Recombinant Autographa californica multiple nuclear polyhedrosis viruses (AcMNPV) have recently been shown to transduce mammalian cells in vitro. Since baculoviruses offer many advantages over viruses currently used in gene therapy, we have tested them for in vivo gene transfer by constructing a baculovirus bearing a nuclear targeted β-galactosidase marker gene (LacZ) under a CMV promoter. Both rabbit aortic smooth muscle cells (RAASMC) and human ECV-304 cells were susceptible to LacZ-baculovirus transduction. Transgene expression was evaluated in vivo by applying 1 × 109 p.f.u. of LacZ-baculoviruses or LacZ-adenoviruses in a silastic collar placed around rabbit carotid arteries in the absence of contact with blood components. As a result, baculoviruses led to transgene expression in adventitial cells in rabbit carotid arteries with efficiency comparable to adenoviruses. The β-galactosidase gene expression was transient staying at a high level for 1 week but disappearing at the 14 day time-point. The arterial structure and endothelium remained intact in the baculovirus-transduced arteries, but macrophage-specific immunostaining detected signs of inflammation comparable to adenoviruses. Baculoviruses are thus able to mediate transient gene transfer in vivo and may become useful tools for gene therapy.
Efficient gene transfer would be a beneficial tool for the treatment of vascular diseases, such as post-angioplasty restenosis, post-bypass atherosclerosis, peripheral atherosclerotic disease, stenosis of vascular prosthesis anastomoses, and thrombus formation12 and a number of different techniques have been developed for this purpose.134567 Periadventitial collars have been used successfully for arterial gene transfer with several vectors and appear to be a useful route for arterial gene transfer during vascular surgery.28 However, there is a continuous need for more facile and efficient gene transfer vectors. In most cases, only a temporary expression of the transgene will be required to achieve a beneficial biological effect in cardiovascular applications.13
Baculoviruses have long been used as biopesticides9 and as tools for efficient recombinant protein production in insect cells.10 They are generally regarded as safe due to the naturally high species specificity and because they are not known to propagate in any non-invertebrate host.11 Although the virions have been shown to enter certain cell lines derived from vertebrate species, no evidence of viral gene expression has been detected using natural viruses.12131415 However, several groups have recently found that the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), containing an appropriate eukaryotic promoter, is able to transfer and express target genes efficiently in several mammalian cell types.1516171819 In addition, Barsoum and colleagues20 have shown that baculovirus having the vesicular stomatitis virus G glycoprotein in its envelope significantly increases the efficiency of transduction of human hepatoma cell lines and broadens the range of mammalian cell types that can be transduced by baculoviruses. Also, stable transduction of mammalian cells by baculoviruses has been achieved by either including an expression cassette encoding a dominant selectable marker into baculovirus genome19 or by hybrid baculovirus–adeno- associated virus vector.21
In this study, we show that baculoviruses are able to mediate periadventitial gene transfer to rabbit carotid arteries with an efficiency comparable to adenoviruses. According to the best of our knowledge, this is the first report of in vivo gene transfer with baculoviruses. The ease of manipulation and rapid construction of recombinant baculoviruses,1022 the lack of cytotoxicity in mammalian cells even at a high multiplicity of infection,171823 an inherent incapability to replicate in mammalian cells,11 and a large capacity for the insertion of foreign sequences101824 make baculoviruses attractive tools for in vivo gene therapy.
Gene transfer in vitro
In order to test the baculovirus stock, RAASMC and ECV-304 cells were transduced at a multiplicity of infection (MOI) of 200 or 1000 p.f.u. per cell in the absence or presence of 10 mM sodium butyrate and the percentage of X-gal-positive cells were counted. The results were compared to cells transduced with LacZ-adenovirus under identical conditions (Table 1). In agreement with published results,19 addition of butyrate to cell cultures increased remarkably the expression of the transgene, especially with baculoviruses. The RAASMC cells seemed to be more susceptible to baculovirus transduction (91% infected at MOI 1000) than ECV-304 cells (21% infected at MOI 1000). Levels of β-galactosidase activity in the RAASMC cells were also measured by a quantitative biochemical assay with o-nitrophenyl β-D- galactopyranoside (ONPG). The results were in line with the X-gal staining showing an increase in the transgene expression after butyrate treatment in the baculovirus-transduced cells (Figure 1).
In vitro toxicity
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)-assay was used to measure the cytotoxicity of virus preparations (Table 2). Neither baculoviruses nor adenoviruses showed any major cytotoxicity to RAASMC cells in the absence of butyrate at MOI 200 or 1000. However, together with butyrate, some cytotoxicity was detected in these cells with baculoviruses at a MOI of 1000. Similar results were obtained with primary WHHL (Watanabe heritable hyperlipidemic) rabbit fibroblasts except that no cytotoxic effects were detected for baculoviruses in the presence of butyrate at a MOI of 1000 (data not shown).
Gene transfer in vivo
In order to determine whether baculoviruses can be used for in vivo gene transfer, NZW rabbits were killed 3, 7 and 14 days after gene transfer. Due to the nuclear targeting of the β-galactosidase expression, intense X-gal staining was located in the nuclei of the transduced cells (Figure 2). The number of cells positive for β-galactosidase activity was calculated from the baculovirus-transduced (1 × 109 p.f.u. per artery) carotid arteries and was compared with similarly treated adenovirus-transduced arteries. Both viruses mediated delivery of the marker gene to the vessel wall. The numbers of transgene- positive cells for baculovirus- and adenovirus-treated arteries at day 3 were 12 ± 5 and 23 ± 7 β-galactosidase positive cells/mm2 of adventitia (11 ± 4 and 19 ± 6/mm2 of the whole artery wall), respectively. At day 7 the corresponding values were 17 ± 6 and 22 ± 7 (15 ± 5 and 18 ± 6). At day 14 the values were 0.1 ± 0 and 0.2 ± 0.1 (0.1 ± 0 and 0.1 ± 0.1). The baculovirus-mediated gene expression was thus transient with a similar efficiency and time pattern as that of the adenovirus-mediated gene transfer. Transgene expression in the arterial wall was also verified with RT-PCR (Figure 3).
The mean value of intima/media ratio (all arteries) was 0.18 ± 0.03 for baculovirus and 0.12 ± 0.01 for adenovirus-treated arteries, which indicates that the procedures did not damage the vessel wall. Histology of the arteries 7 days after gene transfer is shown in Figure 4. No β-galactosidase activity was detected outside adventitia. With both viruses macrophage infiltrates and some T cells were detected in the transduced arteries by RAM-11 and MCA-805 immunostainings, respectively (Figure 4). The arterial structure and endothelium remained intact throughout the experiments (Figure 4). Histological findings at all time-points are summarized in Table 3.
Gene therapy offers an attractive alternative for the local treatment of various cardiovascular diseases including the prevention of post-angioplasty restenosis, post-bypass atherosclerosis, peripheral atherosclerotic vascular disease, stenosis of vascular prosthesis anastomoses and thrombus formation.125 In the present study, we show that baculoviruses can be used for in vivo gene transfer in blood vessels with efficiency comparable to adenoviruses, which are currently the most efficient gene delivery vectors for arterial gene therapy.7
Baculoviruses have several useful properties for gene transfer. In contrast to adenoviruses which cause benign respiratory infections,26 baculoviruses have a restricted host range. Neither gene expression nor DNA replication has been observed in mammalian cells after transduction with natural AcMNPV virus.12131415 Also, baculoviruses do not survive very well in human serum.2327 In terms of biosafety, these are useful properties as compared with adenoviral vectors.28 Another advantage is the ease of baculovirus construction. Baculovirus vectors are not dependent on helper viruses and high titer stocks (109 p.f.u./ml) can be prepared within 2–3 weeks2229 using a transposition-mediated virus genome preparation in Escherichia coli.30 The stability of the viruses at 4°C for at least 6 months further facilitates the virus handling.31 In agreement with that, we have stored virus stocks at 4°C for over 2 months without significant loss of infectivity (data not shown). Since the size of the nucleocapsid is flexible, recombinant baculovirus particles are able to expand to accommodate very large DNA fragments.10 As examples of this feature of baculoviruses, are recent reports of the expression of type 1 poliovirus18 and hepatitis C virus24 as integrated parts of baculovirus genome as well as construction of hybrid baculovirus-adeno- associated virus.21 The hybrid virus was capable of a site- specific integration into human chromosome 19, which is typical for adeno-associated virus.
Sandig and coworkers23 have recently reported unsuccessful attempts to use baculoviruses for in vivo gene delivery in mice and rats by systemic or intraportal application as well as by direct injection into the liver parenchyma. One reason for this is presumably the inactivation of baculoviruses by the classical pathway of serum complement system.2327 The collar-mediated732 local gene delivery method allows gene transfer essentially in the absence of serum thus avoiding deleterious effects of serum components. The method also avoids two other major problems encountered in systemic gene delivery, ie a rapid redistribution of the virus from the injection site and a drop in the local concentration of the virus.
As expected for an episomal DNA molecule,23 the baculovirus-mediated gene delivery led to a transient expression of LacZ in the rabbit artery reaching a high level in 3 days and staying constant for at least 1 week. Baculovirus-mediated gene transfer induced inflammatory responses comparable to adenoviruses in the rabbit carotid artery. It is likely that virion components alone are sufficient to trigger inflammatory responses leading to influx of macrophages.
Our in vitro results with RAASMC and ECV-304 cells support the earlier reports that baculoviruses are able to serve as gene-transfer vectors for transient expression of recombinant proteins in mammalian cells.1516171819 They are also in line with findings that butyrate in culture medium is able to increase markedly the level of baculovirus-mediated gene expression.19 However, the combination of butyrate and high MOI (1000) with baculoviruses seemed to be slightly cytotoxic to RAASMC cells, but not to primary WHHL fibroblasts. An interesting question that remains to be studied is whether the butyrate increased the transduction of cells per se or was the effect solely due to an increased level of expression of the transgene in the transduced cells. The later possibility, which is supported by the results of Condreay et al,19 would have enhanced the detection of X-gal-positive cells and led to an apparent increase in the calculated transduction efficiencies.
Although more work will be needed to characterize the safety and behavior of recombinant baculoviruses in vivo, our results demonstrate that they may become useful tools for gene therapy. The broad knowledge of baculovirus biology10 and AcMNPV genome33 should aid engineering of the improved second-generation viruses for gene transfer applications. The ease of construction and capacity to accept large foreign DNA fragments (>20 kbp) should aid development of baculoviruses with expanded or targeted cell tropism along with more stable, temporal or cell type-specific control of transgene expression.
Materials and methods
Preparation of recombinant baculoviruses
Viruses were constructed by using the transfer vector pFASTBac1 (pFB) (Gibco BRL, Life Technologies, Gaithersburg, MD, USA). A nuclear targeted β-galactosidase (βnt-Gal) cassette with a cytomegalovirus (CMV) promoter was inserted into the StuI site of pFB in reverse orientation with respect to the polyhedrin promoter, generating plasmid pFBCMV-βnt.
Recombinant viruses were generated by using a Bac-To-Bac Baculovirus Expression System (Gibco BRL). Viruses were amplified in Spodoptera frugiperda 9 (Sf9) suspension cultures (SF-900 medium, Gibco BRL) for 3 days using cell density of 2 × 106 cells/ml. For 50 ml of culture 200 μl of primary transfection supernatant was used as an inoculum. To obtain 1 liter of amplified virus 2 ml of amplified virus stock was used as an inoculum. The cell culture medium was centrifuged at 16000 g for 20 min at room temperature to remove cell debris. The clarified supernatant was transferred to ultracentrifuge tubes underlaid with 1.5 ml of 25% sucrose in phosphate-buffered saline (PBS) and the viruses were concentrated by centrifugation (120000 g, 4°C, 1.5 h). The virus pellets were resuspended into 35 ml of ice-cold PBS, transferred into ultracentrifuge tubes containing 3 ml of 25% sucrose in PBS and centrifuged as above. Final virus pellet was resuspended into 10 ml of cold PBS, filtered through the 0.45 μm filter and kept at 4°C protected from light for further use. Virus titer was determined by a plaque assay on Sf9 cells.10 Virus preparations were analyzed for lipopolysaccharide and bacteriological contaminants.34
Preparation of recombinant adenoviruses
Nuclear targeted LacZ encoding adenoviruses (pCMVnls-LacZ Ad5) were constructed and prepared as described earlier.7 Virus preparations were analyzed for replication-competent viruses, lipopolysaccharide and bacteriological contaminants as described.34
Gene transfer in vitro
Rabbit aortic RAASMC6 and human carcinoma/ endothelial cell-like ECV-304 cells (ATTC CRL-1998) were plated at the density of 10000 cells per well (Falcon Culture Slide, Becton Dickinson, Meylan, France). Cells were allowed to attach for 3 h before transduction in serum-free medium (DMEM, 100 units/ml of penicillin and 100 μg/ml of streptomycin, Gibco BRL). Viruses were added to medium at MOIs of 200 or 1000, and cells were incubated for 90 min at 37°C. After transduction, growth medium containing 10% fetal bovine serum was added either with or without 10 mM n-butyric acid (Sigma, St Louis, MD, USA). After 18 h incubation the media were removed and cells were washed three times with PBS. Cells were fixed with 1.25% glutaraldehyde for 15 min and washed three times with PBS. X-Gal (MBI Fermentas, Lithuania) staining solution (1 mg/ml, 2 mM MgCl2, 5 mM K3Fe(CN)6, 5 mM K4Fe(CN)6, 1 × PBS) was added to the cells and incubated for 3 h at 37°C. Cells were then washed with PBS and further fixed with 4% paraformaldehyde (PFA) for 10 min. After washing with PBS, cells were counter-stained with Mayers Carmalum for 5 min. Blue X-gal-positive cells were counted and the transduction efficiencies were expressed as the percentage of positive cells of the total number of cells.
LacZ-encoded β-galactosidase activity in transduced RAASMC cells was measured using a colorimetric substrate, o-nitrophenyl β-D-galactopyranoside (ONPG; Sigma), as described.35
In vitro toxicity assay
Cells were plated in 96-well plates at a density of 20000 cells per well in 100 μl of growth medium consisting of DMEM with 10% fetal bovine serum and antibiotics (100 units/ml of penicillin and 100 μg/ml of streptomycin). Virus transductions were performed as for the transduction efficiency assay and cells were incubated for 48 h at 37°C. Growth medium was removed and cells were washed with PBS. Serum-free DMEM without phenol red containing MTT solution (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide, 5 mg/ml, final concentration 0.3 mg/ml) was then added and cells were incubated for 2 h.36 To dissolve the dark blue formazan crystals, MTT solution was removed, 150 μl of 1 M DMSO was added to each well and mixed thoroughly. Absorbance was measured at 570 nm. Survival percentage was calculated as compared with absorbance of the no virus or no butyrate wells (100% survival).
Male New Zealand White (NZW) rabbits (n = 12; 2.8–3.7 kg) were used. Fentanyl-fluanisone (0.3 ml/kg, s.c.; Janssen Pharmaceutica, Beerse, Belgium) and midazolam (1.5 mg/kg, i.m.; Roche, Basel, Switzerland) were used for anesthesia. Left and right carotid arteries were exposed using midline neck incision. The artery was carefully separated from the surrounding tissue and a 3-cm long silastic collar (MediGene Oy, Kuopio, Finland) was positioned around it.732 Rabbits were re-anesthetized for gene transfer, which was performed 5 days after the installation of the collar exactly in the same way as in our previous study comparing the transfection efficiency of plasmid/liposomes, pseudotyped retroviruses and adenoviruses.7 The collars were opened and filled with 500 μl of the gene transfer solution containing 1 × 109 p.f.u. of adenovirus or baculovirus. In each animal the left carotid artery was used for adenovirus and the right carotid artery for baculovirus treatment. Four rabbits were killed 3, 7 and 14 days after the gene transfer and arteries were removed for histological analyses. All animal procedures were approved by Animal Care and Use Committee, University of Kuopio, Finland.
Collared arteries were divided into three equal parts: the proximal third was immersion-fixed in 4% PFA/15% sucrose (pH 7.4) for 4 h, rinsed in 15% sucrose (pH 7.4) overnight and embedded in paraffin. The medial part was immersion-fixed in 4% PFA/PBS (pH 7.4) for 30 min, rinsed in PBS (pH 7.2) and embedded in OCT compound (Miles Scientific, Naperville, IL, USA). The distal part was snap-frozen in liquid nitrogen and stored at −70°C for mRNA isolation and reverse transcriptase polymerase chain reaction (RT-PCR).
Randomly selected frozen sections (10 μm) from each rabbit were stained with X-gal (MBI Fermentas) for 18 h to identify β-galactosidase-positive cells.6 Gene transfer efficiency was calculated as X-gal-positive cells per mm2 of adventitia or whole artery wall in eight randomly selected sections by two independent observers.7 Mean values ± standard error of mean (s.e.m.) of the results are reported. Paraffin sections were used for immunocytochemical detection of endothelium (CD-31; 1:50 dilution; Dako, Hamburg, Germany), macrophages (RAM-11; 1:100 dilution; Dako), smooth muscle cells (HHF-35; 1:50 dilution; Dako), and T cells (MCA-805; 1:100 dilution; Dako) as described.678 Controls for the immunostainings included sections where the first antibody was omitted and sections incubated with class and species matched immunoglobulins. Morphometry and image analysis were done using hematoxylin–eosin-stained paraffin sections and Image-Pro Plus software with Olympus AX70 microscope (Olympus Optical, Japan).
Total RNA was extracted from transduced carotid artery segments using Trizol Reagent (Gibco-BRL) and treated with excess RQ1 RNAse-free DNAse (Promega, Madison, WI, USA). M-MulV reverse transcriptase (MBI Fermentas) was used for cDNA synthesis. Primers (20 pmol each) for lacZ were designed to distinguish between endogenous and transduced genes by selecting the 5′ primers from the CMV promoter and the 3′ primers from the coding region. Dynazyme polymerase (Finnzymes, Espoo, Finland) was used for amplification.
For lacZ amplification primers were: 5′-TTGGA GGCCTAGGCTTTTGC-3′ for adenovirus (A) and 5′-TTGGCCTAGAGTCGACGGAT-3′ for baculovirus (B) as forward primers, and 5′-TGAGGGGACGACGACAGTAT-3′ for both as a reverse primer. Hot start (95°C 5 min; 58°C 3 min) was followed by 39 cycles, each consisting of 95°C 1 min, 58°C 2 min, 72°C 3 min with the final extension of 10 min at 72°C. Five μl of the first PCR product was used for the second PCR with forward primers 5′-GGTAGAAGACCCCAAGGACTTT-3′ (A) and 5′-CCAAGAAGAAACGCAAAGTG-3′ (B). A reverse primer for both was 5′-CGCCATTCGCC ATTCAG-3′. The first PCR cycle was 95°C for 5 min followed by 20 cycles as in the first PCR.
We thank Ms Irene Helkala, Ms Maiju Jääskeläinen, Ms Mervi Nieminen, Ms Eila Pelkonen, Ms Mervi Riekkinen, and Ms Mari Supinen for expert technical assistance, and Ms Marja Poikolainen for preparing the manuscript. This study was supported by grants from the Academy of Finland, European Union Biomed Program (BMH4 CT-95-0329) and Kuopio University Hospital (EVO grant 5130).
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Virology Journal (2011)