Stable Transgene Expression From HSV Amplicon Vectors in the Brain: Potential Involvement of Immunoregulatory Signals
Authors: Masataka Suzuki, E. Antonio Chiocca, Yoshinaga Saeki
Journal: Molecular Therapy (2008)

The herpes simplex virus (HSV) amplicon is a plasmid-based, infectious gene delivery system that carries up to 150 kilobase (kb) of exogenous DNA. We previously characterized early host responses and stability of transgene expression in mice systemically injected with HSV amplicon vectors. Transgene expression was readily detected primarily in the liver but rapidly declined to undetectable levels within 2 weeks. Molecular analyses revealed induction of type I interferons (IFN) as the primary response, and early transcriptional silencing of the vector followed IFN's activation of signal transducers and activators of transcription 1 (STAT1). In this study, we investigate vector administration by stereotactic injection into the striatum. In the brain, induction of type I IFN was rather modest, and transgene expression lasted more than 1 year despite dose-dependent inflammation and infiltration of immune cells around injection sites. Further analyses revealed dose-dependent upregulation of immunosuppressive cytokines and molecular markers specific to regulatory T cells in the injected brain regions, which supported the immune-privileged properties of the brain parenchyma. Overall, our findings indicate that the spectrum of host responses can differ significantly depending on target organs and administrative routes, and that HSV amplicon vectors hold great potential for gene therapy of chronic neurological disorders. The herpes simplex virus (HSV) amplicon is a plasmid-based, infectious gene delivery system that carries up to 150 kilobase (kb) of exogenous DNA. We previously characterized early host responses and stability of transgene expression in mice systemically injected with HSV amplicon vectors. Transgene expression was readily detected primarily in the liver but rapidly declined to undetectable levels within 2 weeks. Molecular analyses revealed induction of type I interferons (IFN) as the primary response, and early transcriptional silencing of the vector followed IFN's activation of signal transducers and activators of transcription 1 (STAT1). In this study, we investigate vector administration by stereotactic injection into the striatum. In the brain, induction of type I IFN was rather modest, and transgene expression lasted more than 1 year despite dose-dependent inflammation and infiltration of immune cells around injection sites. Further analyses revealed dose-dependent upregulation of immunosuppressive cytokines and molecular markers specific to regulatory T cells in the injected brain regions, which supported the immune-privileged properties of the brain parenchyma. Overall, our findings indicate that the spectrum of host responses can differ significantly depending on target organs and administrative routes, and that HSV amplicon vectors hold great potential for gene therapy of chronic neurological disorders. IntroductionThe herpes simplex virus (HSV) amplicon vector holds considerable promise as a gene-transfer vehicle because of its unique features, including (i) a large transgene capacity to accommodate up to 150 kilobase (kb) of exogenous DNA,1Inoue R Moghaddam KA Ranasinghe M Saeki Y Chiocca EA Wade-Martins R Infectious delivery of the 132 kb CDKN2A/CDKN2B genomic DNA region results in correctly spliced gene expression and growth suppression in glioma cells.Gene Ther. 2004; 11: 1195-1204Crossref PubMed Scopus (54) Google Scholar,2Wade-Martins R Saeki Y Chiocca EA Infectious delivery of a 135-kb LDLR genomic locus leads to regulated complementation of low-density lipoprotein receptor deficiency in human cells.Mol Ther. 2003; 7: 604-612Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar,3Wade-Martins R Smith ER Tyminski E Chiocca EA Saeki Y An infectious transfer and expression system for genomic DNA loci in human and mouse cells.Nat Biotechnol. 2001; 19: 1067-1070Crossref PubMed Scopus (156) Google Scholar (ii) the ability to transduce a wide variety of cell types across a broad range of species, (iii) the ease and flexibility of vector construction,4Kasai K Saeki Y DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.Curr Gene Ther. 2006; 6: 303-314Crossref PubMed Scopus (8) Google Scholar (iv) the availability of several hybrid amplicon vectors to achieve extended transgene expression,5Bowers WJ Mastrangelo MA Howard DF Southerland HA Maguire-Zeiss KA Federoff HJ Neuronal precursor-restricted transduction via in utero CNS gene delivery of a novel bipartite HSV amplicon/transposase hybrid vector.Mol Ther. 2006; 13: 580-588Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar,6Heister T Heid I Ackermann M Fraefel C Herpes simplex virus type 1/adeno-associated virus hybrid vectors mediate site-specific integration at the adeno-associated virus preintegration site, AAVS1, on human chromosome 19.J Virol. 2002; 76: 7163-7173Crossref PubMed Scopus (70) Google Scholar,7Savard N Cosset FL Epstein AL Defective herpes simplex virus type 1 vectors harboring gag, pol, and env genes can be used to rescue defective retrovirus vectors.J Virol. 1997; 71: 4111-4117PubMed Google Scholar,8Sena-Esteves M Saeki Y Camp SM Chiocca EA Breakefield XO Single-step conversion of cells to retrovirus vector producers with herpes simplex virus-Epstein-Barr virus hybrid amplicons.J Virol. 1999; 73: 10426-10439PubMed Google Scholar,9Wang S Vos JM A hybrid herpesvirus infectious vector based on Epstein-Barr virus and herpes simplex virus type 1 for gene transfer into human cells in vitro and in vivo.J Virol. 1996; 70: 8422-8430PubMed Google Scholar,10Wang Y Camp SM Niwano M Shen X Bakowska JC Breakefield XO et al.Herpes simplex virus type 1/adeno-associated virus rep(+) hybrid amplicon vector improves the stability of transgene expression in human cells by site-specific integration.J Virol. 2002; 76: 7150-7162Crossref PubMed Scopus (53) Google Scholar and (v) the limited cytotoxicity, which is due to the lack of viral coding sequences and availability of helper virus–free packaging systems.4Kasai K Saeki Y DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.Curr Gene Ther. 2006; 6: 303-314Crossref PubMed Scopus (8) Google Scholar,11Saeki Y Breakefield XO Chiocca EA Improved HSV-1 amplicon packaging system using ICP27-deleted, oversized HSV-1 BAC DNA.Methods Mol Med. 2003; 76: 51-60PubMed Google Scholar,12Saeki Y Fraefel C Ichikawa T Breakefield XO Chiocca EA Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.Mol Ther. 2001; 3: 591-601Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar However, this vector system requires further characterization, particularly with respect to the stability of its transgene expression and its interaction with hosts in vivo.Recently, we investigated the stability of transgene expression and early host responses in mice after systemic delivery of HSV amplicon vectors.13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google Scholar We intravenously injected C57BL/6 mice with a firefly luciferase (Luc)-encoding HSV/Epstein-Barr virus (EBV) hybrid amplicon vector (pREHZCag-Luc) and evaluated the time course of Luc expression and cytokine/chemokine induction. Immediately after vector administration, Luc expression was readily detected, primarily in the liver. However, transgene expression declined rapidly and was undetectable within 2 weeks. We defined the type I interferon (IFN)-signal transducers and activators of transcription 1 (STAT1) signaling as a critical pathway that is responsible for early silencing of the vector-encoded transgene expression. The transient but robust induction of type I IFNs initiated a cascade of immune responses and suppressed vector-encoded transgene expression at the transcriptional level by the activation of STAT1 and probably by the recruitment of histone deacetylases to the vector DNA.The spectrum of host responses may vary substantially depending on the route of vector administration. Olschowka and colleagues reported only modest and self-limiting innate immune responses in the brains of C57BL/6 mice after stereotactic injection of helper virus–free HSV amplicon vectors.14Olschowka JA Bowers WJ Hurley SD Mastrangelo MA Federoff HJ Helper-free HSV-1 amplicons elicit a markedly less robust innate immune response in the CNS.Mol Ther. 2003; 7: 218-227Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar However, they employed only a low dose [4 × 104 transducing units (TU)] of a LacZ-encoding HSV amplicon vector and did not assess the stability of transgene expression. Most published studies using HSV amplicon vectors have presented only short-term transgene expression in vivo, but Geller's group have reported long-term (up to 14 months) transgene expression in rat brains using HSV amplicon vectors carrying tyrosine hydroxylase neurofilament heavy-chain chimeric promoters.15Sun M Kong L Wang X Holmes C Gao Q Zhang GR et al.Coexpression of tyrosine hydroxylase, GTP cyclohydrolase I, aromatic amino acid decarboxylase, and vesicular monoamine transporter 2 from a helper virus-free herpes simplex virus type 1 vector supports high-level, long-term biochemical and behavioral correction of a rat model of Parkinson's disease.Hum Gene Ther. 2004; 15: 1177-1196Crossref PubMed Scopus (77) Google Scholar,16Zhang GR Wang X Yang T Sun M Zhang W Wang Y et al.A tyrosine hydroxylase-neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors.Brain Res Mol Brain Res. 2000; 84: 17-31Crossref PubMed Scopus (49) Google ScholarIn this study, we extended our investigation of the vector–host interaction to a mouse model of stereotactic intrastriatal injection. To evaluate the correlation of host immune responses with HSV amplicon–mediated gene delivery in the brain, we evaluated the induction of cytokines and chemokines, infiltration of immune cells, and stability of the vector-encoded transgene expression in the brains of C57BL/6 mice after stereotactic intrastriatal injection of three different doses of the pREHZCag-Luc amplicon vector. Our data demonstrate that HSV amplicon vectors can support efficient, long-term transgene expression in the brain and that they hold great potential as vectors for gene therapies of neurological disorders.ResultsInduction of cytokines and chemokines in the brains of mice after stereotactic intrastriatal injection of HSV amplicon vectorsFor this study, the pREHZCag-Luc amplicon plasmid13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google Scholar was packaged into infectious HSV virions using the helper-free system.4Kasai K Saeki Y DNA-based methods to prepare helper virus-free herpes amplicon vectors and versatile design of amplicon vector plasmids.Curr Gene Ther. 2006; 6: 303-314Crossref PubMed Scopus (8) Google Scholar,11Saeki Y Breakefield XO Chiocca EA Improved HSV-1 amplicon packaging system using ICP27-deleted, oversized HSV-1 BAC DNA.Methods Mol Med. 2003; 76: 51-60PubMed Google Scholar,12Saeki Y Fraefel C Ichikawa T Breakefield XO Chiocca EA Improved helper virus-free packaging system for HSV amplicon vectors using an ICP27-deleted, oversized HSV-1 DNA in a bacterial artificial chromosome.Mol Ther. 2001; 3: 591-601Abstract Full Text Full Text PDF PubMed Scopus (186) Google Scholar Because HSV plasmid vectors are replicated and packaged as head-to-tail concatemers to form a ∼150-kb vector genome, each vector particle is expected to contain 8–9 copies of the 17.1-kb seed amplicon sequences. To characterize the immune responses to HSV amplicon vectors in the brain, we stereotactically injected 2 × 106 TU of the packaged pREHZCag-Luc vector into the right striata of adult C57BL/6 mice and determined the levels of mRNA coding for various cytokines and chemokines. At various time points, we isolated total RNA from the vector-injected brain hemispheres and subjected the RNA to quantitative real-time reverse transcriptase PCR analysis (Figure 1). Type I IFNs were transcriptionally upregulated as early as 6 hours after vector injection. IFN-α was minimally induced and disappeared within 24 hours, whereas IFN-β was upregulated as much as 30-fold and its expression remained high for at least a day. In our previous study, in which mice were systemically administered the same amplicon vector, type I IFNs were upregulated ∼100-fold in the livers within an hour.13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google Scholar These inconsistent results may be explained well by the absence in the brain parenchyma of plasmacytoid dendritic cells (pDCs), the principal producers of type I IFNs (especially IFN-α) in response to viral stimuli.17Asselin-Paturel C Boonstra A Dalod M Durand I Yessaad N Dezutter-Dambuyant C et al.Mouse type I IFN-producing cells are immature APCs with plasmacytoid morphology.Nat Immunol. 2001; 2: 1144-1150Crossref PubMed Scopus (864) Google Scholar,18McKenna K Beignon AS Bhardwaj N Plasmacytoid dendritic cells: linking innate and adaptive immunity.J Virol. 2005; 79: 17-27Crossref PubMed Scopus (296) Google Scholar An inflammatory cytokine, tumor necrosis factor-α (TNF-α), was the earliest to be induced, was upregulated 30-fold within an hour, and peaked at 50 times its basal level at 12 hours after injection. The level of TNF-α expression gradually decreased within 3 days but was slightly upregulated again at 6 days after injection. Interestingly, the expression profile of TNF-α in saline-injected brains was indistinguishable from that in amplicon-injected brains for the first 12 hours, indicating that tissue damage associated with needle injection was the primary cause of the TNF-α response. The transcriptional profile of interleukin-12 subunit p40 (IL-12p40) was very similar to that of TNF-α. However, its upregulated expression was seen in amplicon-injected brains, but not in saline-injected controls. Upregulation of IFN-γ (as much as 500-fold at 6 days after amplicon injection) suggested infiltration of a considerable number of lymphocytes into the brain parenchyma. The expression of IFN-γ was maintained at the similar level for a week and decreased gradually to as little as 60-fold within a month after injection (data not shown). Expression of IL-4 remained at basal levels at all time points examined. Among CC chemokines, CCL3, CCL4, and CCL5 were upregulated 10- to 40-fold as early as 6–12 hours after injection. IFN-inducible CXC chemokines, such as CXCL9 and CXCL10, were significantly upregulated, 100- to 500-fold, within 12 hours after injection. Many of these chemokines were upregulated again at 6 days after injection, but their transcriptional levels decreased gradually with time to as little as 4- to 30-fold within a month (data not shown). The double-stranded RNA-dependent protein kinase, a key component of the IFN-mediated antiviral defense mechanism, was moderately upregulated as early as 12 hours but returned to baseline within 6 days after injection.To assess the vector dose dependency of the immune responses, we intrastriatally injected mice with saline or one of three different doses (2 × 104, 2 × 105, and 2 × 106 TU) of the pREHZCag-Luc amplicon vector and determined the expression levels of the cytokines and chemokines at 24 and 144 hours after injection (Figure 2). Many of them, especially IFN-γ, IL-12 subunit p40, CCL5, CXCL9, and CXCL10, revealed their clear dependency on the dose of injected amplicon vector. However, the level of TNF-α at 24 hours increased only modestly with a higher dose of the vector, again indicating little contribution of amplicon vectors to the early phase TNF-α induction in the brain parenchyma. T-helper type I (Th1) cytokines IFN-γ and IL-12 were clearly upregulated in a dose-dependent manner, whereas T-helper type II (Th2) cytokine IL-4 showed very modest induction and little dose dependency. These results may suggest that amplicon vector transduction stimulates Th1 rather than Th2 immune response in the brain.Figure 2Dose-dependent induction of cytokines and chemokines in the brain after intrastriatal injection of herpes simplex virus amplicon vectors. We injected C57BL/6 mice in the right striata with saline or one of three different doses [2 × 104, 2 × 105, or 2 × 106 transducing units (TU)] of pREHZCag-Luc amplicon vector. We extracted total RNA from their injected brain hemispheres at (a) 24 hours or (b) 6 days following injection and determined the levels of mRNA for various cytokines and chemokines. Each value was calculated relative to that of untreated mice. Data are presented as mean + SD (n = 4). The experiment was carried out twice with similar results. *P < 0.05 compared with the results from saline-injected mice. **P < 0.05 compared with the results from mice injected with 2 × 104 TU of amplicon vector. ***P < 0.05 compared with the results from mice injected with 2 × 105 TU of amplicon vector. IFN, interferon; IL, interleukin; IL-12p40, IL-12 subunit p40; TNF-α, tumor necrosis factor-α.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To address whether the observed inflammatory responses were mediated by cellular contaminants in the vector preparations versus amplicon-induced transgene expression, we performed an additional experiment using "mock" vector, which contains a similar amount of cellular contaminants but without HSV components (see Materials and Methods for details). Although mock-injected mice did reveal higher levels of cytokines and chemokines (e.g., IFN-γ, TNF-α, CCL5, CXCL9, and CXCL10) compared to saline-injected mice especially at 6 days after injection (Supplementary Figure S1), amplicon-injected mice expressed significantly higher levels of these cytokines and chemokines. This indicated that, while cellular contaminants alone contribute to some of the observed inflammatory responses in injected brain parenchyma, amplicon mediated a significantly higher level of cyctokines and chemokine expression.Adaptive immune responses after stereotactic intrastriatal injection of HSV amplicon vectorsTo verify whether the upregulated expression of the cytokines and chemokines was associated with the recruitment of immune cells, animals were killed 144 hours after injection, and brain sections were analyzed by immunohistochemistry (Figure 3a). CD4- and CD8-expressing lymphocytes were detected only in amplicon-injected brains. Correlation of the number of CD4- and CD8-positive cells with the dose of injected amplicon vector indicated that the infiltration of lymphocytes depends on immune responses and/or transgene expression mediated by the amplicon vector. Chemokine receptor CXCR3 is selective for CXCR chemokines, CXCL9, CXCL10, and CXCL11 (ref. 19Clark-Lewis I Mattioli I Gong JH Loetscher P Structure-function relationship between the human chemokine receptor CXCR3 and its ligands.J Biol Chem. 2003; 278: 289-295Crossref PubMed Scopus (163) Google Scholar), and known to be expressed on natural killer cells,20Inngjerdingen M Damaj B Maghazachi AA Expression and regulation of chemokine receptors in human natural killer cells.Blood. 2001; 97: 367-375Crossref PubMed Scopus (243) Google Scholar Th1 cells,21Crispe IN Hepatic T cells and liver tolerance.Nat Rev Immunol. 2003; 3: 51-62Crossref PubMed Scopus (563) Google Scholar pDCs,22Colonna M Krug A Cella M Interferon-producing cells: on the front line in immune responses against pathogens.Curr Opin Immunol. 2002; 14: 373-379Crossref PubMed Scopus (205) Google Scholar natural killer T cells,23Wilson SB Delovitch TL Janus-like role of regulatory iNKT cells in autoimmune disease and tumour immunity.Nat Rev Immunol. 2003; 3: 211-222Crossref PubMed Scopus (258) Google Scholar and microglia.24Dijkstra IM Hulshof S van der Valk P Boddeke HW Biber K Cutting edge: activity of human adult microglia in response to CC chemokine ligand 21.J Immunol. 2004; 172: 2744-2747Crossref PubMed Scopus (45) Google Scholar CXCR3 immunoreactivity was relatively low and diffusely distributed in untreated brains. Accumulation of CXCR3-positive cells was evident around the injection site of brains injected with either saline or amplicon. The intensity and density of CXCR3 immunoreactivity as well as the transcriptional level of CXCR3 mRNA in the brain increased proportionately to dose of injected amplicon vector, which suggested the possible responsibility of upregulated CXCL chemokines for recruiting these immune cells to the vector-injected brain regions (Figure 3b).Figure 3Adaptive immune responses to herpes simplex virus amplicon vectors. We intrastriatally injected C57BL/6 mice with saline or three different doses [2 × 104, 2 × 105, or 2 × 106 transducing units (TU)] of pREHZCag-Luc amplicon vector. We killed animals 6 days after injection and subjected their brain samples to immunohistochemical staining (a) using antibodies against CD4, CD8, and CXCR3. Bars = 50 μm. (b) We isolated total RNA from their injected brain hemispheres 6 days following vector injection and determined the levels of mRNA for CXCR3 by real-time reverse transcriptase PCR analysis. Data are presented as mean + SD (n = 4). The experiment was carried out twice with similar results. *P < 0.01 compared with the results from untreated control mice. **P < 0.05 compared with the results from saline-injected mice. ***P < 0.0001 compared with the results from mice injected with 2 × 104 TU of amplicon vector. ****P < 0.005 compared with the results from mice injected with 2 × 105 TU of amplicon vector.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To evaluate systemic humoral responses to HSV amplicon vectors injected intracranially, we determined the titer of anti-HSV neutralizing antibodies. We took serum from each mouse 144 hours after injection, serially diluted serum samples in twofold steps, and incubated them for an hour at 4 °C with 104 TU of the pREHZCag-Luc amplicon vector. We then inoculated the serum–vector mixtures into G16-9 cells and determined the remaining titer (LacZ TU) for each sample. We detected no neutralizing activity in the animals receiving lower doses (2 × 104 and 2 × 105 TU) but noted a slight increase (up to twofold) in the neutralizing titer in the animals with the highest dose (2 × 106 TU). These results indicated that a threshold dose of the amplicon vector may be required to trigger the systemic humoral response. We confirmed that this neutralizing activity was specific to HSV amplicon vectors by comparing the neutralizing activity of serum samples taken from mice injected with an equivalent dose of mock vector. For comparison, we also determined response to neutralizing antibody in mice intravenously injected with 1 × 107 TU of the pREHZCag-Luc amplicon vector. A moderate increase in the neutralizing antibody titer (32-fold) was evidenced by 7 days after vector injection.Transgene expression in the brains of mice intrastriatally injected with HSV amplicon vectorsTo delineate the effects of the host responses on amplicon-mediated transgene expression, we evaluated the copy number of the Luc-encoding vector DNA and the amount of Luc mRNA expressed under the control of the constitutive CAG promoter25Niwa H Yamamura K Miyazaki J Efficient selection for high-expression transfectants with a novel eukaryotic vector.Gene. 1991; 108: 193-199Crossref PubMed Scopus (4550) Google Scholar during the first 4 weeks following vector injection. At five different time points, we isolated total DNA and RNA from the brains injected with three different doses of the amplicon vector. The time-course patterns of vector DNA copies were similar with all three doses, decreasing two- to threefold within the first 24 hours and remaining relatively stable until the end of the 4 weeks (Figure 4a). Considering the tenfold reduction within the first 24 hours in the liver,13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google Scholar clearance of the vector DNA was remarkably low in the brain. These results could suggest that differential mechanisms and/or machineries may be involved in the degradation of the foreign DNA in different tissues and organs. Figure 4b represents the time course of Luc expression in the brain. With a dose of 2 × 105 or 2 × 106 TU, the transcriptional level of Luc peaked at 24 hours and gradually decreased over the following 2 weeks (two- to threefold). In contrast, when the mice were injected with 2 × 104 TU of the vector, Luc expression peaked at 6 days after injection and decreased slightly thereafter. The transcriptional level of the LacZ gene, which is expressed under the control of the viral immediate early (IE) 4/5 promoter, was also determined. Similar to what we found previously in the systemic delivery model,13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google Scholar the level of IE4/5 promoter–driven LacZ mRNA declined drastically during the first 6 days (Supplementary Figure S2). These results indicate that the IE4/5 promoter could become inactivated rather quickly regardless of transduced organs or cell types. We calculated the ratios of Luc mRNA to Luc-encoding vector DNA and plotted them at each time point (Figure 4c). With all doses, the RNA-to-DNA ratio steeply increased during the first 24 hours and remained nearly constant until the end of the 4 weeks. Interestingly, the ratio was slightly but consistently higher with a lower vector dose during the constant phase from 6 to 27 days after injection. This may be associated with the dose-dependent upregulation of cytokine expression and immune cell infiltration in the brain. Indeed, in our previous study of systemic amplicon delivery, induction of cytokines, particularly type I IFNs, and activation of STAT1 signaling were critical in transcriptional silencing of the amplicon-encoded transgene both in vitro and in vivo.13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google ScholarFigure 4Time course of vector DNA copy numbers, firefly luciferase (Luc) mRNA expression, and their ratios in the brains of mice injected with amplicon vectors. We injected C57BL/6 mice in the right striata with 2 × 104 (closed triangle), 2 × 105 (open circle), or 2 × 106 (closed circle) transducing units (TU) of pREHZCag-Luc amplicon vector. We killed animals at five different time points (2 hours and 1, 6, 13, or 27 days after injection); determined the copy numbers of (a) Luc-encoding vector DNA and (b) Luc mRNA transcripts in their injected brain hemispheres by real-time PCR analyses; and (c) calculated the ratio of Luc mRNA to Luc-encoding vector DNA. Data are presented as mean + SD (n = 4). The experiment was carried out twice with similar results.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Stable long-term Luc expression after intrastriatal injection of HSV amplicon vectorsOver the course of a year, using a NightOWL LB 981 bioluminescence imaging system (Berthold Technologies, Oak Ridge, TN), we monitored C57BL/6 mice intrastriatally injected with 2 × 106 TU of the pREHZCag-Luc amplicon vector for Luc expression (Figure 5a). Notably, even at day 385, we detected significant Luc activity in the brain injected with the vector. Three animals were killed at each of four time points (1, 27, 100, and 385 days after injection). We isolated total DNA and RNA from the injected brain hemispheres and determined the copy numbers of Luc-encoding vector DNA and Luc mRNA in each sample by real-time PCR analysis (Figure 5b and c). The total Luc gene copy number in the input dose of the vector was also determined as a reference. Approximately 40% of the input vector DNA was recovered from day 1 through 27 after injection. The copy number of the vector DNA decreased slightly by 100 days and reached ∼10% of the original input by 385 days after injection. The transcriptional level of the Luc transgene also decreased gradually, similar to what was seen with Luc activity. The ratio of Luc mRNA to vector DNA seemed stable throughout this period, indicating stable transcriptional activity of the amplicon vector–encoded Luc transgene delivered directly into the brain tissues. This contrasted with systemic amplicon delivery, wherein Luc expression in the liver drastically declined to undetectable levels within 15 days.13Suzuki M Chiocca EA Saeki Y Early STAT1 activation after systemic delivery of HSV amplicon vectors suppresses transcription of the vector-encoded transgene. 2007; (Mol Ther 15: 2017–2026.)Google