BACKGROUND
herpes simplex virus  vectors are an attractive system for gene transfer into neurons, but the limited levels of long-term expression remain a significant problem.
the numbers of cells expressing recombinant gene products display large decreases within the first few weeks after gene transfer, using either plasmid  or recombinant hsv- <dig> vectors that contain specific viral or neuronal-specific cellular promoters .
interestingly, using hsv- <dig> plasmid vectors, two neuronal type-specific promoters  or preproenkephalin), and chimeric promoters that contain 5' upstream sequences from either of these promoters, support significant levels of long-term expression   <cit> .
although the levels of expression display a significant initial decrease, long-term expression is relatively stable.
consequently, identifying the mechanisms responsible for the initial decrease in expression may provide a foundation for the rational design of hsv- <dig> vectors that support long-term expression.
the reduction in recombinant gene expression might be caused by the mechanisms that repress hsv- <dig> gene expression in hsv- <dig> latency, focusing attention on hsv- <dig> gene regulation.
supporting a role for hsv- <dig> gene regulation in reducing recombinant gene expression, helper virus-containing hsv- <dig> vector systems cause significant cytopathic effects and an inflammatory response, and most of these side effects are caused by hsv- <dig> gene expression from the helper virus  <cit> .
consistent with these results, helper virus-free hsv- <dig> vectors cause substantially less side effects than those observed using helper virus-containing systems  <cit> .
however, helper virus-free hsv- <dig> vectors support minimal, if any, improvement in long-term expression, using a number of viral or neuronal-specific cellular promoters  <cit> .
thus, paradoxically, expression from these promoters remains short-term, even in the absence of almost all  of the hsv- <dig> genome, the latency associated transcript  gene, hsv- <dig> genes, and hsv- <dig> gene expression.
to explain this paradox, we proposed that specific proteins in the hsv- <dig> particle reduce recombinant gene expression  <cit> .
specific proteins in the hsv- <dig> particle have prominent roles in the process of the virus taking control of the cell's biosynthetic machinery .
thus, specific proteins in the hsv- <dig> particle might alter recombinant gene expression.
in an initial study, we examined the effects on recombinant gene expression of five proteins that affect the hsv- <dig> particle  <cit> .
we found that packaging vectors using a mutated vhs or us <dig> resulted in minimal changes in recombinant gene expression; packaging vectors using a mutated us <dig> resulted in improved gene transfer ; and packaging vectors using a mutated ul <dig>  a protein kinase, or vp <dig> supported long-term expression .
these results suggested the hypothesis that specific proteins in the hsv- <dig> particle act by multiple pathways to reduce recombinant gene expression.
thus, interfering with multiple pathways might support higher levels of recombinant gene expression than those obtained by interfering with a single pathway.
specifically, packaging vectors using both mutated ul <dig> and mutated vp <dig> might support higher levels of long-term expression than those obtained by packaging vectors using either mutated ul <dig> or mutated vp <dig>
unfortunately, packaging using mutated vp <dig> lowered the titers ~100-fold, and packaging using both mutated vp <dig> and other mutated hsv- <dig> proteins further lowered the titers to levels that cannot support in vivo gene transfer experiments.
to improve the titers, and to explore the mechanism by which vp <dig> reduces long-term expression, we examined the effects on recombinant gene expression of two hsv- <dig> proteins, ul <dig> and ul <dig>  that modulate the activity of vp <dig>  <cit> .
vectors packaged using an hsv- <dig> cosmid set harboring a deletions of ul <dig>  or ul <dig>  or both, supported both improved gene transfer and long-term expression  <cit> .
vectors packaged using an hsv- <dig> cosmid set harboring deletions in both ul <dig> and ul <dig> supported larger improvements in recombinant gene expression compared to vectors packaged using an hsv- <dig> cosmid set harboring a deletion of either gene alone.
also, packaging using an hsv- <dig> cosmid set harboring the deletion of both ul <dig> and ul <dig> supported higher titers than packaging using a mutated vp <dig>  of note, a second approach to improve the titers using mutated vp <dig> has been reported: a recombinant hsv- <dig> vector containing a mutated vp <dig> was grown to high titer in the presence of equine herpesvirus  gene  <dig>  <cit> , the ehv- <dig> homolog of hsv- <dig> vp <dig>  ehv- <dig> gene  <dig> transactivates hsv- <dig> promoters, thereby restoring the titers to levels similar to those obtained using wild type  vp <dig>  however, ehv- <dig> gene  <dig> is not incorporated into hsv- <dig> particles, so ehv- <dig> gene  <dig> is unlikely to reduce recombinant gene expression.
in the present study, we tested the hypothesis that specific proteins in the hsv- <dig> particle act by multiple pathways to reduce recombinant gene expression.
we examined the long-term expression supported by a hsv- <dig> vector that contained a neuronal-specific promoter and was packaged using an hsv- <dig> cosmid set harboring combinations of mutated proteins that included ul <dig> and specific proteins in the vp <dig> transcriptional complex.
the preferred combination was mutated ul <dig>  ul <dig>  and ul <dig>  and this packaging condition improved both gene transfer and long-term expression.
RESULTS
hsv- <dig> vectors packaged using specific combinations of mutated hsv- <dig> proteins
in this study, we tested the hypothesis that specific proteins in the hsv- <dig> particle act by multiple pathways to reduce recombinant gene expression.
we packaged pnfhlac  <cit> , which contains the neurofilament heavy gene  promoter, using two combinations of mutated hsv- <dig> proteins that included a point mutation in ul <dig>  and a mutated protein in the vp <dig> transcriptional complex, either a linker insertion mutation in vp <dig>  or a deletion of both ul <dig> and ul <dig> .
packagings that used vp16in <dig> also contained ehv- <dig> gene  <dig> to improve the titers  <cit> .
control packaging conditions included i) ul13g, ii) vp16in <dig> and ehv- <dig> gene  <dig>  iii) Δul46/ <dig>  or iv) standard conditions , as the negative control.
the numbers of infectious vector particles  in these vector stocks were determined by 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside  staining at  <dig> hours after transduction of baby hamster kidney  fibroblast cells.
the results  showed that pnfhlac/vp16in <dig>  gene <dig>  ul13g  reduced the titers  <dig> -fold, and the other packaging conditions supported titers that were similar  to those obtained using standard packaging conditions .
next, we determined the titers of vector genomes ; we isolated dna from the vector stocks and performed pcr using primers from the lac z gene  <cit> .
as a measure of the packaging efficiency, we calculated the ratio of the physical titer to the biological titer  for each vector stock.
the results  showed that packaging pnfhlac in the presence of any of these mutated hsv- <dig> proteins, alone or in combination, resulted in similar ratios of the physical titer to the biological titer compared to packaging using standard conditions.
thus, these mutated hsv- <dig> proteins, alone or in specific combinations, did not cause large changes in the packaging efficiency.
three rats were analyzed for condition and time point; the means ± sds are shown.
athe titers of each vector stock after purification and concentration.
vg/ml is vector genomes/ml; ivp/ml is infectious vector particles/ml.
bthe efficiency of gene transfer is the number of positive cells at  <dig> days/the amount of vector injected.
the relative efficiency of gene transfer is the efficiency of gene transfer with a specific condition/the efficiency of gene transfer with wt packaging.
cvector stock diluted  <dig> -fold before gene transfer.
dfor these two packaging conditions, the titers and the x-gal cell counts for  <dig> days through  <dig> months were previously reported in  <cit>  and are included here only to support comparisons.
the x-gal cell counts at  <dig> months have not been previously reported.
the levels of long-term expression supported by hsv- <dig> vectors packaged using specific combinations of mutated hsv- <dig> proteins
these vector stocks were microinjected into the striatum, the rats were sacrificed  <dig> days, or  <dig> weeks, or  <dig>   <dig>  or  <dig> months after gene transfer, and x-gal staining was performed.
control rats that received pbs lacked x-gal positive striatal cells, but faint x-gal staining was observed in small numbers of cells that lined the brain vasculature .
sections from rats sacrificed at  <dig> days after gene transfer with each of these vector stocks contained numerous x-gal positive cells proximal to the injection sites.
high power views revealed that some of these cells contained neuronal morphology, including positive cell bodies with proximal processes, and more distal positive processes that did not appear to be associated with a cell body were also observed .
hsv- <dig> is known to infect axon terminals and be retrogradely transported to the cell body  <cit> , and small numbers of positive cells were observed at distant sites, including specific areas of neocortex, that contain neurons that project to the striatum .
subsequent analyses focused on the x-gal positive striatal cells, the majority of the x-gal positive cells.
to determine the efficiency of gene transfer, we quantified the number of positive cells at  <dig> days after gene transfer divided by the amount of vector that was injected.
to quantify any changes in the efficiency of gene transfer caused by packaging using specific mutated hsv- <dig> protein, we compared the efficiencies of gene transfer supported by pnfhlac packaged using specific mutated hsv- <dig> protein to the efficiency of gene transfer supported by pnfhlac/wt .
additionally, this ratio should correct for any underestimation of the titers of these vector stocks due to the use of fibroblast cells in the tittering .
the results  showed that pnfhlac/Δul46& <dig>  ul13g supported a  <dig> -fold increase in the relative efficiency of gene transfer, and we previously observed that pnfhlac/Δul46& <dig> supported a  <dig> -fold increase  <cit> .
vector stocks that were packaged using either vp16in <dig>  gene <dig> or ul13g, or both, supported modest, if any, increases in the efficiency of gene transfer, similar to previous results from packagings that used either vp16in <dig> or ul13g  <cit> .
in comparing two specific packaging conditions, the efficiency of gene transfer in the striatum may not correlate with the titers obtained using cultured fibroblast cells because of specific aspects of striatal anatomy and physiology, such as the extracellular matrix, that are absent from the cultures of fibroblast cells.
to quantify long-term expression, we compared the numbers of x-gal positive cells at  <dig> weeks, or  <dig>   <dig>  or  <dig> months after gene transfer to the numbers of positive cells at  <dig> days.
this comparison should be independent of any variability in either the titering procedure or the gene transfer process, because this calculation uses the number of positive cells at  <dig> days for the initial value in the comparison.
for the control condition, pnfhlac/wt, by  <dig> weeks after gene transfer the numbers of x-gal positive cells had declined to 14% of the numbers of positive cells observed at  <dig> days, by  <dig> month the number of positive cells was <3% of that at  <dig> days, and at  <dig> or  <dig> months no positive cells were observed .
{the data using pnfhlac/wt and pnfhlac/Δul46& <dig>  from  <dig> days through  <dig> months, was collected in parallel with another study and has been previously reported  <cit> .}
the decline in expression we observed with pnfhlac/wt is similar to that we have reported in two previous studies that examined pnfhlac/wt  <cit> .
vector stocks that were packaged using each of the three mutated hsv- <dig> proteins alone supported x-gal positive cells at  <dig> or  <dig> months after gene transfer , times when no positive cells were observed using pnfhlac/wt.
cell counts showed that packaging using each of the three mutated hsv- <dig> proteins alone supported increases in % of x-gal positive cells at  <dig> weeks, or  <dig>   <dig>  or  <dig> months after gene transfer compared to pnfhlac/wt .
at  <dig> weeks after gene transfer, the stabilities of expression supported by these  <dig> packaging conditions ranged from ~ <dig> to 50%, but these  <dig> packaging conditions supported similar stabilities of expression at  <dig>   <dig>  or  <dig> months.
the stabilities of expression observed using pnfhlac/ul13g or pnfhlac/vp16in <dig>  gene <dig> were similar to those observed in a previous study that used pnfhlac/ul13g or pnfhlac/vp16in <dig>  <cit> .
the two stocks of pnfhlac that were prepared using specific combinations of mutated hsv- <dig> proteins supported x-gal positive cells at  <dig> or  <dig> months after gene transfer .
interestingly, the two vector stocks that were packaged using specific combinations of mutated hsv- <dig> proteins supported higher percentages of long-term expression than those supported by pnfhlac packaged using each of the mutated hsv- <dig> proteins alone .
pnfhlac/Δul46& <dig>  ul13g supported somewhat higher percentages of long-term expression than pnfhlac/vp16in <dig>  gene <dig>  ul13g at each of the time points examined.
nonetheless, the percentages of long-term expression exhibited declines during the period that was examined  for each packaging condition .
we determined the statistical significance of the changes in the percentages of long-term expression using a two way analysis of variance .
the anova showed a statistically significant effect of packaging condition .
subsequent pairwise comparisons showed that the pnfhlac/Δul46& <dig>  ul13g condition supported significantly higher levels of long-term expression compared to each of the other five conditions, including pnfhlac/vp16in <dig>  gene <dig>  ul13g, at each of the four time points .
also, subsequent pairwise comparisons suggested that the pnfhlac/vp16in <dig>  gene <dig>  ul13g condition supported higher levels of long-term expression compared to each of the three conditions that used a single mutated hsv- <dig> protein, at each of the four time points .
additionally, the anova showed a statistically significant effect of time .
subsequent pairwise comparisons showed that the percentages of long-term expression declined for comparing each time point to the previous time point, for each packaging condition .
the levels of neuronal-specific expression supported by these vector stocks
because a specific altered vector particle might potentially support a different cell type specificity of transduction and/or expression than wt vector particles, we confirmed that each of these vector stocks targeted expression to neurons.
we performing double staining using antibodies against either e. coli β-galactosidase  or a neuronal marker, neun, on rats sacrificed at  <dig> days after gene transfer .
additionally, to establish long-term, neuronal-specific expression, we performed the same assay on rats that received either pnfhlac/Δul46& <dig>  ul13g or pnfhlac/vp16in <dig>  gene <dig>  ul13g and were sacrificed at  <dig> months after gene transfer .
cell counts showed that each of these vector stocks supported ~90% neuronal specific expression at  <dig> days after gene transfer, and pnfhlac packaged using each combination of mutated hsv- <dig> proteins supported ~90% neuronal specific expression at  <dig> months after gene transfer .
packaging
β-gal-ir was detected using a rabbit anti-e. coli β-gal antibody that was visualized with a fluorescein isothiocyanate-conjugated goat anti-rabbit igg, and neun-ir was detected in the same sections using a mouse monoclonal anti-neun antibody that was visualized with a rhodamine isothiocyanate-conjugated goat anti-mouse igg.
discussion
hsv- <dig> gene expression is hypothesized to shut off recombinant gene expression from hsv- <dig> vectors.
however, using this helper virus-free hsv- <dig> vector system, expression from many promoters remains short-term, even though this vector system does not contain any hsv- <dig> genes.
to resolve this paradox, we hypothesized that specific hsv- <dig> proteins in the vector particle shut off recombinant gene expression; in support of this hypothesis, we previously reported that packaging using specific mutated hsv- <dig> proteins improves long-term expression  <cit> .
these results suggested the hypothesis that specific proteins in the hsv- <dig> particle act by multiple pathways to reduce recombinant gene expression; thus, interfering with multiple pathways might support higher levels of recombinant gene expression than those obtained by interfering with a specific single pathway.
the data in this study support this hypothesis and suggest that multiple hsv- <dig> proteins act together to shut off recombinant gene expression by at least two different pathways.
one pathway contains the ul <dig> protein kinase, and a second pathway contains the vp <dig> transcriptional complex.
of note, the preferred packaging condition was pnfhlac packaged using mutated ul <dig>  ul <dig>  and ul <dig>
titers
vector particles produced by packaging using a specific mutated hsv- <dig> protein might have a reduced capability to bind to and/or enter cells.
a reduction in the transduction efficiency would likely be detected as an increased ratio of vector genomes  to biological titer ), for a specific packaging condition.
in contrast, the vg/ivp ratio was similar for the standard condition  and each packaging condition that used a mutated hsv- <dig> protein, including the two conditions that used specific combinations of mutated hsv- <dig> proteins.
thus, the vector particles produced by these different packaging conditions appear to have similar transduction efficiencies.
similarly, we previously found that packaging vectors in the presence of mutations in each of seven different hsv- <dig> proteins  did not affect the transduction efficiency  <cit> .
the two vector stocks that were prepared using specific combinations of mutated hsv- <dig> proteins contained sufficient titers to support gene transfer into the rat brain.
when used individually, the mutated ul <dig> had modest effects on the titers, and the mutated ul <dig> and ul <dig> had little or no effects on the titers, in either vector packaging or the growth of hsv- <dig> viruses that contain each mutation  <cit> .
consistent with these observations, the pnfhlac/Δul46& <dig>  ul13g condition supported titers that were similar to the standard packaging condition .
in contrast, packaging pnfhlac in the presence of a mutated vp <dig> reduced the titers ~100-fold  <cit> , and a hsv- <dig> virus that contained this mutated vp <dig> displayed reduced growth  <cit> .
use of ehv- <dig> gene  <dig> was previously shown to improve the growth of hsv- <dig> viruses that contain a mutated vp <dig>  <cit> .
analogous to those results, pnfhlac packaged using both a mutated vp <dig> and ehv- <dig> gene  <dig>  and either wt or mutated ul <dig>  yielded titers that were within an order of magnitude of the titers obtained using the standard packaging condition.
improved gene expression from pnfhlac packaged using combinations of mutated hsv- <dig> proteins
we previously found that packaging pnfhac in the presence of either a mutated ul <dig> or specific components of the vp <dig> transcriptional complex improved long-term expression  <cit> .
there is no known interaction between ul <dig> and the vp <dig> transcriptional complex.
thus, these observations suggested the hypothesis that there are at least two pathways by which specific proteins in the hsv- <dig> particle reduce recombinant gene expression.
thus, packaging vectors in the presence of both mutated ul <dig> and a mutated component of the vp <dig> complex would further improve long-term expression, compared to previous results  <cit> .
the results of this study support this hypothesis.
pnfhlac packaged using either of two combinations of mutated proteins that included both mutated ul <dig> and a mutated component of the vp <dig> complex supported higher levels of long-term expression than the levels supported by pnfhlac packaged using any of these mutated hsv- <dig> proteins, individually.
the longest time point examined in this study was  <dig> months, because that time was sufficient to establish improved long-term expression; however, we have previously shown that hsv- <dig> vectors support long-term expression for at least  <dig> months  <cit> .
interestingly, pnfhlac/Δul46& <dig>  ul13g supported a higher level of long-term expression than pnfhlac/vp16in <dig>  gene <dig>  ul13g.
this observation is difficult to explain in the absence of more detailed mechanistic information about how ul <dig>  ul <dig>  and vp <dig> interact.
pnfhlac/Δul46& <dig>  ul13g also improved the efficiency of gene transfer, consistent with previous results with pnfhlac/Δul46& <dig>  the capability of more complex packaging conditions to further improve recombinant gene expression, as shown here, is encouraging for future approaches to obtain additional improvements in expression.
potential mechanisms that reduce recombinant gene expression from hsv- <dig> vectors
currently available information suggests that ul <dig> and vp <dig> may act by different mechanisms to reduce recombinant gene expression.
in regards to ul13: substrates for ul <dig> include a number of hsv- <dig> proteins   <cit> ) and at least two cellular proteins, elongation factor 1δ  <cit>  and rna polymerase ii  <cit> .
ul <dig> and cdc <dig> target the same site in elongation factor 1δ  <cit> .
in regards to vp16: the hsv- <dig> oris fragment in pnfhlac contains the immediate early  4/ <dig> promoter and adjacent taatgarat elements  <cit> , binding sites for the vp <dig> transcriptional complex  <cit> .
although pnfhlac contains three polyadenylation sites between the ie and nfh promoters, increased transcription from the ie 4/ <dig> promoter might interfere with transcription from the nf-h promoter.
alternatively, wt hsv- <dig>  but not in <dig>  activates the c-jun n-terminal kinase/stress-activated protein kinase  <cit> , raising the possibility that this signaling pathway might reduce the activity of the nf-h promoter in pnfhlac.
although packaging using both a mutated ul <dig> and a mutated component of the vp <dig> transcriptional complex improved recombinant gene expression, the levels of expression still declined over time, suggesting that additional, as yet unknown, factors contribute the decline in expression.
the tegument in the hsv- <dig> virus particle contains ~ <dig> proteins, and  <dig> of these proteins have been examined for effects on recombinant gene expression.
two tegument proteins had minimal effects on expression, and specific mutations in each of five tegument proteins improved the efficiency of gene transfer and/or long-term expression  <cit> .
it remains to be determined if other tegument proteins affect recombinant gene expression.
other potential factors that might reduce recombinant gene expression include specific proteins in the hsv- <dig> vector particle capsid or envelope, or the transduction–vector particle disassembly  process may be detected by specific cellular signaling pathways.
CONCLUSIONS
in conclusion, the preferred combination of mutated hsv- <dig> proteins was mutated ul <dig>  ul <dig>  and ul <dig>  vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for  <dig> months, the longest time examined.
these advances will benefit specific gene therapy applications of hsv- <dig> vectors.

 herpes simplex virus vectors are an attractive system for gene transfer into neurons, but the limited levels of long-term expression remain a significant problem. the numbers of cells expressing recombinant gene products display large decreases within the first few weeks after gene transfer, using either plasmid or recombinant hsv- vectors that contain specific viral or neuronal-specific cellular promoters. interestingly, using hsv- plasmid vectors, two neuronal type-specific promoters or preproenkephalin), and chimeric promoters that contain 5' upstream sequences from either of these promoters, support significant levels of long-term expression. although the levels of expression display a significant initial decrease, long-term expression is relatively stable. consequently, identifying the mechanisms responsible for the initial decrease in expression may provide a foundation for the rational design of hsv- vectors that support long-term expression. the reduction in recombinant gene expression might be caused by the mechanisms that repress hsv- gene expression in hsv- latency, focusing attention on hsv- gene regulation. supporting a role for hsv- gene regulation in reducing recombinant gene expression, helper virus-containing hsv- vector systems cause significant cytopathic effects and an inflammatory response, and most of these side effects are caused by hsv- gene expression from the helper virus. consistent with these results, helper virus-free hsv- vectors cause substantially less side effects than those observed using helper virus-containing systems. however, helper virus-free hsv- vectors support minimal, if any, improvement in long-term expression, using a number of viral or neuronal-specific cellular promoters. thus, paradoxically, expression from these promoters remains short-term, even in the absence of almost all of the hsv- genome, the latency associated transcript gene, hsv- genes, and hsv- gene expression. to explain this paradox, we proposed that specific proteins in the hsv- particle reduce recombinant gene expression. specific proteins in the hsv- particle have prominent roles in the process of the virus taking control of the cell's biosynthetic machinery. thus, specific proteins in the hsv- particle might alter recombinant gene expression. in an initial study, we examined the effects on recombinant gene expression of five proteins that affect the hsv- particle. we found that packaging vectors using a mutated vhs or us resulted in minimal changes in recombinant gene expression; packaging vectors using a mutated us resulted in improved gene transfer; and packaging vectors using a mutated ul a protein kinase, or vp supported long-term expression. these results suggested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression. thus, interfering with multiple pathways might support higher levels of recombinant gene expression than those obtained by interfering with a single pathway. specifically, packaging vectors using both mutated ul and mutated vp might support higher levels of long-term expression than those obtained by packaging vectors using either mutated ul or mutated vp unfortunately, packaging using mutated vp lowered the titers ~100-fold, and packaging using both mutated vp and other mutated hsv- proteins further lowered the titers to levels that cannot support in vivo gene transfer experiments. to improve the titers, and to explore the mechanism by which vp reduces long-term expression, we examined the effects on recombinant gene expression of two hsv- proteins, ul and ul that modulate the activity of vp. vectors packaged using an hsv- cosmid set harboring a deletions of ul or ul or both, supported both improved gene transfer and long-term expression. vectors packaged using an hsv- cosmid set harboring deletions in both ul and ul supported larger improvements in recombinant gene expression compared to vectors packaged using an hsv- cosmid set harboring a deletion of either gene alone. also, packaging using an hsv- cosmid set harboring the deletion of both ul and ul supported higher titers than packaging using a mutated vp of note, a second approach to improve the titers using mutated vp has been reported: a recombinant hsv- vector containing a mutated vp was grown to high titer in the presence of equine herpesvirus gene, the ehv- homolog of hsv- vp ehv- gene transactivates hsv- promoters, thereby restoring the titers to levels similar to those obtained using wild type vp however, ehv- gene is not incorporated into hsv- particles, so ehv- gene is unlikely to reduce recombinant gene expression. in the present study, we tested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression. we examined the long-term expression supported by a hsv- vector that contained a neuronal-specific promoter and was packaged using an hsv- cosmid set harboring combinations of mutated proteins that included ul and specific proteins in the vp transcriptional complex. the preferred combination was mutated ul ul and ul and this packaging condition improved both gene transfer and long-term expression.  hsv- vectors packaged using specific combinations of mutated hsv- proteins in this study, we tested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression. we packaged pnfhlac, which contains the neurofilament heavy gene promoter, using two combinations of mutated hsv- proteins that included a point mutation in ul and a mutated protein in the vp transcriptional complex, either a linker insertion mutation in vp or a deletion of both ul and ul. packagings that used vp16in also contained ehv- gene to improve the titers. control packaging conditions included i) ul13g, ii) vp16in and ehv- gene iii) Δul46/ or iv) standard conditions, as the negative control. the numbers of infectious vector particles in these vector stocks were determined by 5-bromo-4-chloro-3-indoyl-β-d-galactopyranoside staining at hours after transduction of baby hamster kidney fibroblast cells. the results showed that pnfhlac/vp16in gene ul13g reduced the titers -fold, and the other packaging conditions supported titers that were similar to those obtained using standard packaging conditions. next, we determined the titers of vector genomes; we isolated dna from the vector stocks and performed pcr using primers from the lac z gene. as a measure of the packaging efficiency, we calculated the ratio of the physical titer to the biological titer for each vector stock. the results showed that packaging pnfhlac in the presence of any of these mutated hsv- proteins, alone or in combination, resulted in similar ratios of the physical titer to the biological titer compared to packaging using standard conditions. thus, these mutated hsv- proteins, alone or in specific combinations, did not cause large changes in the packaging efficiency. three rats were analyzed for condition and time point; the means ± sds are shown. athe titers of each vector stock after purification and concentration. vg/ml is vector genomes/ml; ivp/ml is infectious vector particles/ml. bthe efficiency of gene transfer is the number of positive cells at days/the amount of vector injected. the relative efficiency of gene transfer is the efficiency of gene transfer with a specific condition/the efficiency of gene transfer with wt packaging. cvector stock diluted -fold before gene transfer. dfor these two packaging conditions, the titers and the x-gal cell counts for days through months were previously reported in and are included here only to support comparisons. the x-gal cell counts at months have not been previously reported. the levels of long-term expression supported by hsv- vectors packaged using specific combinations of mutated hsv- proteins these vector stocks were microinjected into the striatum, the rats were sacrificed days, or weeks, or or months after gene transfer, and x-gal staining was performed. control rats that received pbs lacked x-gal positive striatal cells, but faint x-gal staining was observed in small numbers of cells that lined the brain vasculature. sections from rats sacrificed at days after gene transfer with each of these vector stocks contained numerous x-gal positive cells proximal to the injection sites. high power views revealed that some of these cells contained neuronal morphology, including positive cell bodies with proximal processes, and more distal positive processes that did not appear to be associated with a cell body were also observed. hsv- is known to infect axon terminals and be retrogradely transported to the cell body, and small numbers of positive cells were observed at distant sites, including specific areas of neocortex, that contain neurons that project to the striatum. subsequent analyses focused on the x-gal positive striatal cells, the majority of the x-gal positive cells. to determine the efficiency of gene transfer, we quantified the number of positive cells at days after gene transfer divided by the amount of vector that was injected. to quantify any changes in the efficiency of gene transfer caused by packaging using specific mutated hsv- protein, we compared the efficiencies of gene transfer supported by pnfhlac packaged using specific mutated hsv- protein to the efficiency of gene transfer supported by pnfhlac/wt. additionally, this ratio should correct for any underestimation of the titers of these vector stocks due to the use of fibroblast cells in the tittering. the results showed that pnfhlac/Δul46& ul13g supported a -fold increase in the relative efficiency of gene transfer, and we previously observed that pnfhlac/Δul46& supported a -fold increase. vector stocks that were packaged using either vp16in gene or ul13g, or both, supported modest, if any, increases in the efficiency of gene transfer, similar to previous results from packagings that used either vp16in or ul13g. in comparing two specific packaging conditions, the efficiency of gene transfer in the striatum may not correlate with the titers obtained using cultured fibroblast cells because of specific aspects of striatal anatomy and physiology, such as the extracellular matrix, that are absent from the cultures of fibroblast cells. to quantify long-term expression, we compared the numbers of x-gal positive cells at weeks, or or months after gene transfer to the numbers of positive cells at days. this comparison should be independent of any variability in either the titering procedure or the gene transfer process, because this calculation uses the number of positive cells at days for the initial value in the comparison. for the control condition, pnfhlac/wt, by weeks after gene transfer the numbers of x-gal positive cells had declined to 14% of the numbers of positive cells observed at days, by month the number of positive cells was <3% of that at days, and at or months no positive cells were observed. {the data using pnfhlac/wt and pnfhlac/Δul46& from days through months, was collected in parallel with another study and has been previously reported.} the decline in expression we observed with pnfhlac/wt is similar to that we have reported in two previous studies that examined pnfhlac/wt. vector stocks that were packaged using each of the three mutated hsv- proteins alone supported x-gal positive cells at or months after gene transfer, times when no positive cells were observed using pnfhlac/wt. cell counts showed that packaging using each of the three mutated hsv- proteins alone supported increases in % of x-gal positive cells at weeks, or or months after gene transfer compared to pnfhlac/wt. at weeks after gene transfer, the stabilities of expression supported by these packaging conditions ranged from ~ to 50%, but these packaging conditions supported similar stabilities of expression at or months. the stabilities of expression observed using pnfhlac/ul13g or pnfhlac/vp16in gene were similar to those observed in a previous study that used pnfhlac/ul13g or pnfhlac/vp16in. the two stocks of pnfhlac that were prepared using specific combinations of mutated hsv- proteins supported x-gal positive cells at or months after gene transfer. interestingly, the two vector stocks that were packaged using specific combinations of mutated hsv- proteins supported higher percentages of long-term expression than those supported by pnfhlac packaged using each of the mutated hsv- proteins alone. pnfhlac/Δul46& ul13g supported somewhat higher percentages of long-term expression than pnfhlac/vp16in gene ul13g at each of the time points examined. nonetheless, the percentages of long-term expression exhibited declines during the period that was examined for each packaging condition. we determined the statistical significance of the changes in the percentages of long-term expression using a two way analysis of variance. the anova showed a statistically significant effect of packaging condition. subsequent pairwise comparisons showed that the pnfhlac/Δul46& ul13g condition supported significantly higher levels of long-term expression compared to each of the other five conditions, including pnfhlac/vp16in gene ul13g, at each of the four time points. also, subsequent pairwise comparisons suggested that the pnfhlac/vp16in gene ul13g condition supported higher levels of long-term expression compared to each of the three conditions that used a single mutated hsv- protein, at each of the four time points. additionally, the anova showed a statistically significant effect of time. subsequent pairwise comparisons showed that the percentages of long-term expression declined for comparing each time point to the previous time point, for each packaging condition. the levels of neuronal-specific expression supported by these vector stocks because a specific altered vector particle might potentially support a different cell type specificity of transduction and/or expression than wt vector particles, we confirmed that each of these vector stocks targeted expression to neurons. we performing double staining using antibodies against either e. coli β-galactosidase or a neuronal marker, neun, on rats sacrificed at days after gene transfer. additionally, to establish long-term, neuronal-specific expression, we performed the same assay on rats that received either pnfhlac/Δul46& ul13g or pnfhlac/vp16in gene ul13g and were sacrificed at months after gene transfer. cell counts showed that each of these vector stocks supported ~90% neuronal specific expression at days after gene transfer, and pnfhlac packaged using each combination of mutated hsv- proteins supported ~90% neuronal specific expression at months after gene transfer. packaging β-gal-ir was detected using a rabbit anti-e. coli β-gal antibody that was visualized with a fluorescein isothiocyanate-conjugated goat anti-rabbit igg, and neun-ir was detected in the same sections using a mouse monoclonal anti-neun antibody that was visualized with a rhodamine isothiocyanate-conjugated goat anti-mouse igg. discussion hsv- gene expression is hypothesized to shut off recombinant gene expression from hsv- vectors. however, using this helper virus-free hsv- vector system, expression from many promoters remains short-term, even though this vector system does not contain any hsv- genes. to resolve this paradox, we hypothesized that specific hsv- proteins in the vector particle shut off recombinant gene expression; in support of this hypothesis, we previously reported that packaging using specific mutated hsv- proteins improves long-term expression. these results suggested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression; thus, interfering with multiple pathways might support higher levels of recombinant gene expression than those obtained by interfering with a specific single pathway. the data in this study support this hypothesis and suggest that multiple hsv- proteins act together to shut off recombinant gene expression by at least two different pathways. one pathway contains the ul protein kinase, and a second pathway contains the vp transcriptional complex. of note, the preferred packaging condition was pnfhlac packaged using mutated ul ul and ul titers vector particles produced by packaging using a specific mutated hsv- protein might have a reduced capability to bind to and/or enter cells. a reduction in the transduction efficiency would likely be detected as an increased ratio of vector genomes to biological titer ), for a specific packaging condition. in contrast, the vg/ivp ratio was similar for the standard condition and each packaging condition that used a mutated hsv- protein, including the two conditions that used specific combinations of mutated hsv- proteins. thus, the vector particles produced by these different packaging conditions appear to have similar transduction efficiencies. similarly, we previously found that packaging vectors in the presence of mutations in each of seven different hsv- proteins did not affect the transduction efficiency. the two vector stocks that were prepared using specific combinations of mutated hsv- proteins contained sufficient titers to support gene transfer into the rat brain. when used individually, the mutated ul had modest effects on the titers, and the mutated ul and ul had little or no effects on the titers, in either vector packaging or the growth of hsv- viruses that contain each mutation. consistent with these observations, the pnfhlac/Δul46& ul13g condition supported titers that were similar to the standard packaging condition. in contrast, packaging pnfhlac in the presence of a mutated vp reduced the titers ~100-fold, and a hsv- virus that contained this mutated vp displayed reduced growth. use of ehv- gene was previously shown to improve the growth of hsv- viruses that contain a mutated vp. analogous to those results, pnfhlac packaged using both a mutated vp and ehv- gene and either wt or mutated ul yielded titers that were within an order of magnitude of the titers obtained using the standard packaging condition. improved gene expression from pnfhlac packaged using combinations of mutated hsv- proteins we previously found that packaging pnfhac in the presence of either a mutated ul or specific components of the vp transcriptional complex improved long-term expression. there is no known interaction between ul and the vp transcriptional complex. thus, these observations suggested the hypothesis that there are at least two pathways by which specific proteins in the hsv- particle reduce recombinant gene expression. thus, packaging vectors in the presence of both mutated ul and a mutated component of the vp complex would further improve long-term expression, compared to previous results. the results of this study support this hypothesis. pnfhlac packaged using either of two combinations of mutated proteins that included both mutated ul and a mutated component of the vp complex supported higher levels of long-term expression than the levels supported by pnfhlac packaged using any of these mutated hsv- proteins, individually. the longest time point examined in this study was months, because that time was sufficient to establish improved long-term expression; however, we have previously shown that hsv- vectors support long-term expression for at least months. interestingly, pnfhlac/Δul46& ul13g supported a higher level of long-term expression than pnfhlac/vp16in gene ul13g. this observation is difficult to explain in the absence of more detailed mechanistic information about how ul ul and vp interact. pnfhlac/Δul46& ul13g also improved the efficiency of gene transfer, consistent with previous results with pnfhlac/Δul46& the capability of more complex packaging conditions to further improve recombinant gene expression, as shown here, is encouraging for future approaches to obtain additional improvements in expression. potential mechanisms that reduce recombinant gene expression from hsv- vectors currently available information suggests that ul and vp may act by different mechanisms to reduce recombinant gene expression. in regards to ul13: substrates for ul include a number of hsv- proteins ) and at least two cellular proteins, elongation factor 1δ and rna polymerase ii. ul and cdc target the same site in elongation factor 1δ. in regards to vp16: the hsv- oris fragment in pnfhlac contains the immediate early 4/ promoter and adjacent taatgarat elements, binding sites for the vp transcriptional complex. although pnfhlac contains three polyadenylation sites between the ie and nfh promoters, increased transcription from the ie 4/ promoter might interfere with transcription from the nf-h promoter. alternatively, wt hsv- but not in activates the c-jun n-terminal kinase/stress-activated protein kinase, raising the possibility that this signaling pathway might reduce the activity of the nf-h promoter in pnfhlac. although packaging using both a mutated ul and a mutated component of the vp transcriptional complex improved recombinant gene expression, the levels of expression still declined over time, suggesting that additional, as yet unknown, factors contribute the decline in expression. the tegument in the hsv- virus particle contains ~ proteins, and of these proteins have been examined for effects on recombinant gene expression. two tegument proteins had minimal effects on expression, and specific mutations in each of five tegument proteins improved the efficiency of gene transfer and/or long-term expression. it remains to be determined if other tegument proteins affect recombinant gene expression. other potential factors that might reduce recombinant gene expression include specific proteins in the hsv- vector particle capsid or envelope, or the transduction–vector particle disassembly process may be detected by specific cellular signaling pathways.  in conclusion, the preferred combination of mutated hsv- proteins was mutated ul ul and ul vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for months, the longest time examined. these advances will benefit specific gene therapy applications of hsv- vectors. 
these results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all of the hsv- genome, hsv- genes, and hsv- gene expression. these results suggested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression. implications of these results for strategies to further improve long-term expression are discussed. moreover, long-term expression will benefit specific gene therapy applications. a hsv- vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. vector particles containing this combination of mutated hsv- proteins improve recombinant gene expression. to test this hypothesis, we examined combinations of mutated proteins that included both ul and specific components of the vp transcriptional complex. for supporting long-term expression, the preferred combination of mutated hsv- proteins was mutated ul ul and ul vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for months, the longest time examined. to resolve this paradox, we hypothesized that specific proteins in the hsv- virus particle shut off recombinant gene expression. herpes simplex virus gene expression is thought to shut off recombinant gene expression from hsv- vectors; however, in a helper virus-free hsv- vector system, a number of promoters support only short-term expression. in two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated hsv- proteins. we found that vectors packaged using mutated ul, or vp or ul and/or ul supported improved long-term expression, and vectors packaged using mutated ul and/or ul also supported improved gene transfer.
 herpes simplex virus gene expression is thought to shut off recombinant gene expression from hsv- vectors; however, in a helper virus-free hsv- vector system, a number of promoters support only short-term expression. these results raise the paradox that recombinant gene expression remains short-term even in the absence of almost all of the hsv- genome, hsv- genes, and hsv- gene expression. to resolve this paradox, we hypothesized that specific proteins in the hsv- virus particle shut off recombinant gene expression. in two earlier studies, we examined the effects on recombinant gene expression of packaging vectors using specific mutated hsv- proteins. we found that vectors packaged using mutated ul, or vp or ul and/or ul supported improved long-term expression, and vectors packaged using mutated ul and/or ul also supported improved gene transfer. these results suggested the hypothesis that specific proteins in the hsv- particle act by multiple pathways to reduce recombinant gene expression. to test this hypothesis, we examined combinations of mutated proteins that included both ul and specific components of the vp transcriptional complex.  a hsv- vector containing a neuronal-specific promoter was packaged using specific combinations of mutated proteins, and the resulting vector stocks were tested in the rat striatum. for supporting long-term expression, the preferred combination of mutated hsv- proteins was mutated ul ul and ul vectors packaged using this combination of mutated proteins supported a higher efficiency of gene transfer and high levels expression for months, the longest time examined.  vector particles containing this combination of mutated hsv- proteins improve recombinant gene expression. implications of these results for strategies to further improve long-term expression are discussed. moreover, long-term expression will benefit specific gene therapy applications. 