ANALYSIS OF A MULTI-MUTANT HERPES-SIMPLEX VIRUS TYPE-1 FOR GENE-TRANSFER INTO SYMPATHETIC PREGANGLIONIC NEURONS AND A COMPARISON TO ADENOVIRUS VECTORS

A non-replicating triple-mutant herpes simplex virus (14H Delta vhsZ) expressing the bacterial marker enzyme beta-galactosidase, was assesse d for neurotropism and cytopathic effects as a vector for gene transfe r into differentiated phaeochromocytoma 12 cells in vitro and into spi nal sympathetic neurons in vivo. In the in vivo study, the 14H Delta 3 vhsZ was injected into the adrenal gland of hamsters. For comparison, an evaluation of two adenovirus vectors, AdCA17lacZ and AdCA36lacZ, wa s performed. Infection of the differentiated phaeochromocytoma 12 cell s by 14H Delta 3vhsZ resulted in intense beta-galactosidase staining i n 80-90% of the cells without changes in cell morphology, detected by light microscopy, after a period of four days. No cytoskeletal disrupt ion was detected by immunocytochemistry for the neurofilament protein and no apoptosis was demonstrated by the Hoescht stain for nuclear chr omatin in virus-infected cells in comparison to mock-infected control cells. Two to three days after adrenal inoculation with 14H Delta 3vhs Z, beta-galactosidase was detected in 240 preganglionic neurons per ha mster (n=8), a number equal to about 25% of the population of targeted neurons. The beta-galactosidase reaction product extended throughout the normal kite-shaped neuronal somata and extensive dendritic arbour. The number decreased to 120 by five days (n=3) and to two by eight da ys (n=4). This decrease was presumably due to loss of expression of th e marker gene and not to cell death because, at eight days, the number of sympathetic preganglionic neurons in the nucleus intermediolateral is, pars principalis, that were immunoreactive for the neurotransmitte r enzyme choline acetyltransferase, and demonstrated nicotinamide aden ine dinucleotide phosphate-diaphorase activity, were the same on the i nfected left side of the cord as on the uninfected right side. Inflamm atory cells surrounded some of the infected neurons at five days but b y eight days the infiltrate was reduced. Infection of differentiated p haeochromocytoma 12 cells by AdCA17lacZ and AdCA36lacZ also resulted i n marker gene expression in a large proportion of the cells (80-90%) i n the absence of cytopathic effects. In contrast, four days after adre nal injection of AdCA17lacZ or AdCA36lacZ (n=5 for each) only an avera ge of three preganglionic neurons per hamster expressed beta-galactosi dase activity, despite clear adrenal infection. AdCA17lacZ and AdCA36l acZ both produced light patches of staining confined to the neuronal s oma. These neurons had normal morphology but sometimes were surrounded by an inflammatory infiltrate. In conclusion, the non-replicating her pes simplex virus, 14H Delta 3vhs2, had minimal cytotoxic effects in n eurons, in vitro or in vivo, and was efficiently transported from the adrenal gland to infect many sympathoadrenal preganglionic neurons. In contrast, very few neurons demonstrated beta-galactosidase activity a fter injection into the adrenal gland of AdCA17lacZ and AdCA36lacZ. Th erefore, 14H Delta 3vhsZ is a more suitable vector than either of the adenovirus vectors tested for eliciting short-term changes in pregangl ionic neuron gene expression. (C) 1998 IBRO. Published by Elsevier Sci ence Ltd.