Transient immunosuppression by FK506 permits a sustained high-level dystrophin expression after adenovirus-mediated dystrophin minigene transfer to skeletal muscles of adult dystrophic (mdx) mice.

Adenovirus (AV)-mediated gene transfer into skeletal muscles of adult immune-competent animals has been limited by the fact that a cell-mediated immune attack of the host against transduced muscle fibers prevented efficient long-term transgene expression. More recently, various immunomodulating strategies have been shown to improve the longevity of transgene expression after AV-mediated gene transfer. In this study we treated adult dystrophic (mdx) mice with daily subcutaneous injections of the immunosuppressive drug FK506 (tacrolimus) over 5, 10, 30 and 60 days after AV-mediated dystrophin gene transfer and compared the transduction level with saline-injected mdx controls. We show that daily FK506 treatment after AV-mediated dystrophin gene transfer into adult mdx muscle results in the maintenance of the initial transgene expression for at least 2 months, even when FK506 treatment was discontinued after 1 month. This is in keeping with the marked reduction of inflammatory infiltrates and the reduced activation level (inducible nitric oxide synthase) of macrophages in adenoviral recombinant (AVR)-injected muscles of FK506-treated animals. Moreover, we find that FK506 efficiently suppresses the humoral immune response against both the vector proteins and the transgene protein product (dystrophin). Furthermore, we demonstrate that continuous FK506 treatment over 30 days significantly improves the efficiency of gene transfer when the same vector is readministered to an animal which had been transduced 20 days earlier. In conclusion, the data suggest that sensitization by the initial antigenic load of the AVR application plays a pivotal role in triggering the humoral and cellular immune response of the host, which can be significantly counteracted by relatively short-term immunosuppressive treatment. These findings have important implications for the design of future human trials for gene replacement therapy in Duchenne muscular dystrophy.