In this study, we tested the hypothesis that specific proteins in the HSV-1 particle act by multiple pathways to reduce recombinant gene expression. We packaged pNFHlac 7, which contains the neurofilament heavy gene (NF-H) promoter, using two combinations of mutated HSV-1 proteins that included a point mutation in U_L13 (U_L13g, abolishes protein kinase activity 14) and a mutated protein in the VP16 transcriptional complex, either a linker insertion mutation in VP16 (VP16in14 2122) or a deletion of both U_L46 and U_L47 (ΔU_L46/47 161719). Packagings that used VP16in14 also contained EHV-1 gene 12 to improve the titers 20. Control packaging conditions included i) U_L13g, ii) VP16in14 and EHV-1 gene 12, iii) ΔU_L46/47, or iv) standard conditions (wt, no mutated HSV-1 proteins), as the negative control. The numbers of infectious vector particles (IVP/ml) in these vector stocks were determined by 5-bromo-4-chloro-3-indoyl-β-D-galactopyranoside (X-gal) staining at 24 hours after transduction of baby hamster kidney (BHK) fibroblast cells. The results (Table 1) showed that pNFHlac/vp16in14, gene12, ul13g (nomenclature, vector followed by the mutated proteins used in packaging) reduced the titers 7.2-fold, and the other packaging conditions supported titers that were similar (≤ 3-fold reduction) to those obtained using standard packaging conditions (pNFHlac/wt). Next, we determined the titers of vector genomes (VG/ml); we isolated DNA from the vector stocks and performed PCR using primers from the

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These vector stocks were microinjected into the striatum, the rats were sacrificed 4 days, or 2 weeks, or 1, 2, or 3 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 (not shown). Sections from rats sacrificed at 4 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 (pNFHlac/ul13g, Figure 1A; pNFHlac/VP16in14, gene12, Figure 1D; pNFHlac/Δul46&47, ul13g, Figure 1G; pNFHlac/VP16in14, gene12, ul13g, Figure 1J). HSV-1 is known to infect axon terminals and be retrogradely transported to the cell body 23, and small numbers of positive cells were observed at distant sites, including specific areas of neocortex, that contain neurons that project to the striatum (not shown). 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 4 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-1 protein(s), we compared the efficiencies of gene transfer supported by pNFHlac packaged using specific mutated HSV-1 protein(s) to the efficiency of gene transfer supported by pNFHlac/wt (relative efficiency of gene transfer). 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 (see methods). The results (Table 1) showed that pNFHlac/Δul46&47, ul13g supported a 5.7-fold increase in the relative efficiency of gene transfer, and we previously observed that pNFHlac/Δul46&47 supported a 9.9-fold increase 19. Vector stocks that were packaged using either VP16in14, gene12 or ul13g, or both, supported modest, if any, increases in the efficiency of gene transfer, similar to previous results from packagings that used either VP16in14 or ul13g 14. 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 2 weeks, or 1, 2, or 3 months after gene transfer to the numbers of positive cells at 4 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 4 days for the initial value in the comparison. For the control condition, pNFHlac/wt, by 2 weeks after gene transfer the numbers of X-gal positive cells had declined to 14% of the numbers of positive cells observed at 4 days, by 1 month the number of positive cells was <3% of that at 4 days, and at 2 or 3 months no positive cells were observed (Table 1, Figure 2). {The data using pNFHlac/wt and pNFHlac/Δul46&47, from 4 days through 2 months, was collected in parallel with another study and has been previously reported 19.} The decline in expression we observed with pNFHlac/wt is similar to that we have reported in two previous studies that examined pNFHlac/wt 17.

Vector stocks that were packaged using each of the three mutated HSV-1 proteins alone supported X-gal positive cells at 2 or 3 months after gene transfer (pNFHlac/ul13g, Figure 1B and 1C; pNFHlac/VP16in14, gene12, Figure 1E and 1F), times when no positive cells were observed using pNFHlac/wt. Cell counts showed that packaging using each of the three mutated HSV-1 proteins alone supported increases in % of X-gal positive cells at 2 weeks, or 1, 2, or 3 months after gene transfer compared to pNFHlac/wt (Table 1, Figure 2). At 2 weeks after gene transfer, the stabilities of expression supported by these 3 packaging conditions ranged from ~30 to 50%, but these 3 packaging conditions supported similar stabilities of expression at 1, 2, or 3 months. The stabilities of expression observed using pNFHlac/ul13g or pNFHlac/VP16in14, gene12 were similar to those observed in a previous study that used pNFHlac/ul13g or pNFHlac/VP16in14 14.

The two stocks of pNFHlac that were prepared using specific combinations of mutated HSV-1 proteins supported X-gal positive cells at 2 or 3 months after gene transfer (pNFHlac/Δul46&47, ul13g, Figure 1H and 1I; pNFHlac/VP16in14, gene12, ul13g, Figure 1K and 1L). Interestingly, the two vector stocks that were packaged using specific combinations of mutated HSV-1 proteins supported higher percentages of long-term expression than those supported by pNFHlac packaged using each of the mutated HSV-1 proteins alone (Table 1, Figure 2). pNFHlac/Δul46&47, ul13g supported somewhat higher percentages of long-term expression than pNFHlac/VP16in14, gene12, ul13g at each of the time points examined. Nonetheless, the percentages of long-term expression exhibited declines during the period that was examined (2 weeks to 3 months) for each packaging condition (Table 1, Figure 2).

We determined the statistical significance of the changes in the percentages of long-term expression using a two way analysis of variance (ANOVA). The ANOVA showed a statistically significant effect of packaging condition (p < 0.001). Subsequent pairwise comparisons showed that the pNFHlac/Δul46&47, ul13g condition supported significantly higher levels of long-term expression compared to each of the other five conditions, including pNFHlac/VP16in14, gene12, ul13g, at each of the four time points (12 of these 20 comparisons p < 0.001; 6 of the comparisons p < 0.05; 1 comparison p = 0.056; 1 comparison p > 0.10). Also, subsequent pairwise comparisons suggested that the pNFHlac/VP16in14, gene12, ul13g condition supported higher levels of long-term expression compared to each of the three conditions that used a single mutated HSV-1 protein, at each of the four time points (7 of these 12 comparisons p < 0.05; 4 of the comparisons p < 0.10; 1 comparison p > 0.10). Additionally, the ANOVA showed a statistically significant effect of time (p < 0.001). 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 (p < 0.05).

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 (β-gal) or a neuronal marker, NeuN, on rats sacrificed at 4 days after gene transfer (Table 2; pNFHlac/Δul46&47, ul13g, Figure 3A–C; pNFHlac/VP16in14, gene12, ul13g, Figure 3G–I). Additionally, to establish long-term, neuronal-specific expression, we performed the same assay on rats that received either pNFHlac/Δul46&47, ul13g or pNFHlac/VP16in14, gene12, ul13g and were sacrificed at 2 months after gene transfer (Figure 3D–F and 3J–L, Table 2). Cell counts showed that each of these vector stocks supported ~90% neuronal specific expression at 4 days after gene transfer, and pNFHlac packaged using each combination of mutated HSV-1 proteins supported ~90% neuronal specific expression at 2 months after gene transfer (Table 2).

Vector particles produced by packaging using a specific mutated HSV-1 protein(s) 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 (VG) to biological titer (infectious vector particles (IVP)), for a specific packaging condition. In contrast, the VG/IVP ratio was similar for the standard condition (pNFHlac/wt) and each packaging condition that used a mutated HSV-1 protein, including the two conditions that used specific combinations of mutated HSV-1 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-1 proteins (U_L13, vhs, U_L46, U_L47, VP16, U_S3, or U_S11) did not affect the transduction efficiency 1419.

The two vector stocks that were prepared using specific combinations of mutated HSV-1 proteins contained sufficient titers to support gene transfer into the rat brain. When used individually, the mutated U_L13 had modest effects on the titers, and the mutated U_L46 and U_L47 had little or no effects on the titers, in either vector packaging or the growth of HSV-1 viruses that contain each mutation 141617. Consistent with these observations, the pNFHlac/Δul46&47, ul13g condition supported titers that were similar to the standard packaging condition (pNFHlac/wt). In contrast, packaging pNFHlac in the presence of a mutated VP16 reduced the titers ~100-fold 14, and a HSV-1 virus that contained this mutated VP16 displayed reduced growth 22. Use of EHV-1 gene 12 was previously shown to improve the growth of HSV-1 viruses that contain a mutated VP16 20. Analogous to those results, pNFHlac packaged using both a mutated VP16 and EHV-1 gene 12, and either wt or mutated U_L13, yielded titers that were within an order of magnitude of the titers obtained using the standard packaging condition.

We previously found that packaging pNFHac in the presence of either a mutated U_L13 or specific components of the VP16 transcriptional complex improved long-term expression 1419. There is no known interaction between U_L13 and the VP16 transcriptional complex. Thus, these observations suggested the hypothesis that there are at least two pathways by which specific proteins in the HSV-1 particle reduce recombinant gene expression. Thus, packaging vectors in the presence of both mutated U_L13 and a mutated component of the VP16 complex would further improve long-term expression, compared to previous results 1419.

The results of this study support this hypothesis. pNFHlac packaged using either of two combinations of mutated proteins that included both mutated U_L13 and a mutated component of the VP16 complex supported higher levels of long-term expression than the levels supported by pNFHlac packaged using any of these mutated HSV-1 proteins, individually. The longest time point examined in this study was 3 months, because that time was sufficient to establish improved long-term expression; however, we have previously shown that HSV-1 vectors support long-term expression for at least 14 months 2. Interestingly, pNFHlac/Δul46&47, ul13g supported a higher level of long-term expression than pNFHlac/VP16in14, gene12, ul13g. This observation is difficult to explain in the absence of more detailed mechanistic information about how U_L46, U_L47, and VP16 interact. pNFHlac/Δul46&47, ul13g also improved the efficiency of gene transfer, consistent with previous results with pNFHlac/Δul46&47. 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.

Currently available information suggests that U_L13 and VP16 may act by different mechanisms to reduce recombinant gene expression. In regards to U_L13: Substrates for U_L13 include a number of HSV-1 proteins (ICP0, ICP22, gE, and VP22 (U_L49) 24252627) and at least two cellular proteins, elongation factor 1δ 28 and RNA polymerase II 2930. U_L13 and cdc2 target the same site in elongation factor 1δ 31. In regards to VP16: The HSV-1 ori_s fragment in pNFHlac contains the immediate early (IE) 4/5 promoter and adjacent TAATGARAT elements 7, binding sites for the VP16 transcriptional complex 32. Although pNFHlac contains three polyadenylation sites between the IE and NFH promoters, increased transcription from the IE 4/5 promoter might interfere with transcription from the NF-H promoter. Alternatively, wt HSV-1, but not in1814, activates the

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Although packaging using both a mutated U_L13 and a mutated component of the VP16 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-1 virus particle contains ~15 proteins, and 7 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 1419. 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-1 vector particle capsid or envelope, or the transduction–vector particle disassembly (uncoating) process may be detected by specific cellular signaling pathways.

Restriction endonucleases were obtained from New England Biolabs and Roche. G418, Dulbecco's modified minimal essential medium, fetal bovine serum, OPTI-MEM I, and lipofectamine were obtained from Invitrogen. X-Gal was purchased from Sigma. Rabbit anti-

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pNFHlac 7 contains the mouse NF-H promoter (0.6 kb fragment 35). pNFHlac supports only short-term expression following packaging using standard conditions (no mutated HSV-1 proteins); rats sacrificed at 4 days after gene transfer contained many X-gal positive cells, but few, if any, positive cells were observed in rats sacrificed at 1 or 2 months 17. Plasmid pC12 contains EHV-1 gene 12 under the control of the cytomegalovirus IE promoter (20 gift from Dr. Coffin).

Cosmid set C (cos6, cos14, cos28, cos48, cos56, 36) represents the HSV-1 genome, and the a sequence was deleted from the two cosmids that contain it (cos6Δa, cos48Δa 12). cos6Δa/ul13g 14 contains a point mutation in U_L13 that abolishes protein kinase activity 14; HSV-1 particles contain U_L13 2737. cos56/vp16in14 14 contains a linker insertion mutation in VP16 2122 that abolishes enhancer activity but supports assembly of HSV-1 particles. cos56/Δul46/47 19 contains a deletion of both U_L46 and U_L47 17; U_L46 and U_L47 are components of VP16 transcriptional complex and are present in HSV-1 particles 161732.

The culture conditions for BHK21 cells or 2-2 cells 38 have been described 14. Helper virus-free packaging 12 was performed using a modified protocol 39 that improves the titers and cosmids that contain specific mutated HSV-1 proteins, or no mutated HSV-1 proteins (standard or wt conditions). Packaging procedures that used EHV-1 gene 12 employed the standard DNA transfection conditions 1239 with a 1:1 (μg:μg) ratio of pNFHlac and pC12 20. Packaging using VP16in14 reduces the titers ~100-fold 14, and EHV-1 gene 12 was added to improve the titers. Vector stocks were purified as described 40.

The titers of vector genomes (VG)/ml were determined by extracting DNA from the vector stocks and determining the amounts of pNFHlac DNA using PCR and primers that recognize the

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These studies were approved by the W. Roxbury VA Hospital IACUC. Stereotactic injections (3 μl/site, 2 sites) were used to deliver vector stocks into the striatum of male Sprague Dawley rats (150–175 gm; anterior-posterior (AP) +0.8, medial-lateral (ML) +2.5, dorsal-ventral (DV) -5.5; AP +0.8, ML -2.5, DV -5.5). AP is relative to bregma, ML is relative to the sagittal suture, and DV is relative to the bregma-lambda plane 41. Vector stocks were injected using a micropump (model 100, KD Scientific); 3 μl of vector stock was injected over 8 minutes, the needle was maintained in place for an additional 5 minutes, and the needle was slowly withdrawn over approximately 5 minutes. At 4 days to 3 months after gene transfer, the rats were perfused, the brains were sectioned, and X-gal staining was performed 1 at room temperature, pH 7.9, for 3 hours. Alternatively, to localize β-gal to neurons, rabbit anti-E. coli β-gal was visualized with a fluorescein isothiocyanate-conjugated goat anti-rabbit IgG, and mouse monoclonal anti-NeuN was visualized with a rhodamine isothiocyanate-conjugated goat anti-mouse IgG 1.

Coronal sections (25 μm) that included the striatum were prepared. Every 4th section was analyzed for recombinant gene expression, and ~12 of these sections contained either the X-gal or the β-gal-immunoreactivity (IR) positive cells. Low power (10×) was used to identify the positive cells, and high power (40×) was used to perform the cell counts. To verify the accuracy of the cell counts, each section was counted at least two times, on different days, and the values differed by <10% for each section. ANOVA followed by multiple pairwise comparisons was used to analyze the statistical significance of differences in the numbers of X-gal cells.