Strategies to increase longevity of adenoviral-mediated transgene expression in muscle

Adenoviral vectors are reasonably efficient at transducing a wide variety of tissues including lung, liver, and muscle. However, transgene expression is limited by a combination of innate immune responses, apoptosis, and cell-mediated immunity against vector-encoded proteins including the transgene. Recently, tumor necrosis factor (TNF) has been implicated as a critical cytokine in anti-adenoviral immune responses based on the fact that adenoviral-mediated transgene expression in TNF knockout mice. However, TNF knockout mice lack germinal centers in their lymph nodes and thus, it remains unclear whether these findings could be generalized to a therapeutic model. To investigate this point, we pre-treated mice with a single dose of either a polyclonal goat-anti rat TNF antibody or non-immune IgG prior to the administration of 10⁹ pfu of AdCMVLacZ into the tibialis anterior (TA) muscle. A subgroup of mice were treated with 10⁹ pfu of AdCMVLacZ and AdTNF-R, which encodes a TNF inhibitor consisting of a TNF-R:Fc fusion protein. Mice were sacrificed at 4, 14, or 28 days. At sacrifice, the TA was harvested, incubated in a PBS sucrose gradient, frozen in OCT and then sectioned. Sections were stained with X-gal solution and then counter-stained with safranin, or H and E. A blinded pathologist scored stained sections for gene transfer and inflammation. Mice receiving the anti-TNF had significantly less muscle injury as measured by the number of central nuclei. Moreover, there was significantly less inflammation at both 4 and 14 days. All TA muscles from the anti-TNF group demonstrated gene expression at 28 days whereas all control mice were negative beyond 14 days. These data demonstrate that TNF is a critical cytokine to anti-adenoviral immune responses in muscle. We speculate that transient blockade of TNF bioactivity may be used therapeutically to prolong transgene expression in vivo.