Co-expression of the complement regulatory proteins human DAF and CD59 with an IRES-mediated dicistronic mammalian vector enhances their cell protective effects.

The human complement regulatory proteins (hCRPs) decay accelerating factor (DAF/CD55) and protectin CD59 transfected into non-human cells could confer protection against human complement. The combination of DAF and CD59 would be an effective strategy to help overcome host complement-induced hyperacute rejection in xenotransplantation. We constructed a dicistronic mammalian expression vector pcDNA3-CD59IRESDAF by using the internal ribosomal entry sites (IRES) of the encephalomyocarditis virus (EMCV). RT-PCR, Western blotting and immunofluorescence microscopic analysis demonstrated that the EMCV IRES allowed for efficient co-expression of hCD59 and hDAF on the surface of NIH/3T3 cells transfected stably with pcDNA3-CD59IRESDAF. Human complement-mediated cytolysis assays showed that co-expressed DAF and CD59 proteins could provide more significant protection against complement-mediated cytolysis than either hCD59 or hDAF alone. These results suggest that IRES containing polycistronic vector should improve the efficiency and effectiveness of multi-gene delivery and that the construct pcDNA3-CD59IRESDAF vector has potential therapeutic value for effectively controlling complement activation and for preventing hyperacute rejection in clinical gene therapy.