Most of the previous studies in which cytokine DNA plasmids were delivered by systemic administration exhibited only marginal therapeutic effects, if any, in the EAE model. One strategy to overcome this limitation would be to determine the optimal delivery route leading to significant beneficial effects both in early (prophylactic) and late (therapeutic) treatments. To address this issue, we directly compared the effects of intrasplenic (i.s.) and intramuscular (i.m.) electro-transfer of interleukin-4 (IL-4) DNA in the rat experimental allergic encephalomyelitis (EAE) model. In the preventive experiment, rats received i.m. (25 or 150 microg) or i.s. (25 microg) administration of IL-4 DNA followed by in vivo electroporation the day before MBP immunization. In the late treatment experiment, rats were treated with i.m. (150 microg) or i.s. (25 microg) administration of IL-4 DNA with electroporation 10 days after MBP immunization. As a control the same amount of vector DNA was used. Macroscopic analysis indicated that the onset of moderate to severe EAE in rats treated with i.s. transfer of 25 microg of IL-4 DNA was prevented on a significant level compared with i.m. 25 microg of the IL-4 DNA transfer group or the control group in the preventive experiments. More importantly, i.s. transfer of 25 microg of IL-4 DNA considerably suppressed the severity of EAE in late treatment experiments while i.m. transfer of 150 microg of IL-4 DNA had little effect. The MBP-specific expression of IFN-gamma from stimulated splenocytes was considerably decreased by the i.s. IL-4 DNA transfer group both in the preventive and therapeutic experiments while i.m. transfer had this effect only in the preventive protocol. Histological analysis showed that spinal cord inflammation was considerably reduced in the i.s. IL-4 DNA transfer group. These data provide the first demonstration that i.s. electro-transfer of IL-4 DNA is more effective both in the prevention and modulation of EAE than i.m. transfer and that i.s. electro-gene transfer may present a new approach to cytokine therapy in autoimmune diseases.