Treatment with Soluble TNFR1 to Eliminate Ad Inflammation in Lung and Liver

The rationale for use of soluble TNF receptor as a modulator of adenovirus inflammation stems from the observation that TNFa is one of the principal mediators of inflammation after adenovirus gene therapy [41-43]. Neutralization of TNFa with TNFa inhibitors, such as soluble TNFR1 (sTNFRl) greatly reduces tissue injury and cell death after endotoxin and other inflammatory agents. This is a rational approach, since adenovirus takes advantage of TNFa as an immune mediator to promote expression of several immunosubversive proteins supporting its escape from immunosurveil-lance [5], The interaction of TNFa with its receptor is a strong virulence factor for inflammation and elimination of virus infection. The E3-gpl9 K protein not only prevents CTL recognition of Ad-infected fibroblasts by sequestering

MHC class I proteins in the endoplasmic reticulum, but also E3 proteins 10.4 K, 14.5 K, and 14.7 K function to protect infected cells from TNFa cytolysis. Transgenic mice that express the E3 gene encoding these proteins have been shown to exhibit decreased pulmonary infiltration after intranasal inoculation [42]. Peng et al. [43] showed that adenovirus gene transfer of an sTNFR results in effective blockade of tumor necrosis factor activity and also prolongs the gene therapy. Therefore, neutralization of TNFa is a rational approach to decrease chronic inflammation as well as prolonged transgene expression.

Certain cytokines such as TNFa can result in rapid clearance of adenovirus or viral therapy. Administration of anti-inflammatory cytokines, such as IL-10, can reduce inflammation and prolong gene therapy [44], We have evaluated the effect of the treatment with a novel TNF-binding protein (TNF-bp), a polyethylene-glycol (PEG)-linked dimer of sTNFRl, on inflammation of the lung and viral clearance after intranasal administration of AdCMVLacZ (1 x 1010 pfu) [45, 46] (Fig. 3, see color insert). Three days after intranasal administration, there was a moderate inflammatory infiltrate in the lungs of control (CT)-treated C57BL/6-+/4- mice, which peaked at day 7 and was nearly resolved by day 30 (Fig. 3A). In contrast, 3 days after administration of AdCMVLacZ, there was no evidence of an inflammatory infiltrate in the lungs of TNF-bp-treated C57BL/6-+/+ mice and only minimal evidence of infiltration was observed from day 3 through day 30. We next determined the expression of LacZ adenovirus gene-therapy product. The results indicate that the expression of p-galactosidase (ยก3-Gal) in control-treated C57BLI6-+/+ mice was high at day 3, but was considerably reduced by day 30 (Fig. 3B). The expression of the (3-Gal in TNF-bp-treated C57BL/6-4-/+ mice was equivalent at day 3 but, in contrast to the control-treated mice, the expression of |3-Gal remained high in the lung through day 30. These results indicate that there is greatly decreased inflammatory disease and prolonged gene expression in AdCMVLacZ-infected mice treated with TNF-bp compared to vehicle-treated mice. The results also indicate that TNFa is a key factor in the pathogenesis of inflammation in AdCMVLacZ-virus-infected mice. Thus, the TNF-bp PEG-linked dimer may be therapeutically useful in reducing the inflammatory response to adenovirus gene therapy.

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