tested our vectors for transgene expression in rodent brain. While we detected variable persistence of gene expression from the latency locus, we were surprised to observe vigorous long-term reporter gene expression from one other locus despite the absence of gene expression from this locus in non-neuronal cells. These findings demonstrate that transgene expression in neurons is operatively different from that in non-neuronal cells and suggest that multiple loci can be used for expression of foreign genes in the nervous system. In addition, our data raise the prospect that our highly defective HSV vector system will be applicable as a safe delivery tool for large and multiple therapeutic genes to a wide range of non-neuronal tissues.