|Budget Amount *help
¥13,300,000 (Direct Cost: ¥13,300,000)
Fiscal Year 2002: ¥6,400,000 (Direct Cost: ¥6,400,000)
Fiscal Year 2001: ¥6,900,000 (Direct Cost: ¥6,900,000)
Among a number of techniques for gene transfer in vivo, the direct injection of plasmid DNA into muscle is simple, inexpensive and safe.
We attempted to treat viral myocarditis with cytokine gene therapy by transferring an inhibitory cytokine, IL-1 receptor antagonist (IL-1ra) or viral KL-10 (vIL-10), by in vivo electroporation, a new method for gene transfer into muscle, Four-week-old male DBA/2 mice were inoculated intraperitoneally with 10 PFU of encephalomyocarditis virus. Immediately after virus inoculation, an expression plasmid carrying IL-1ra or vIL-10 was injected into tibialis anterior muscles followed by electroporation. Serum levels of IL-1ra and vIL-10 reached 10.5 and 2.3 ng/ml, respectively, on day 5, when gene expression reached its peak.
Histopathological examination showed that myocardial cellular infiltration was improved in mice treated with IL-1ra or vIL-10 compared with the control group. On day 14 after the onset of myocarditis, transfer of IL-1ra or vIL-10 express
ion plasmid had significantly improved the survival rates of the animals. The expression of TNF-α was decreased to 0.60-fold (p < 0.005) and inducible nitric oxide synthase (iNOS) 0.43-fold (p < 0.005) by IL-1ran treatment, and the expression of IFN-γ in the heart was decreased to 0.35-fold (p < 0.05), and iNOS 0.21-fold (p < 0.005), by vIL-10 relative to the controls.
To increase the efficacy of gene transfer, 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 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 decreased the mortality in a mouse viral myocarditis model. 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 gene. Less