Nonspecific Inflammation Inhibits Adenovirus-Mediated Pulmonary Gene Transfer and Expression Independent of Specific Acquired Immune Responses

Abstract

Replication-deficient adenovirus vectors (Avs) have shown high-efficiency gene transfer in a variety of animal models, but demonstrated lower than expected efficiency in the intensely inflammatory milieu of the respiratory tract of individuals with cystic fibrosis (CF). Specific acquired immune responses directed at adenovirus capsid proteins are known to limit the duration of transgene expression and the effectiveness of vector readministration. In these models, however, nonspecific inflammation is also frequently noted to accompany specific immune responses. Because inflammation can occur early after Av administration, we hypothesized that inflammation may block Av-mediated gene transfer in the lung independent of specific immune responses. To evaluate this hypothesis, we measured pulmonary gene transfer and expression in the absence or presence of the potent antiinflammatory agent dexamethasone. To address and eliminate concerns over the potentially confounding effects of systemic, vector-specific acquired immune responses, evaluations were confined to a 3-day period following Av administration and were carried out, in parallel, in normal and immunodeficient (athymic) mice. Dexamethasone significantly reduced Av-associated inflammation in all animals as measured by a significant reduction of blinded, quantitative lung histopathology scores and by reduced proinflammatory cytokine release. Concomitant with reduced inflammation, gene transfer efficiency was significantly increased in both normal and immunodeficient animals as measured by transgene product activity (β-galactosidase) in total lung homogenates 3 days after vector administration. This finding could not be explained by a direct effect of dexamethasone on transgene specific activity. To begin to understand the molecular mechanisms of Av-induced inflammatory responses, lung levels of the chemoattractive chemokines MIP-2, MIP-1α, and MCP-1 were quantified. All were elevated significantly in Av-exposed animals. Dexamethasone reduced levels of MCP-1 and MIP-1α, but not MIP-2, consistent with the observed pattern of inflammatory cell changes. Expression of several proinflammatory cytokines including TNF-α, IL-6, IL-1β, and IFN-γ were also elevated in Av-exposed animals and modulated by dexamethasone. These observations demonstrate that nonspecific inflammation is an important determinant of the efficiency of in vivo pulmonary gene transfer and expression independent of specific immune responses and may have important implications for human gene therapy for diseases of the lung. Adenovirus vectors have important potential for in vivo gene therapy. However, both animal studies and human clinical trials have demonstrated relatively low-efficiency gene transfer to airway epithelium in vivo and have also demonstrated induction of acquired, specific immune responses as well as nonspecific inflammatory host responses. While acquired immune responses to Av have been studied extensively, fewer studies have focused on nonspecific innate host responses. In this study, we evaluated the effect of nonspecific inflammation on the efficiency of pulmonary gene transfer and expression. To eliminate concerns about effects on acquired immune host responses, studies were also carried out in immunodeficient animals. Dexamethasone blocked inflammation in both normal and immunodeficient mice and significantly improved gene transfer and expression in the lung. These results demonstrate an important role for inflammation in reducing the effectiveness of in vivo gene transfer and expression to the lung.