Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis

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Abstract

Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe. Although combining direct DNA injection with in vivo electroporation increases the efficiency of gene transfer into muscle, applications of this method have remained limited because of the relatively low expression level. To overcome this problem, we developed a plasmid vector that expresses a secretory protein as a fusion protein with the noncytolytic immunoglobulin Fc portion and used it for electroporation-mediated viral interleukin 10 (vIL-10) expression in vivo. The fusion cytokine vIL-10/mutFc was successfully expressed and the peak serum concentration of vIL-10 was almost 100-fold (195 ng/ml) higher than with a non-fusion vIL-10 expression plasmid. The expressed fusion cytokine suppressed the phytohemagglutinin-induced IFN-γ production by human peripheral blood mononuclear cells and decreased the mortality in a mouse viral myocarditis model as effectively as vIL-10 expression. These results demonstrate that the transfer of plasmid DNA expressing a noncytolytic Fc-fusion cytokine is useful to deliver enhanced levels of cytokine without altering general biological activities. This simple and efficient system should provide a new approach to gene therapy for human diseases and prove very useful for investigating the function of newly discovered secretory protein genes.

Introduction

Recently, various cytokines and growth factors have been cloned in succession and their functions in vivo are intensively explored in many laboratories. However, there are several limitations to the extent to which their functions can be analyzed by the administration of recombinant protein. First, the half-life of these cytokines or growth factors is generally so short1 that high doses need to be administered repeatedly to obtain an effective concentration of cytokine. Such administration leads to the repetitive fluctuation between extremely high peak levels to basal levels of cytokine in the serum and may cause untoward systemic effects. Second, there is a risk that recombinant proteins are contaminated with impurities such as lipopolysaccharide, especially when the recombinant protein is obtained from bacteria. Such impurities, although present at low levels, can provoke unexpected immunological responses.2 Little attention has been given to these points in the past, and it is unlikely that all previous findings reflect the physiological functions of native cytokines or growth factors in vivo.345 In addition, these administrations require a large quantity of recombinant protein which takes time and considerable laborious procedures to prepare, therefore they might not be practical for use as a therapy from an economic point of view.

Recent progress in molecular biotechnology makes it possible to introduce exogenous genes into animal tissues and achieve efficient expression of the transferred gene. Viruses (adenovirus, retrovirus, adeno-associated virus and others) are widely used as a carrier to introduce exogenous genes into animal cells. While these vectors can efficiently transfer exogenous genes into target cells, it is still possible that the protein encoded by viral genes could induce immunological responses that might be inappropriately attributed to cytokine function.

On the other hand, direct injection of plasmid DNA into skeletal muscle has several advantages over transfer using viral vectors. A large quantity of highly purified plasmid DNA is easily and inexpensively obtained. Because there are fewer size constraints than with current viral vectors, plasmid vectors can carry larger genes. Most importantly, because there is less likelihood of inducing an immunological response than with viral vectors, plasmid vectors are suitable for analyzing the function of gene products that are related to the immune system. Several groups have shown the long-term systemic delivery of the products of transferred genes using this method and observed the function of the gene products in vivo (reviewed in Ref. 6).

Although combining direct injection with in vivo electroporation increases the efficiency of gene transfer into muscle,7 the application of this method is still limited because of the relatively low expression level of the transferred gene. To overcome this problem, we developed a plasmid vector that expresses secretory proteins as a noncytolytic immunoglobulin fusion protein and used it for in vivo electroporation-mediated transfer and expression of viral interleukin 10 (vIL-10) in vivo. Because we recently showed a therapeutic effect of the in vivo gene transfer of the immunosuppressive cytokine vIL-10 in a mouse viral myocarditis model,8 we tested the gene transfer of the noncytolytic vIL-10 fusion protein in the same model. We show that elevated expression of the cytokine can be achieved using this plasmid DNA-produced Fc-fusion protein and that this fusion protein does maintain the function of the native cytokine both in vitro and in vivo.

Results

Expression of transferred gene

pCAGGS-vIL10 and pCAGGS-vIL10/mutFc are plasmid vectors expressing vIL-10, and a fusion protein of vIL-10 and the Fc portion of mouse IgG2a with a noncytolytic mutation, respectively, under the cytomegalovirus immediate–early enhancer-chicken β actin hybrid (CAG) promoter (Figure 1). As shown in Figure 2, immunoblotting of the culture supernatant from BMT-10 cells transfected with pCAGGS-vIL10 revealed the successful expression of a 16-kDa protein, the correct size for vIL-10. Immunoblotting of the culture supernatant from BMT-10 cells transfected with pCAGGS-vIL10/mutFc showed that the band representing vIL-10 protein migrated under reducing conditions as a single 48-kDa band, which indicated that vIL-10 was fused with the Fcγ2a protein (32 kDa). Under non-reducing conditions, it migrated at a molecular size of 96 kDa, which indicated that the fusion protein existed as a homodimer, as expected.

Figure 1
figure1

Construction of fusion protein expression vector. (a) Site-directed mutagenesis of mouse Fcγ2a cDNA. Mutations were introduced in the CH2 domain of the Fcγ2a fragment to replace Glu 318, Lys 320, Lys 322 with Ala residues, and Leu 235 with Glu (see Materials and methods). XhoI–NotI sites were introduced just before the hinge sequence, to use as a cloning site for the cDNA to be fused with Fcγ2a. (b) Structure of the noncytolytic fusion cytokine vIL10/mutFc expression plasmid. Plasmid pCAGGS-mutFc was constructed by inserting the mutated Fcγ2a cDNA (described above) between the CAG promoter and a 3’-flanking sequence of the rabbit β-globin gene on the pCAGGS expression vector. The fragment encoding the vIL-10 cDNA has an XhoI site just before the start codon and a NotI site replacing its stop codon, and was introduced at the XhoI–NotI site of pCAGGS-mutFc to create the vIL-10/mutFc expression vector (pCAGGS-vIL10/mutFc).

Figure 2
figure2

Western blot analysis of the pCAGGS-vIL10/mutFc transfectant. The culture supernatant from BMT-10 cells transfected with pCAGGS (lane 1), pCAGGS-vIL10 (lane 2), and pCAGGS-vIL10/mutFc (lane 3) was subjected to SDS-PAGE under reducing conditions, and the supernatant from pCAGGS-vIL10/mutFc was also run under non-reducing conditions (lane 4). Recombinant vIL-10 was simultaneously loaded in lane r.

In vitro assay

To confirm that the fusion protein retains the biological activities of vIL-10, a cytokine synthesis inhibitory factor (CSIF) assay was performed with supernatants from transfected BMT-10 cells using human peripheral blood mononuclear cells (PBMCs). The results (Figure 3) showed that supernatant from pCAGGS-vIL10-transfected cells dose-dependently inhibited the phytohemagglutinin (PHA)-induced IFN-γ production by human PBMCs. Supernatant from pCAGGS-vIL10/mutFc-transfected cells inhibited the production of IFN-γ to a similar extent. Supernatant from non-transfected cells (mock) or pCAGGS (empty vector)-transfected cells had no effect in this assay. The vIL-10 concentrations as measured by ELISA were almost the same for the supernatants from both the pCAGGS-vIL10- and pCAGGS-vIL10/mutFc-transfected cells (not shown) and the supernatants showed similar levels of activity in the CSIF assay Figure 3. These data suggested that the fusion protein produced from pCAGGS-vIL10/mutFc possesses almost equivalent biological function as vIL-10 in vitro.

Figure 3
figure3

In vitro assay of fusion protein vIL10/mutFc. PBMCs were activated with PHA in the presence of the indicated concentrations of the supernatants from pCAGGS-, pCAGGS-vIL10-, pCAGGS-vIL10/mutFc-, mock-transfected BMT-10 cells, or recombinant vIL-10. After 72 h, the PBMC culture supernatants were harvested and assayed for human IFN-γ. *P < 0.001 versus the pCAGGS group. **P < 0.05 versus the pCAGGS-vIL10 (1%) group. ***P < 0.05 versus the pCAGGS-vIL10/mutFc (1%) group.

Gene transfer by in vivo electroporation

To examine whether the electroporation-mediated transfer of each expression vector could deliver the protein in vivo, 50 μg of plasmid (a total of 100 μg/mouse) was injected into the bilateral tibialis anterior muscles of a DBA/2 mouse (n = 5 for each group) (Figure 4). In the pCAGGS-vIL10 group, the serum level of vIL-10 reached approximately 2 ng/ml between days 5 to 8 followed by a gradual decrease. In contrast, in the pCAGGS-vIL10/mutFc group, the serum level of vIL-10 reached 6 ng/ml at day 2 and 195 ng/ml at day 5, which was almost 100-fold higher than the peak levels of the pCAGGS-vIL10 group. Note that these concentrations were determined by a vIL-10-specific ELISA and indicate the equivalent amounts of vIL-10 epitope recognized by the monoclonal antibody used in this ELISA. vIL-10 in the serum could be detected by ELISA for more than 2 weeks after a single injection in both groups. Control mice similarly treated with the empty vector did not show a detectable level of vIL-10. These results show that in vivo electroporation with pCAGGS-vIL10/mutFc produced a significantly higher expression of vIL-10 than is obtained by the same procedure using a plasmid expressing native vIL-10.

Figure 4
figure4

Time course of the serum concentration of vIL-10 after the in vivo electroporation-mediated gene transfer of pCAGGS-vIL10 and pCAGGS-vIL10/mutFc. Male DBA/2 mice were injected with 100 μg of pCAGGS-vIL10 (n = 5; open bars) or pCAGGS-vIL10/mutFc (n = 5; closed bars) into the bilateral TA muscle and electric pulses were delivered. The serum vIL-10 concentrations were examined by ELISA.

vIL-10 gene transfer for myocarditis

We next applied the in vivo electroporation technique to cytokine gene therapy for experimental myocarditis. Four-week-old DBA/2 male mice were inoculated with 10 p.f.u. of encephalomyocarditis virus (EMCV). EMCV-inoculated mice were immediately subjected to electroporation-mediated transfer of pCAGGS (empty vector-treated group), pCAGGS-vIL10 (vIL-10-treated group), pCAGGS-vIL10/mutFc (vIL-10/mutFc-treated group) plasmid DNA (100 μg/mouse), or carrier alone (PBS-treated group). Without treatment, fewer than 20% of these mice survived 14 days after inoculation. The empty vector-treated and PBS-treated groups showed almost the same survival rate as the untreated mice (not shown), indicating that the electroporation itself or vector alone had no effect. The vIL-10-treated group showed a significantly higher survival rate than the empty vector- or PBS-treated group (P < 0.05) (Figure 5). Similarly vIL-10/mutFc transfer also decreased the mortality of EMCV-inoculated mice (P < 0.05), with a tendency towards better survival than that seen for the vIL-10-treated group. In the vIL-10-treated, vIL-10/mutFc-treated, and control groups, heart sections were histologically evaluated on day 5, a time point at which inflammation had developed but no mice had died (Figure 6). The hearts of the vIL-10-treated and vIL-10/mutFc-treated groups showed small focal inflammation, while those of the empty vector-treated group showed massive inflammation. These data suggest that the gene transfer of vIL-10/mutFc had a therapeutic effect for viral myocarditis that was as good or better than that of vIL-10.

Figure 5
figure5

Survival curve after EMCV inoculation. Analysis of the survival curve revealed that the pCAGGS-vIL10-treated group (n = 16) showed significantly higher survival than the empty vector (n = 16) or the PBS-treated groups (n = 16) (P < 0.05). vIL-10/mutFc gene transfer (n = 16) also decreased the mortality of mice inoculated with EMCV (P < 0.05).

Figure 6
figure6

Histological examination. Figures show a representative Azan-Mallory staining of the heart sections at day 5 after EMCV inoculation for each group. The hearts of the pCAGGS-vIL10-treated and pCAGGS-vIL10/mutFc-treated groups showed small focal inflammation (stained light blue, which indicates inflammation), while that of the empty vector-treated group showed massive inflammation.

Cytokine expression in the heart

To clarify the mechanism of the favorable effects of vIL-10 and vIL-10/mutFc on viral myocarditis, the expression levels of inflammatory genes in the heart at day 5 were measured by the real-time quantitative PCR method (Figure 7). In the vIL-10-treated mice, the expression of IFN-γ and iNOS was repressed to 25% and 27%, respectively, of the levels seen in the empty vector-treated mice (IFN-γ, P < 0.01; iNOS, P < 0.01), while in the vIL-10/mutFc-treated mice, IFN-γ and iNOS were repressed to 23% and 35%, respectively, of the levels seen in the empty vector-treated mice (IFN-γ, P < 0.01; iNOS, P < 0.01). These data suggested that vIL-10/mutFc suppressed the expression of inflammatory genes in the heart by a similar mechanism as that of vIL-10.

Figure 7
figure7

Cytokine expression in the heart. Total RNA was extracted from the hearts of mice that had been treated with pCAGGS-vIL10, pCAGGS-vIL10/mutFc, or empty vector. RNA was reverse-transcribed and subjected to real-time quantitative PCR to determine the levels of mRNA of cytokines in the heart. In pCAGGS-vIL10-treated mice, the expression levels of IFN-γ and iNOS in the heart were significantly decreased (n = 5, vIL10-treated group versus empty vector-treated group, P < 0.01). In a similar fashion, pCAGGS-vIL10/mutFc-treated mice showed significantly decreased expression of IFN-γ and iNOS in the heart (n = 5, P < 0.01).

Discussion

Cytokines generally have a very short circulating half-life in vivo. For example, the circulating half-life time of IL-10 and IL-2 is <20 min9 and <5 min,10 respectively. Therefore repeated administration has been necessary to observe the effects of cytokines in vivo. However, the repeated administration of cytokine results in its fluctuation from extremely high peak levels to basal levels in the serum, which may cause untoward systemic effects. On the other hand, the intramuscular injection of plasmid DNA can provide a long-lasting delivery of proteins into the systemic circulation.1112 Furthermore, we have reported that combining direct DNA injection with in vivo electroporation markedly increases the efficiency of gene transfer into muscle. Mice that were transfected with IL-5 expression plasmid using the electroporation technique showed higher serum levels than did mice that received only DNA injection, and this effect persisted for 3 weeks.7 However, further improvements are needed to apply this method for cytokine delivery to larger animals.

Fusing cytokines,13 the extracellular regions of receptors,1415 or adhesion molecules16 to immunoglobulin has been observed to prolong their half-life in vitro and in vivo. One explanation for this is that the increased size conferred by the Fc region evades kidney clearance,1 and another is that the fusion protein is itself more stable.17 Applying this advantage of Fc fusion proteins to in vivo electroporation-mediated gene transfer, we achieved a high level of vIL-10 expression in the serum. We have observed a similar effect using other Fc-fused cytokines (unpublished data).

Fcγ2a, the portion of the Fc region that is fused to the cytokine, is able to bind effectively to cells expressing the high-affinity FcγRI receptor. It also possesses a complement (C1q) binding domain, and thus is able to facilitate Ab-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) activity. These activities may influence the immune system and lead to inappropriate interpretation of the experimental results on the function of cytokines. Thus, we have introduced mutations at both the C1q- and FcγRI-binding sites of the Fcγ2a domain by site-directed mutagenesis as done in previous reports.181920 These reports showed that the specific mutation introduced in the Fcγ2a CH2 domain markedly attenuated the Fcγ receptor I-binding activities18 and that the mutation in the C1q-binding site greatly diminished the ability of the Fcγ2a domain to activate complement.1920

To confirm that the fusion protein possessed the original function of the cytokine, we performed a CSIF assay.21 vIL-10 is considered to be a captured cellular cytokine of the Epstein–Barr virus and shares many of the anti-inflammatory properties of cellular IL-10.22 It inhibits IFN-γ production by stimulated mouse Th1 clone and also by stimulated human PBMCs.2324 Our CSIF assay showed that the expressed fusion protein vIL-10/mutFc suppressed the IFN-γ production of PHA-stimulated human PBMCs in a dose-dependent manner, to the same extent as that seen with vIL-10. Taken together, our results showed that pCAGGS-vIL10/mutFc expressed a fusion protein that has the same biological activities of vIL-10 in vitro.

Recently, Nakano et al8 have shown that in vivo gene transfer of vIL-10 has a therapeutic effect on EMCV-induced murine myocarditis. This effect was attributable to the inhibition of inflammatory cytokines, including IFN-γ, in the heart. To investigate whether expressed vIL-10/mutFc has the same effect as vIL-10 in vivo, we applied it to this myocarditis model.25 In our experiment, in vivo gene transfer of vIL-10/mutFc suppressed the expression of inflammatory cytokines in the heart in a manner similar to that of vIL-10, and prevented the mortality of myocarditis. These results show that vIL-10/mutFc has the same activity as vIL-10 in the EMCV myocarditis model. Although the gene transfer of pCAGGS-vIL10/mutFc showed a significantly higher expression of vIL-10 than the transfer of pCAGGS-vIL10, we could not see a significant difference in the survival rate between the vIL-10- and vIL-10/mutFc-treated groups. Thus, the enhanced expression of vIL-10 by pCAGGS-vIL10/mutFc did not further improve the preventive effect of pCAGGS-vIL10 on myocarditis. However, the fusion protein vIL10/mutFc had no additional untoward effects in vivo.

Although a higher peak concentration of serum vIL-10 was achieved by the in vivo electroporation of pCAGGS-vIL10/mutFc than by pCAGGS-vIL10, the stabilizing effect of Fc did not increase the duration of its expression, contrary to our expectations. As vIL-10 is not an innate protein, the high level of vIL-10/mutFc expression might activate the immune system against vIL-10, resulting in its rapid elimination.

In summary, we developed a plasmid vector expressing secretory proteins as a fusion protein with the Fc portion of immunoglobulin, and by applying this vector to vIL-10 expression by in vivo electroporation, we achieved an improved expression of vIL-10 without altering its activity. This system is suitable for the analysis of the in vivo function of cytokines and may provide a new approach to gene therapy for human diseases. Furthermore, because this method enables the delivery of a gene product that is unstable in vivo with low cost and with a minimum of labor, it should be very useful for investigating the functions of new candidates discovered from the human genome project.

Materials and methods

Plasmid DNA

Murine Fcγ2a cDNA was cloned by RT-PCR from the total RNA extracted from an IgG2a-secreting hybridoma cell line (a gift from Dr M Miyasaka, Osaka University, Osaka, Japan). To obtain the noncytolytic vIL-10/mutFc construct, PCR-mediated site-directed mutagenesis26 was used to replace the C1q-binding motif Glu318, Lys320, Lys322 with Ala residues. Similarly, Leu235 was replaced with Glu to inactivate the Fcγ receptor I-binding site. XhoI and NotI restriction sites were introduced upstream of the Fcγ2a sequence Figure 1a. These sites were used as cloning sites in the fusion protein expression plasmid. Plasmid pCAGGS-mutFc was constructed by inserting the above fragment between the CAG promoter and a 3’-flanking sequence of the rabbit β-globin gene on the pCAGGS expression vector.27 The vIL-10 cDNA was cloned from pcDSRα-BCRF28 by PCR with vIL-10 specific synthetic oligonucleotides. The 5’ oligonucleotide primer included XhoI restriction site just before the start codon (ATG), and the 3’ oligonucleotide primer included a mutation to eliminate the stop codon (TGA) and to modify the C-terminal Ala codon to accommodate the creation of a unique NotI site. The resulting vIL-10 fragment was introduced into the XhoI–NotI site of pCAGGS-mutFc described above to produce pCAGGS-vIL10/mutFc Figure 1b. Consequently, this plasmid bears a gene encoding a fusion product comprised of the cytokine component and the mutated Fcγ2a component intervened by three Ala residues. Plasmid pCAGGS-vIL10 was constructed by inserting the vIL-10 cDNA into the EcoRI site of the pCAGGS expression vector as described.8 All the fragments, including the sites of mutagenesis, were confirmed by sequencing. Plasmids were grown in Escherichia coli strain DH10B, extracted by the alkaline lysis method, and purified by two cycles of ethidium bromide-CsCl equilibrium density gradient ultracentrifugation.29 Plasmids were further purified by isopropanol precipitation, phenol extraction, phenol/chloroform extraction, and ethanol precipitation.

SDS-PAGE and Western blotting

pCAGGS-vIL10, pCAGGS-vIL10/mutFc, and pCAGGS were transfected into BMT-10 cells,30 with Lipofectamine2000 (GIBCO BRL, Gaithersburg, MD, USA). At day 2 after transfection, the culture supernatants were mixed with a one-third volume of SDS sample buffer (75 mM Tris-HCl, pH 6.8, 6% SDS, 15% glycerol, and 0.015% bromophenol blue) with or without 15% 2-mercaptoethanol, heated at 98°C for 5 min, and subjected to SDS-PAGE on a 12% polyacrylamide gel. Recombinant vIL-10 was simultaneously loaded on to the gel. Following electrophoresis, the samples were transferred on to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was incubated at room temperature for 3 h with biotinylated rat anti-IL-10 mAb (JES3-12G8; PharMingen, San Diego, CA, USA). After washing, the membrane was then incubated at room temperature for 1 h with streptoavidin-horseradish peroxidase conjugate (PharMingen), and was processed for autoradiography using chemiluminescence techniques (ECL kit; Amersham, Arlington Heights, IL, USA), according to the manufacturer's instructions.

In vitro assay

Human peripheral mononuclear cells were isolated from the buffy coats of a healthy donor by centrifugation over Ficoll–Paque (Amersham Pharmacia, Uppsala, Sweden) and cultured at 106 cells per ml with varying amounts of transfected BMT-10 supernatants or recombinant vIL-10 for 72 h in RPMI 1640 medium with 10% FCS, with or without 5 ng/ml PHA (Sigma, St Louis, MO, USA). Cultures were prepared in triplicate in 96-well plates, 200 μl per well. IFN-γ in culture supernatants was measured by an ELISA kit (Endogen, Woburn, MA, USA), according to the manufacturer's instruction.

In vivo electroporation

Mice were anesthetized with sodium pentobarbital. Plasmid DNA (50 μl in each side at a concentration of 1.0 μg/μl, for a total of 100 μg/mouse) was injected into the bilateral tibialis anterior muscles followed by in vivo electroporation as described previously.7 Briefly, a pair of electrode needles with a 5-mm gap was inserted into the muscle to a depth of 5 mm to encompass the DNA injection sites. Six pulses were delivered to the bilateral muscles by an electric pulse generator (Electro Square Porator T820M; BTX, San Diego, CA, USA) at a rate of one pulse/s, with each pulse being 50 ms in duration. In the infection study, DBA/2 mice were first inoculated intraperitoneally with 10 p.f.u. of the M variant of EMCV diluted with PBS, and gene transfer by in vivo electroporation was immediately performed.

ELISA

Serum samples were obtained from the tail veins of the mice. The concentration of vIL-10 was assayed as follows:31 96-well plates were coated with 2 μg/ml of the rat anti-IL-10 mAb (JES3-9D7; PharMingen) at 4°C overnight, and washed with PBS containing 1% bovine serum albumin at room temperature for 1 h. After additional washing with PBS containing 0.05% Tween (PBS/Tween), appropriately diluted samples were added to the wells. The plates were incubated at 4°C overnight and washed with PBS/Tween. Biotinylated rat anti-IL-10 mAb (2 μg/ml) (JES3-12G8; PharMingen) was added to the wells, and the plates were incubated at room temperature for 3 h with agitation. After washing with PBS/Tween, diluted streptavidin-horseradish peroxidase conjugate was added to the wells. The plates were incubated at room temperature for 30 min and washed with PBS/Tween. Substrate (o-phenylenediamine) was added to the wells and absorbance at 490 nm was measured on a microplate reader. Recombinant vIL-10 (PharMingen) was used as a standard, and the linear range of this ELISA system was from 30 to 2000 pg/ml. This ELISA system does not detect murine IL-10.

Murine model of myocarditis

Four-week-old inbred male DBA/2 mice were inoculated intraperitoneally with 10 p.f.u. of the M variant of EMCV diluted in phosphate-buffered saline (PBS). The day of inoculation was defined as day 0.

Histological examination

The hearts were fixed in 10% formalin, embedded in paraffin, sectioned, and stained with Azan-Mallory's stain.

Real-time PCR

Total RNA was extracted from the hearts of mice by the acid guanidine-phenol-chloroform method. The RNA was reverse-transcribed using an oligo-dT primer and Superscript II reverse transcriptase (GIBCO BRL, Gaithersburg, MD, USA). To determine the levels of the mRNA of cytokines in the heart, real-time quantitative PCR32 was performed using the ABI PRISM 7700 System (PE Biosystems, Foster City, CA, USA). The primer pairs and probes used for these analyses were as follows: IFN-γ forward primer, 5’-TCAAGTGGCATAGATGTGGAAGAA-3’; IFN-γ backward primer, 5’-TGGCTCTGCAGGATTTTCATG-3’; IFN-γ probe, 5’-TCACCATCCTTTTGCCAGTTCCTCCAG-3’; iNOS forward primer, 5’-CAGCTGGGCTGTACAAACCTT-3’; iNOS backward primer, 5’-CATTGGAAGTGAAGCGTTTCG-3’; iNOS probe, 5’-CGGGCAGCCTGTGAGACCTTTGA-3’; GAPDH forward primer, 5’-TTCACCACCATGGAGAAGGC-3’; GAPDH backward primer, 5’-GGCATGGACTGTGGTCATGA-3’; GAPDH probe, 5’-TGCATCCTGCACCACCAACTGCTTAG-3’.

These probes were designed to encompass the intron sequences to distinguish the appropriate PCR products from the products amplified from contaminating genomic DNA. The 5’ and 3’ ends of the probes were labeled with 6-carboxyfluorescein and 6-carboxytetramethylrhodamine, respectively.

Statistical analysis

Survival was analyzed by the Kaplan–Meier method. Statistical comparisons of survival rate were performed by the log-rank test. Student's t test was used to compare the level of cytokines and expression of cytokines in the heart between groups.

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Acknowledgements

We thank Dr Satwant Narula at Schering-Plough Research Institute for the generous gift of pcDSRα-BCRF. This work was supported by a grant from the Research for the Future Program (JSPS-RFTF97I00201) of the Japan Society for the Promotion of Science (JSPS). This work was also supported by a grant from the Ministry of Education, Science, Sports and Culture.

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Correspondence to J Miyazaki.

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Adachi, O., Nakano, A., Sato, O. et al. Gene transfer of Fc-fusion cytokine by in vivo electroporation: application to gene therapy for viral myocarditis. Gene Ther 9, 577–583 (2002) doi:10.1038/sj.gt.3301691

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Keywords

  • gene therapy
  • electroporation
  • naked DNA
  • fusion protein
  • viral IL-10
  • myocarditis

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