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. 2010 Feb 23;18(5):987–992. doi: 10.1038/mt.2010.25

Screening of Complement Inhibitors: Shielded Baculoviruses Increase the Safety and Efficacy of Gene Delivery

Minna U Kaikkonen 1,2, Antti I Maatta 1, Seppo Ylä-Herttuala 1,3,4, Kari J Airenne 1
PMCID: PMC2890102  PMID: 20179675

Abstract

One of the major obstacles in the use of baculovirus vectors for in vivo gene transfer is the virus inactivation by serum complement. In this study, we investigated the effect of decay-accelerating factor (DAF), factor H (FH)–like protein-1 (FHL-1), C4b-binding protein (C4BP), and membrane cofactor protein (MCP) on protection of baculovirus vectors from the complement-mediated inactivation. Complement regulatory proteins were displayed on baculovirus surface as fusions to membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein. This strategy resulted in abundant expression of recombinant proteins on the viral envelope while viral titers comparable to control virus were reached. The surface-modified vectors exhibited complement resistance in vitro, DAF showing the highest level of protection. Intraportal delivery of DAF-displaying baculovirus resulted in increased survival and enhanced gene expression in immunocompetent mice. Mice receiving DAF-displaying baculovirus also exhibited lower level of liver inflammation as evidenced by aspartate aminotransferase (AST). In line with this, macrophages treated with DAF baculovirus produced lower levels of inflammatory cytokines IL-1β, IL-6, and IL-12p40 compared to control virus. These results suggest that DAF-display can protect the vector against complement inactivation but also reduce complement-mediated inflammation injury. In conclusion, complement shielded baculovirus vectors represent attractive tools for effective in vivo gene delivery.

Introduction

Baculoviruses are gaining popularity as potential vectors for gene transfer technology.1 The appeal of the system lies in its capacity to carry large DNA inserts, low pathogenicity and toxicity, and ease of production of high virus titers. Several recent studies have implicated the potential therapeutic use of baculoviruses for the treatment of cancer2 and in tissue engineering of bone3 and cartilage.4

Considerable progress has been made in elucidating the biology of baculovirus vectors, but limitations regarding the efficacy of these vectors in vivo have slowed their widespread applications in the field of gene therapy. One major obstacle is the inactivation of baculovirus by serum complement.5 Strategies to inactivate the complement system at the time of transduction,5,6,7 to deliver viruses into immunoprivileged areas,8,9,10 and to use methods that minimize the exposure to the complement11,12 have been pursued to overcome the inactivation problem. Yet in another approach, Hüser et al. created a complement-resistant baculovirus vector by displaying a decay-accelerating factor (DAF) fused to the baculovirus envelope protein gp64 (ref. 13). However, the DAF-GP64 vector production resulted in low titers.13,14

Regulation of complement activation is mediated by a family of complement receptor and regulatory proteins, including DAF (CD55),15 C4b-binding protein (C4BP),16 factor H (FH),17 FH-like protein-1 (FHL-1),18 complement receptor 1 (CR1; CD35), and membrane cofactor protein (MCP; CD46).15 They function by acting as cofactors for the factor I–dependent cleavage of C3b or by the decay of the complement pathway convertases. Of these factors, we investigated the effect of DAF, MCP, FHL-1, and C4BP on the protection of baculovirus vectors from the complement-mediated inactivation. To overcome the problem of low viral titers often associated with gp64-based fusion proteins, these regulators of the complement cascade were fused to membrane anchor of the vesicular stomatitis virus-G (VSV-G) protein. This strategy resulted in abundant expression of recombinant proteins on the viral envelope while viral titers comparable to control virus [1010 to 1011 plaque-forming units (PFUs)/ml] were reached. The surface-modified vectors exhibited complement resistance in vitro, DAF-VSV-GED showing the highest level of protection. Finally, we demonstrated the utility of DAF-display in rescuing mice from experimentally lethal doses of vector (5 × 108 to 2.5 × 109 PFUs) while enabling efficient gene expression after portal vein injection. These novel baculovirus vectors represent a means to surmount the inactivation by the complement that previously has hampered their effective use in gene therapy applications.

Results

Generation and characterization of complement-resistant baculoviruses

To allow for efficient display of complement regulatory proteins on the baculovirus surface without compromising viral titers, the proteins were fused to the 21-amino acid membrane anchor of VSV-G (VSV-GED, EctoDomain).19 Similarly to previous studies, DAF protein deprived of its membrane anchor,13,20 the smallest functional fragments of FHL-1 (SCR 1–4),21 and C4BP (CCP1–5)22 were chosen as fusion partners. A soluble fragment containing SCR domains 1–4 and the serine/threonine-rich (STP domain) region where heavy O-glycosylation occurs15 was used to produce an MCP-fusion protein. To achieve codisplay of several complement regulatory proteins on baculovirus surface, an equal amount of PFUs of the secondary viruses displaying DAF, FHL-1, C4BP, or MCP fused to VSV-GED was used to co-infect Sf9 cells. Vectors codisplaying DAF+C4BP, DAF+FHL-1+C4BP, and DAF+MCP were created to study the synergistic effects of these proteins (codisplay indicated by +).

To analyze the incorporation of complement regulatory proteins on the baculovirus envelope, the gradient-purified viruses were probed with VSV-G antibody recognizing a luminal domain of VSV-G (Figure 1). The correct-sized FHL-1 and C4BP fusion proteins (37 and 46 kd) were detected in the respective virus concentrates. The size of DAF and MCP fusion proteins did not correspond to expected size of 44 kd, but higher molecular weight products of ~64 and ~55 kd were detected, respectively. This is most likely due to extensive post-translational modifications, e.g., N- and O-linked glycosylation.23,24

Figure 1.

Figure 1

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Immunoblot analysis of the complement-resistant baculoviruses. Display of DAF (~64 kd), FHL-1 (37 kd), C4BP (46 kd), and MCP (~55 kd) fusion proteins on the surface of gradient-purified viruses was detected using anti-VSV-G antibody. Codisplay of DAF+C4BP, DAF+FHL-1+ C4BP, and DAF+MCP was achieved by co-infection of Sf9 cells with equal amounts of plaque-forming units of the respective secondary viral preparations. The nonsurface-modified control (Ctrl) virus was used as a negative control for staining. C4BP, C4b-binding protein; DAF, decay-accelerating factor; FHL-1, factor H like protein-1; MCP, membrane cofactor protein.

The secondary virus preparations (30 ml) exhibited titers ranging from typical 3.1 × 107 to 1.2 × 109 PFUs/ml, whereas titers ranging from 3.0 × 1010 to 1.1 × 1011 PFUs/ml were reached after 500-fold concentration of the large-scale productions (500 ml) for the surface-modified viruses. This suggested that no adverse effects were associated with foreign protein incorporation on the virus envelope.

Complement resistance of surface-modified baculoviruses in vitro

To investigate the stability of the surface-modified baculovirus vectors, we determined the vector survival after preincubation with native serum from mouse, rat, and human (Figure 2). As baculovirus pseudotyping per se has been shown to exhibit greater resistance to animal serum inactivation compared to the unmodified baculovirus, VSV-G and VSV-GED pseudotyped viruses were included in the study.25 Viruses were incubated with untreated or heat-inactivated serum for 1 hour, and residual infectivity was determined following transduction of HepG2 cells. The vector survival was determined as the percentage of β-galactosidase activity resulting from vector incubation with untreated compared with heat-treated sera.

Figure 2.

Figure 2

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Transduction efficiency of baculovirus vectors in the presence of complement. Stability of recombinant baculoviruses in the presence of (a) mouse, (b) rat, and (c) human serum. The vector survival was determined as the percentage of LacZ enzyme activity resulting from vector preincubation with nonheat-inactivated sera compared with heat-inactivated sera. Error bars correspond to the SD of 3–5 independent transductions. The data from the surface-modified baculoviruses were compared to the control virus using one-way analysis of variance followed by Dunnett's multiple comparison test. *P < 0.05, **P < 0.01. C4BP, C4b-binding protein; DAF, decay-accelerating factor; FHL-1, factor H like protein-1; MCP, membrane cofactor protein.

In line with previous studies, human and rat serum resulted in significant reductions in the transduction efficiency of the nonsurface-modified control virus (~1%), whereas mouse (14%) and rabbit serum (data not shown) possessed weaker inactivation effects.11,25 The virus inactivation was rapid with a major loss in survival during the first 15 minutes (data not shown). Almost all the surface-modified baculoviruses exhibited an increased survival in mouse serum compared to nonsurface-modified control virus (Figure 2a). In rat serum, DAF increased the vector survival by over tenfold, whereas C4BP, MCP, and DAF+MCP resulted in four- to fivefold increase (Figure 2b). In human serum, however, only DAF and DAF+C4BP showed significant serum resistance, increasing the transduction efficacy by 18- and 13-fold, respectively. Based on these results, DAF was chosen for in vivo studies (Figure 2c).

Enhanced survival of mice receiving DAF-displaying baculovirus

Previous studies have shown substantial toxicity associated with portal administration of baculovirus that can, in part, be reversed by soluble complement receptor 1 (sCR1).7 To study whether DAF-display would translate into similar protecting effect, we injected increasing doses of viruses to portal vein of C57Bl6 mice (5 × 108, 1 × 109, or 2.5 × 109 PFUs). The results showed that irrespective of the dose, only 30% of the animals receiving the nonsurface-modified control virus survived, whereas the survival was increased from 30 to 100% toward the lower dosage when DAF-displaying vector was used (Table 1). The animals that are deceased lived for no longer than 24–48 hours after delivery.

Table 1.

Survival and clinical chemistry values of mice receiving three experimental doses of nonsurface-modified control virus or DAF-displaying baculovirus by intraportal administration

graphic file with name mt201025t1.jpg

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Clinical chemistry was used to study the effects of intraportal delivery of baculovirus on C-reactive protein, lactate dehydrogenase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine. No significant differences were seen in the C-reactive protein and creatinine values between the serum samples taken before vector delivery and 5 days after gene transfer. AST was found to be significantly increased in both groups receiving the high dose of 2.5 × 109 PFUs and in the control virus group receiving 1 × 109 PFUs (Table 1). In these animals, also signs of liver necrosis were detected (data not shown). However, no alterations in ALT or lactate dehydrogenase were seen in any of the groups (Table 1).

To investigate induction of the early inflammatory-based events involving innate immunity, we studied the mRNA expression of proinflammatory cytokines in mouse macrophage cell line, RAW 264.7, 6 hours after baculovirus inoculation. The results showed a minor reduction of IL-6 and IL-12p40 mRNA induction, 1.2- and 1.3-fold, respectively, in cells treated with DAF compared to the nonsurface-modified control virus (Figure 3). However, a more notable fivefold reduction was seen in IL-β mRNA induction after DAF inoculation compared to control (Figure 3). Differences in inflammatory cytokine levels were significant between the viruses when the virus amount was equalized to protein amount (20 µg/ml; Figure 3) or PFUs (2 × 108 PFUs/ml; data not shown). No significant differences were detected in the mRNA levels of TNF-α and IFN-α between the virus-treated cells (data not shown).

Figure 3.

Figure 3

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Induction of IL-6, IL-12p40, and IL-β mRNA levels in RAW 264.7 cells after 6-hour treatment with DAF or nonsurface-modified control baculovirus (20 µg/ml). Data represent mean fold changes in relative mRNA expression after virus treatment compared to the nontreated cells ± SD of three independent experiments. The data from the DAF-displaying baculovirus was compared to the control virus using Student's t-test. *P < 0.05, ***P < 0.001. DAF, decay-accelerating factor.

Gene transfer efficiency in the presence of complement in vivo

Next, we evaluated the gene transfer efficacy from the liver tissues after portal vein injection. The highest level of β-galactosidase expression was detected after injection of 2.5 × 109 PFUs of DAF-displaying baculovirus when blue cells were found evenly distributed throughout the liver tissue (Figure 4a). Positive cells were also found at the endothelial lining of blood vessels (Figure 4b). Only a few positive cells were detected with the highest dose of nonsurface-modified control virus (Figure 4c). At the intermediate dose (1 × 109 PFUs), only few positive cells were detected in both study groups making quantitative comparisons difficult (Figure 4d,e). Therefore, quantitative measurements of the β-galactosidase activity were performed from the liver lysates of the animals receiving 5 × 108 to 1 × 109 PFUs. DAF-displaying virus exhibited a fourfold higher enzyme levels compared to the control virus that showed levels close to the background values measured from animals receiving intraportal injection of saline (Figure 4f). Very few LacZ-expressing cells were detected with the lowest viral dose used, and the values for β-galactosidase activity were close to the background enzyme activity in both study groups preventing further comparisons (Figure 4f).

Figure 4.

Figure 4

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Expression of nuclear-localized LacZ in the liver sections of mice receiving 100 µl of baculovirus vector intraportally. Five days after gene transfer, the organs were harvested and assayed for β-galactosidase activity using X-gal staining. After injection, 2.5 × 109 PFUs of DAF-displaying baculovirus LacZ expression was seen (a) uniformly distributed over the liver tissue and (b) at the epithelial lining of the blood vessels. (c) High dose of nonsurface-modified control virus (2.5 × 109 PFUs) resulted in only single blue cells around the tissue sections. After intermediate doses (1 × 109 PFUs) of (d) DAF baculovirus and (e) control virus, only rare blue cells were seen. LacZ+ cells are indicated by arrowheads. Original magnification ×60-fold. (f) β-galactosidase enzyme activity measured from the liver lysates of animals receiving 1 × 109 or 5 × 108 PFUs of DAF-displaying vector or nonsurface-modified control virus. The background represents enzyme activity measured from mice receiving intraportal injection of saline. Means ± SD. DAF, decay-accelerating factor; PFU, plaque-forming unit.

Discussion

Previous studies have shown that baculoviral gene transfer is strongly reduced in the presence of serum complement.5 One of the most prominent strategies to overcome this is to modify the surface of the virions recognized by the factors of the complement cascade. Accordingly, VSV-G pseudotyping is suggested to confer greater resistance to animal serum inactivation compared to the unmodified control baculovirus.25 Barsoum et al. hypothesized that VSV-G-pseudotyped baculovirus conferred resistance to complement, imparting the ability to perform gene transduction into mouse hepatocytes following tail vein injection.26 Pieroni et al. demonstrated increased gene delivery into mouse quadriceps after direct intramuscular injection of VSV-G-modified baculovirus, partially bypassing the complement system.27 In further support of this idea, we observed the complement-protective effect of VSV-G and VSV-GED pseudotyping in mouse serum. However, in line with previous results, only minor vector survival was seen after preincubation with rat and human serum, highlighting the importance of alternative strategies.25

Viruses, bacteria, and parasites have developed many efficient strategies to avoid clearance and destruction by complement. Several enveloped viruses, such as human immunodeficiency virus, passively sequester DAF and MCP into the envelopes of newly emerging viruses, thereby inhibiting the host's ability to regulate complement.28,29 C4BP and FHL-1 on the other hand are captured by several bacterial pathogens, such as Streptococcus pyogenes, subsequently leading to downregulation of complement activation.30,31 To test the ability of these complement regulators in conferring resistance to baculovirus vectors, the functional domains of DAF, FHL-1, C4BP, and MCP were fused to VSV-GED and displayed on viral surface. In fact, the sequence of FHL-1 (CCP1-5) is identical to the nucleotide sequence of FH, except for the 12 last nucleotides, thus allowing extrapolation of the result to FH.18 CR1 was excluded from the study due to its big molecular weight of over 200 kd.

The activation of the classical pathway of the complement system is thought to be the major cause for baculovirus inactivation in human serum.5 However, a more recent study has suggested that both alternative and classical pathways are important, the latter being mediated by the binding of naturally occurring IgM molecules to baculovirus envelope.7 Of the complement regulatory proteins studied, DAF and MCP act on both pathways either by promoting the decay of the C3-convertases on the complement cascade (DAF)32 or by catalyzing the permanent inactivation of C3-convertases via proteolytic cleavage by factor I.33 FHL-1 and C4BP, on the other hand, possess both of these activities but function only on one of the pathways, FHL-1 on the alternative pathway and C4BP on classical pathway, respectively.16,18 Based on our results, only DAF confers protection to the human complement in vitro, thus preventing elucidation of the role of different complement pathways on baculovirus inactivation. Previous studies done with lentiviral vectors have also evidenced DAF as the most effective in complement protection compared to MCP or CD59 (ref. 34). Combination of DAF with FHL-1, C4BP, and MCP did not confer synergism in complement resistance, but rather the serum stability was decreased when the amount of DAF displayed on the baculovirus surface was diminished. However, our results might suggest that membrane-associated complement regulatory proteins, such as DAF and MCP, may function better in protecting baculovirus from complement injury, than the naturally secreted proteins FHL-1 and C4BP.35

Activation of complement and Toll-like receptor (TLR) systems results in the production of several biologically active molecules that contribute to inflammation. In this study, no acute inflammation or trauma to kidneys was seen as indicated by C-reactive protein and creatinine values. However, increase in AST levels was detected with high concentrations of the vector indicating liver toxicity or inflammation. This was not accompanied by increases in ALT and lactate dehydrogenase suggesting that AST might be the best factor for predicting inflammation.36 With DAF-displaying baculovirus, AST levels were normal even at virus dose of 1 × 109 PFUs, whereas with the control virus, a further decrease to 5 × 108 PFUs was needed. This is in line with previous studies where no activation of hepatic enzymes was seen using viral dose of 1 × 108 PFUs.37 Strikingly, even at the lowest dose studied, the control virus showed only 30% survival in contrast to 100% of the DAF-displaying baculovirus. In addition, DAF-display allowed more efficient transduction of liver cells. Similar effect on animal survival and transgene expression has previously been shown in a study using coadministration of soluble complement inhibitor sCR1 while injecting baculovirus vector to mouse portal vein.7

Moreover, our results lead us to speculate a central role for DAF in protecting mice from complement-mediated inflammatory injury. In fact, there is mounting evidence of the role of DAF in modulating the interaction between TLR signaling and complement. In these studies, the deletion of DAF in mice was shown to render the animals more susceptible to complement-mediated injury.38,39 In addition to activating the classical complement pathway, baculovirus has also been shown to stimulate immune cells via TLR9/MyD88-dependent signaling pathway leading to induction of inflammatory cytokines IL-6, TNF-α, and IL-12 (ref. 40). According to recent study, these cytokines are also induced in whole human blood.41 A recent report has shown that DAF was able to prevent TLR ligand–induced complement activation in vivo, thus leading to the production of lower levels of inflammatory cytokines, such as IL-6, TNF-α, and IL-1β.42 In the same study, DAF was also shown to play a role in TLR9 by affecting the production of IL-12. This hypothesis was strongly supported by our data, showing that macrophages treated with DAF exhibit lower levels of IL-1β, IL-6, and IL-12p40 mRNA compared to the control virus. Whether C4BP, FHL-1, and MCP also decrease the TLR ligand–induced complement activation should also be studied even though their efficacy in complement resistance in vitro was not as prominent as for DAF.

Previously, a recombinant baculovirus-displaying DAF fused to the baculovirus major envelope glycoprotein gp64 was constructed in an effort to achieve protection against complement.13 This modification allowed 22 and 40% protection against rat and human sera, respectively. This is in line with the 12 and 28 survival percentages seen in our study, but dissimilarities in the experimental setup preclude direct comparison of the data. However, the efficacy of this vector was only shown in neonatal mice that share an immature immune system. Also, only low titers of these viruses were achieved even after final purification (108 PFUs/ml) inhibiting studies in bigger animals. In our study, we show for the first time the efficacy of a complement shielded virus in an immunocompetent animal while demonstrating no compromise in viral titers. Moreover, our results suggest that DAF can reduce the inflammatory injury mediated by complement and TLR signaling. These novel vectors might prove broadly useful for gene therapy applications to tissues requiring high vector exposure to the complement and further encourage the development of baculovirus vectors with reduced innate immune response.

Materials and Methods

Generation of the recombinant baculoviruses. Each fragment of the complement regulatory proteins was cloned to a PstI-site on the plasmid previously used to display VSV-G EctoDomain (VSV-GED) on baculovirus surface under the control of polyhedrin promoter.19 Gene encoding DAF was amplified by PCR from the DNA obtained from a sequence-verified I.M.A.G.E. cDNA (#3460621; Geneservice, Cambridge, UK) using primers Fwd-5′-ATACTGCAGGACTGTGGCCTTCCCCCAGATGTACC-3′ and Rev-5′-ATACTGCAGACCTGAAGTGGTTCCACTTCCTTTATTTGG-3′. FHL-1 functional short consensus repeat domains (SCR) 1–4 were amplified from cDNA extracted from human lung fibroblasts (MRC-5) using the primers Fwd-5′-TTCTCTGCAGGAAGATTGCAATGAACTTCCTCCAAG-3′ and Rev-5′-TTTTCTGCAGTTTTTCTTCACATGAAGGCAACG-3′. Complement control domains (CCP) 1–5 of C4BP were amplified from cDNA extracted from human hepatoma cells (HepG2) using primers Fwd-5′-TTTTCTGCAGAATTGTGGTCCTCCACCCAC-3′ and Rev-5′-TTTTCTGCAGGCCGCCCGCCTCACATCCTTGGTATGG-3′. To obtain soluble recombinant human MCP, a DNA fragment comprising the extracellular region was amplified from MRC-5 cells by PCR using primers Fwd-5′-CTGCAGTGTGAGGAGCCACCAACA-3′ and Rev-5′-CTGCAGTATTCCTTCCTCAGGT-3′. The VSV-G-displaying baculovirus was cloned by replacing VSV-GED with VSV-G using primers Fwd-5′-GGAAGTTCACCATAGTTTTTCCAC-3′ and Rev-5′-AGATCTTTACTTTCCAAGTCGGTTCA-3′. Transfer vector used to produce the nonsurface-modified control virus (Ctrl) was deleted of genes under polyhedrin promoter. All the baculovirus transfer vectors contained nuclear-targeted LacZ reporter gene under cytomegalovirus promoter19 and were verified by sequencing.

The production of recombinant baculoviruses was performed using Bac-to-Bac baculovirus expression system (Invitrogen, Carlsbad, CA). Production of primary (2 ml) and secondary stocks (30 ml) of viruses was followed by a large-scale production (500 ml) that was further concentrated as described earlier12 and analyzed for lipopolysaccharide and bacteriological contaminants. For codisplay of complement factors, an equal amount of plaque-forming units (PFUs) of the secondary virus stocks in 1 ml was used to infect Sf9 cells in large-scale production flask.

SDS-PAGE and immunoblot analysis. Gradient-purified viruses were diluted in reducing sample buffer and denaturated at 95 °C for 5 minutes prior to SDS-PAGE and immunoblotting. Samples were subjected to 12.5% SDS-PAGE, blocked with 5% BSA-TBS, transferred onto a nitrocellulose membrane (Trans-Blot; Bio-Rad, Hercules, CA) and probed with rabbit anti-VSV-G (Bethyl Laboratories, Montgomery, TX). The primary antibody was detected with alkaline phosphatase–conjugated secondary antibody (1:2,000; Bio-Rad), followed by a color reaction (NBT/BCIP; Roche, Basel, Switzerland).

Complement assay. HepG2 cells were seeded at 50,000 cells per well on 24-well plates in their recommended medium. After 24 hours, the medium was removed and fresh medium containing serum-virus dilutions was added. Purified baculoviruses representing a multiplicity of infection of 400 were diluted 1:2 with native sera from human, rabbit, rat, and mouse (Sigma-Aldrich, St Louis, MO) in a total of 30 µl and incubated at 37 °C for 1 hour before transduction. The corresponding treatment with heat-inactivated sera served as a control (56 °C, 1 hour). After 48-hour incubation at 37 °C, 5% CO2, cells were trypsinized, lysed and analyzed for β-galactosidase activity according to the luminescent β-galactosidase enzyme assay (Clontech-Takara Bio Europe, Saint-Germain-en-Laye, France) manual. The luminescence was measured with black luminometer 96-well plates (Black Isoplate TC; Wallac, Turku, Finland) and Victor2 luminometer (Wallac). Coomassie Plus protein assay (Bio-Rad) was used to equalize the protein amounts from lysed cell samples according to the manufacturer's instructions.

Portal vein injections to mice. Male C57Bl/6J mice, 8- to 10-week-old, were anesthetized intraperitoneally with a combination of Rompun (Orion Pharma, Espoo, Finland) and Ketalar combination (Pfizer Animal Health, Espoo, Finland). Vectors were injected into the portal vein in a total volume of 100 µl using a 30 G needle. Five days after gene transfer, the animals were killed, and liver samples were collected. The liver samples were processed for LacZ analysis, as described below. Blood samples were collected at the baseline and at the time of killing for clinical chemistry.

Analysis of inflammatory cytokine mRNA levels from macrophages. RAW 264.7 were seeded in 24-well plates (0.5 × 106 cells/well). After 24 hours, RAW cells were inoculated with baculoviruses (20 µg/ml) for 6 hours. Total RNA was isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany), and cDNA synthesis was perfomed using SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). SYBR Green (Applied Biosystems, Foster City, CA) RT-PCR was used to quantify the levels of mouse IL-6, IL-12p40, and IL-1β using primers obtained from the Primer Bank (http://pga.mgh.harvard.edu/primerbank/). Samples were amplified in ABI PRISM 7500 Real-Time PCR System (Applied Biosystems) under the following conditions: 10 minutes at 95 °C, followed by 40 cycles of 15 seconds at 95 °C, 15 seconds at 60 °C, and 30 seconds at 72 °C. Input amounts of cDNAs were corrected by amplification of the GAPDH mRNA. Ratios of target gene and GAPDH expression (relative gene expression) were calculated. The fold changes in mRNA expression were obtained by dividing the relative gene expression levels of AcMNPV-treated cells with that of control cells with no treatment.

Analysis of LacZ expression from liver. Tissue samples (2–3 samples/animal/tissue) were homogenized using the Precellys 24 bead grinder homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). The tissue samples were transferred to a tube containing Precellys ceramic beads (Bertin Technologies) and 1 ml of T-PER Tissue Protein Extraction Reagent (Pierce, Rockford, IL) containing Halt Protease Inhibitor Cocktail (Pierce). The tissues were homogenized using a protocol of 5,000 rpm, 2 × 50 seconds, 20-second break. β-galactosidase activity was determined according to the luminescent β-galactosidase enzyme assay as described above.

For histological analysis, frozen liver tissues were embedded in Tissue-Tek O.C.T. Compound (Electron Microscopy Sciences, Fort Washington, PA) and cut to 7 µm cryosections. The LacZ expression was analyzed as described.9

Statistical analysis. Statistical analyses were performed by GraphPad Prism 5 (GraphPad Software, San Diego, CA). The data were analyzed using Student's t-test or one-way analysis of variance followed by Dunnett's post hoc test. The α-level for significance level was set as P value <0.05.

Acknowledgments

We thank Tarja Taskinen, Joonas Malinen, and Jaana Siponen for excellent technical assistance. We are grateful to Jere Pikkarainen, Taina Vuorio, and Svetlana Laidinen for the help with animal work. We also thank Emilia Makkonen for her valuable help in virus characterization. This work was supported by Ark Therapeutics Ltd and EU Baculogenes program (LHSBCT-2006-037541).

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